cytoskeleton 

Lecture Outline Overview of the Major Functions of the Cytoskeleton 

I. Cytoskeleton - composed of 3 well-defined filamentous structures that form an elaborate interactive network; has functions analogous to that of skeleton (support cell & play key role in mediating cell movements)

 A. Each of the 3 cytoskeletal filaments is a polymer of protein subunits connected to one another by weak, noncovalent bonds 

1. Such construction lends itself to rapid assembly & disassembly, which is dependent upon complex cellular regulation

2. Each cytoskeletal element has distinct mechanical properties

B. The 3 types of cytoskeletal elements

1. Microtubules (MTs) - hollow, rigid cylindrical tubes with walls composed of tubulin subunits

2. Microfilaments (MFs) - solid, thinner structures composed of actin

3. Intermediate filaments (IFs) - tough, ropelike fibers composed of a variety of related proteins

C. Until recently, it was widely held that the cytoskeleton was a strictly eukaryotic invention that was absent from prokaryotic cells, however……

1. Numerous prokaryotes contain tubulin- & actin-like proteins that polymerize into cytoplasmic filaments that carry out cytoskeletal-like activities

2. Proteins distantly related to those of intermediate filaments have also been discovered in certain prokaryotes

3. Thus, it appears that all 3 types of cytoskeletal elements had their evolutionary roots in prokaryotic structures

II. Function in number of interrelated activities – they are highly dynamic; can do rapid & dramatic reorganization; often require accessory proteins that are not part of filaments

A. Dynamic scaffold; provides structural support that helps determine cell shape (ex.: RBC spectrin-actin) & resist forces that tend to deform it

1. The flat, rounded shape of many cultured cells depends on a radial array of MTs in cell cytoplasm

B. Internal framework that is responsible for positioning organelles in cell interior (ex.: polarized epithelial cells - organelles arranged in defined pattern along an axis from the apical to the basal end of the cell)

C. A network of tracks that direct the movement of materials & organelles within the cell

1. Delivery of mRNA molecules to specific parts of cell

2. Movement of membranous carriers from ER to Golgi complex

3. Transport of vesicles containing neurotransmitters down the length of nerve cell - carry vesicles from synthesis site to axon terminal

4. Peroxisome transport over tracks of microtubules; the two are closely associated as shown by labeling peroxisomes with GFP & microtubules with fluorescent red dye attached to tubulin antibodies

D. Force-generating apparatus - move cells from place to place (cilia, flagella, pseudopodia)

1. Single-celled organisms - crawl over surface of solid substratum or propel themselves through aqueous environment with aid of specialized locomotor organelles (cilia & flagella protruding from cell surface)

2. Multicellular animals have variety of cells capable of independent locomotion - sperm, white blood cells, fibroblasts, highly motile tip of growing axon (its movement resembles crawling blood cell)

E. mRNA anchoring sites that facilitate translation into polypeptides

1. Extract cell with nonionic detergent —> much of translation machinery (including mRNA) stays behind with detergent-insoluble cytoskeleton, so it is tied to cytoskeleton

F. Separation of chromosomes during mitosis & meiosis & cytokinesis (splitting parent cell into two daughter cells); essential component of cell's division machinery

The Study of the Cytoskeleton

I. Cytoskeleton is currently actively studied due largely to the development of techniques that allow a coordinated morphological, biochemical & molecular approach; thus, we know:

A. Much about the families of proteins that make up the cytoskeleton

B. How their subunits are organized into their respective fibrous structures

C. The molecular motors that generate the forces that allow the cytoskeleton to engage in motile activities

D. The dynamic qualities that control the spatial organization, assembly & disassembly of the various elements of the cytoskeleton

II. The use of live-cell fluorescence imaging – traditional light microscopy is tool used to learn about cell organization within fixed stained tissues & to observe the motile activities of living cells

A. New technique allows us to view things below resolution limit of regular light scopes (too small); used to study dynamics of cytoskeleton in live cells – fluorescence microscope; known as live-cell imaging

B. Make cytoskeletal structure protein subunits fluorescent – in vitro, covalently link small fluorescent dye to cytoskeleton (tubulin or keratin) subunit (conjugation)

1. Microinject labeled subunits into living cell & they are incorporated into polymeric form of protein, the growing cytoskeleton (microtubule or intermediate filament)

2. Follow fluorescent structure location over time as cell participates in normal activities

3. Unlike most other high-resolution techniques, cells can remain alive during observation

4. Shows dramatic changes in length & orientation of individual microtubules & other cytoskeletal elements

C. Can also fluorescently label proteins & make them in the cell by fusing their gene with that of the green fluorescent protein; makes what is called a fusion protein

D. If inject particularly small amounts of fluorescently labeled proteins, cytoskeletal filaments are no longer uniformly labeled, but instead contain irregularly spaced fluorescent speckles

1. They serve as fixed markers to follow dynamic changes in length or orientation of filament

2. The technique is called fluorescence speckle microscopy

E. Can also be used to detect location in cell of protein present in very low concentration

1. Make antibodies specific for protein in question; they bind this protein with high affinity

2. Antibodies are useful since they can distinguish between closely related variants (isoforms) of protein

3. Inject antibody into living cell —> see location & also discover function; since antibodies can interfere with normal function when bound to protein

4. Thus, if cell stops performing particular function, that protein is probably involved in it

5. Can also get location by adding fluorescent antibody to fixed cells or tissue sections

F. Resolution has become much better recently with development of confocal fluorescence microscope

1. It allows one to focus at a chosen level in the cell without background interference from fluorescent structures at other levels in the specimen

III. The use of video microscopy & focused laser beams & in vitro mobility assays

A. Advantages of using video camera, video tape recorder and television monitor

1. Digital video image has exceptional contrast; can be enhanced by computer processing

2. Can see, observe & photograph usually invisible objects that are well below light scope resolution, like 25-nm MTs & membrane vesicles (40 nm)

3. Video microscopy was used to visualize the growth & shrinkage of individual MTs as they gained or lost subunits in vivo; contributed to the understanding of their dynamic properties

B. High resolution video microscopy has led to development of a technique for detecting activity of individual protein molecules acting as molecular motors (in vitro motility assays)

1. Single-molecule assays have allowed researchers to make measurements not possible with standard biochemical techniques that average the results on large numbers of molecules

2. Attach MTs to glass cover slip, place micrometer-sized beads coated with motor proteins directly onto MTs with focused laser beams

3. The laser beams are shone through objective lens of a microscope, producing a weak attractive force near the point of focus; they can grasp microscopic objects & are thus called optical tweezers

4. Using ATP for energy under appropriate conditions, can watch bead move along MT on TV screen revealing the size of individual steps taken by motor protein

5. Can also trap a single bead & measure minute forces (a few piconewtons; pN) generated by single motor as it “tries” to move against force of optical tweezers (optical trap) holding it in position

IV. Development of techniques for working with single molecules has coincided with creation of new mechanical engineering field (nanotechnology)

A. Nanotechnologists involved in development of tiny nanomachines (10 - 100 nm size range); capable of performing specific activities in submicroscopic world; there are many perceived uses

B. May play role in medicine; someday might be introduced into human body where they could perform specific task, like finding & destroying individual cancer cells

C. Molecular motors are such nanosized machines evolved by Nature; investigators already working with them

V. The use of cells with altered gene expression - study phenotype of cells in which a particular polypeptide is either absent or nonfunctional; excellent way to study the function of a particular polypeptide

A. With development of recombinant DNA technology, scientists can create DNA molecules with the desired alteration & then incorporate the altered DNA into the genomes of lab animals or cultured cells

1. Don't have to wait for random mutations to test effect of absent or nonfunctional proteins

2. Now that genomes of numerous eukaryotes have been sequenced, can study role of any genes that might be required for a particular function (like cytoskeletal operation)

B. Analysis of fruit fly genome indicates that they possess genes encoding ~100 proteins that either function as motor proteins or interact with them

1. Any one of these genes can be isolated, modified & genetically inactivated

2. Gene inactivation effects on organism or cell are usually studied in 1 of 2 experimental approaches: gene knockout experiments & cells overexpressing a dominant negative mutant protein

C. Gene knockout animals (usually mice) lack a particular gene

1. Sometimes knockout mice may show little or no effects of a missing protein - tells little about the protein's possible function, besides the fact that it is nonessential

2. Sometimes mice lacking a particular gene may show highly specific defects, which strongly suggests that the missing protein plays an important role in the defective process

3. Knockout mice often die during early stages of development, but cells from these abnormal embryos can be isolated & cultured, allowing identification of the molecular deficiency

4. Example: mice lacking the motor protein cytoplasmic dynein do not develop past ~8 days of embryonic life; cells from embryo had fragmented Golgi complex dispersed throughout cytoplasm

a. Conclude that cytoplasmic dynein plays essential role in positioning Golgi complex in cell

D. Cells that overexpress dominant negative mutant protein - cells produce large amounts of nonfunctional protein; cells of this type generally produced by transfection

1. Cause cells to take up altered DNA & incorporate it into their chromosomes

2. Once cells are genetically modified, mutant protein (or protein fragment) either competes with normal protein or interferes in some other way with its function, causing cell to exhibit mutant phenotype

3. Example: treat control pigment cell from amphibian Xenopus with hormone that induces pigment granule dispersion into cell's peripheral processes; in the whole animal, this lightens the skin

a. If overexpress mutant version of motor protein (kinesin II) after treating with same hormone, pigment granules do not disperse —> suggests that kinesin II is responsible for granule movement

Microtubules (MTs): General Information, Structure and Function

I. Structure and composition - hollow, relatively rigid, tubular structures; found in most eukaryotic cells (mitotic spindle of dividing cell, core of cilia & flagella)

A. Structure - outer diameter of 24 nm; wall thickness of ~5 nm; may extend across cell length or breadth

1. Microtubule wall composed of globular proteins arranged in longitudinal rows (protofilaments)

2. Protofilaments are aligned parallel to tubule long axis

3. In cross section, seen to consist of 13 protofilaments aligned side-by-side in circular pattern within wall

4. Noncovalent interactions between adjacent protofilaments are thought to play an important role in maintaining microtubule structure

B. Each protofilament is assembled from dimeric building blocks (one a-tubulin & one b-tubulin subunit; a heterodimer); the tubulin dimers are organized in linear array along length of protofilament

1. The two types of globular tubulin subunits have similar 3D structure & fit tightly together

2. Protofilaments are asymmetric (a-tubulin at one end, b-tubulin at other); each assembly unit has 2 nonidentical components (heterodimer)

3. All protofilaments of microtubule have same polarity; thus so does full tubule (plus- & minus-end)

4. Plus end - fast-growing (b-tubulins on tip); minus end - slow-growing (a-tubulins on tip)

5. Structural polarity of microtubules is important factor in growth of these structures & their ability to participate in directed mechanical activities

II. MT-associated proteins (MAPs) - MTs assemble in vitro from purified tubulin, but MAPs are found with MTs isolated from cells; most found only in brain tissue; MAP4 has wide distribution in non-neuronal mammal cells

A. MAPs typically have one domain that attaches to MT side & another domain that projects outward as filament from MTs surface

B. Some MAPs are seen in EM as cross-bridges connecting MTs to each other, thus maintaining their parallel alignment; MAPs generally increase MT stability & promote their assembly

C. MT-binding activity of various MAPs is controlled mostly by addition & removal of phosphate groups from particular amino acid residues; example - Alzheimer’s disease (AD)

1. Abnormally high phosphorylation of one particular MAP (tau) is implicated in development of several fatal neurodegenerative diseases like AD

2. Brain cells of people with these diseases contain strange, tangled filaments (neurofibrillary tangles) made of excessively phosphorylated tau molecules that cannot bind to MTs; may help kill nerve cells

3. People with one of these diseases, a type of inherited dementia called FTDP-17, carry mutations in tau gene, indicating that alteration of tau protein is primary cause of disorder

III. Functions of MTs – structural supports & organizers

A. MTs are stiff enough to resist forces that might compress or bend the fiber, enabling them to provide mechanical support, like steel girders in tall building or poles in structure of tent

B. Cytoplasmic MT distribution in cell helps to determine cell shape

1. In cultured animal cells, MTs extend in radial array outward from area around nucleus, giving these cells their rounded, flattened shape

2. In contrast, MTs of columnar epithelial cells are typically oriented with their long axis parallel to long axis of cell; this configuration suggests that MTs help support the cell's elongated shape

C. Role of MTs as skeletal elements is evident in certain highly elongated cellular processes (axons of nerve cells & axopods of heliozoan protests, cilia & flagellae)

1. Nerve cell axon is filled with MTs that are oriented parallel to axon long axis; in mature axons, these MTs serve as tracks for movement of vesicles & other cytoplasmic particles

a. In developing embryo, MTs play key role in maintaining axon's extended shape as it slowly grows out of central NS into peripheral embryonic tissues

b. Axonal outgrowth followed in cell culture; nerve cell with growing axon treated with MT-disrupting drugs (colchicine [CO], nocodazole [NO]) —> outgrowth stops, collapses back to rounded cell body

2. Long, slender axopodial processes of heliozoan protists show MT role in maintenance of cell shape

a. Each axopod contains core structure composed of large numbers of MTs arranged in spiral with individual MTs traversing entire length of process

b. Treatments that promote the disassembly of the MTs lead to collapse of the axopodia

D. In plant cells, MTs play similar role; affect cell shape indirectly by influencing cell wall formation; during interphase, most of plant cell's MTs found just below plasma membrane forming a distinct cortical zone

1. Cortex MTs thought to affect movement of cellulose-synthesizing enzymes in cell membrane, which, in turn, affects cell wall growth & shape

2. Cellulose microfibrils of innermost cell wall layer assemble in orientation parallel to cortex MTs below

3. Cellulose microfibril orientation plays key role in determining cell growth characteristics & thus shape

4. In most cells, newly made cellulose microfibrils & coaligned MTs are arranged perpendicular (transversely) to cell long axis, like hoops around barrel

5. Since cellulose microfibrils resist lateral expansion, turgor pressure exerted by the cell is directed to cell ends —> cell elongates

E. MTs also thought to have role in maintenance of cell internal organization (organelle placement)

1. Treatment of cells with MT-disrupting drugs can seriously affect location of membranous organelles, particularly the Golgi complex

2. Golgi complex is typically located near center of animal cell, just outside nucleus; treatment of cells with NO or CO can disperse Golgi elements into cell's peripheral regions

3. When drug is removed & MTs reassemble, Golgi membranes return to normal position in cell interior

IV. Functions of MTs – MTs as agents of intracellular motility

A. Macromolecules & organelles move around cell in directed manner from place to place (intracellular motility)

1. Transport of materials from one membrane compartment to another depends on presence of MTs since specific disruption of MTs halts movements

B. Example: axonal transport – intracellular movement in nerve cells relies on highly organized arrangement of MTs & other cytoskeletal filaments; axon may stretch from spinal cord to tip of finger or toe

1. Proteins made in neuron cell body (rounded part of cell; neuron manufacturing center residing within spinal cord) move down axon in vesicles (neurotransmitters, etc.)

2. Inject labeled amino acids into cell body —> incorporated into labeled proteins that move into axon & gradually travel down its length

3. Many different materials, including neurotransmitters, are compartmentalized within membranous vesicles in ER & Golgi complex of cell body & then transported down length of axon

4. Different materials move at different rates; fastest rate is 5 µm/sec (400 mm/day); vesicles seen attached to MTs

C. Structures & materials moving toward neuron terminals from cell body move in anterograde direction

1. Other structures, like endocytic vesicles that form at neuron terminals & carry regulatory factors from target cells, move from synapse to cell body in the opposite or retrograde direction

2. Defects in both anterograde & retrograde transport have been linked to several neurological diseases

D. Axons filled with bundles of MTs, MFs & IFs interconnected in various ways

1. Evidence suggests that both anterograde & retrograde movement are mediated primarily by MTs; video microscopy shows vesicles moving along axon MTs either toward or away from cell body

2. Confirmed by EM of axons; molecular motors move vesicles along the MTs that serve as tracks

3. MTs are primarily passive structures that serve as tracks for a large number of motor proteins that generate the forces required to move objects within a cell

Microtubules: General Information on Microtubular Motors & In Vitro Motility

I. Motor proteins of cell - convert chemical energy stored in ATP into mechanical energy that is used to generate force or to move cellular cargo attached to the motor

A. Types of cellular cargo transported by these molecular motors include: ribonucleoprotein particles, vesicles, organelles (mitochondria, lysosomes, chloroplasts), chromosomes & other cytoskeletal filaments

B. A single cell may contain dozens of different motor proteins, each specialized for moving a particular type of cargo in a particular cell region

II. Collectively, motor proteins are grouped into 3 broad superfamilies: myosins, kinesins, dyneins

A. Kinesins & dyneins move along MTs; myosins move along MFs; none known that use IFs as tracks

B. Motor proteins move unidirectionally along their cytoskeletal track in a stepwise manner from one binding site to the next

III. As they move along, they undergo a series of conformational changes that constitute a mechanical cycle

A. Steps of mechanical cycle are coupled to steps of a chemical (or catalytic) cycle, which provide energy necessary to fuel the motor's activity

B. Steps include motor binding ATP, ATP hydrolysis, product (ADP & Pi) release & new ATP molecule binds binding/hydrolysis of 1 ATP moves motor a number of nanometers along track

1. As motor protein moves to successive sites along cytoskeletal polymer, both cycles repeated many times

2. This pulls the cargo over considerable distances

C. Molecular-sized motors, unlike human-made machines, are greatly influenced by their environment

1. Motor proteins have virtually no momentum (inertia) & are subjected to tremendous frictional resistance from their environment

2. Motor proteins come to a stop almost immediately once energy input has ceased

Microtubules: The Molecular Motors of Microtubules – Kinesins

I. Kinesins move vesicles/organelles from cell body to synaptic knobs; isolated in 1985 from cytoplasm of squid giant axons; the smallest & best understood microtubular motors

A. Kinesin is a tetramer constructed of 2 identical heavy chains & 2 identical light chains; a large protein that has several parts, including:

1. A pair of globular heads that bind a MT & generate force by hydrolyzing ATP

2. Each head (motor domain) is connected to a neck, a rodlike stalk & a fan-shaped tail that binds cargo to be hauled

3. Surprisingly, the motor domain of kinesin is strikingly similar in structure to that of myosin despite the fact that kinesin is a much smaller protein & the two types of motors operate over different tracks

4. Kinesins & myosins almost certainly evolved from a common ancestral protein present in a primitive eukaryotic cell

B. In vitro mobility assay – kinesin (purified)-coated beads move along individual MTs toward polymer "+" end (axon tip); it is a "+" end-directed MT motor; therefore, kinesin is responsible for anterograde movement

1. All MTs of axon are oriented with"-" ends facing cell body & "+" ends facing synaptic terminals

2. Thus kinesin transports vesicles, organelles & other cargo toward synaptic terminals

II. A single kinesin molecule moves through ATP-dependent cross-bridge cycle along single MT protofilament (rate proportional to [ATP]; up to a maximal velocity of ~1 µm/sec)

A. At low ATP concentrations, kinesins move slowly enough to see their movement in distinct steps

1. Each step is ~8 nm in length, which is also the spacing between tubulin dimers along protofilament & requires hydrolysis of a single ATP molecule; thus it moves 2 globular subunits (or 1 dimer at a time)

B. It is now generally accepted that kinesin moves by a "hand-over-hand" mechanism – basically similar to a person climbing a rope

1. The 2 heads alternate in taking the leading & lagging positions without an accompanying rotation of the stalk & cargo every step

C. Kinesin molecule movement is highly processive both in vitro & in vivo

1. This means motor protein tends to move long distances along individual MT without falling off (>1 µm)

2. 2-headed kinesin molecule can do this since at least one of the heads is attached to MT at all times

3. Such a motor protein is well adapted for independent, long-distance transport of small parcels of cargo

D. 2 heads of kinesin behave in coordinated manner, so that they are always present at different stages in their chemical & mechanical cycles at a given time

1. When one head binds to MT, the resulting conformational changes in adjacent neck region of motor protein cause the other head to move forward toward the next binding site on the protofilament

2. Catalytic activity of the head leads to a large-scale, swinging movement of the neck seen when the neck is attached to a molecule of GFP rather than to a second kinesin head

3. A kinesin molecule walks along a MT, hydrolyzing one ATP with each step

III. Conventional kinesin (kinesin-1; discovered in 1985) is only one member of a superfamily of related proteins (KRPs; kinesin-related proteins) or kinesin-like proteins (KLPs)

A. Mammalian genome sequence analysis leads to estimate that mammals make ~45 different KLPs, most of which can be found in brain

B. Heads of KLPs have related amino acid sequences, reflecting their common evolutionary ancestry & their similar role in moving along MTs

C. In contrast, KLP tails have diverse sequences, reflecting variety of cargo these different motors haul; a number of different proteins identified as potential adaptors that link specific KLPs & their cargoes

IV. Like kinesin-1, most KLPs move toward the "+" end of MT to which they are bound

A. One small subfamily (called kinesin-14), including the widely studied Drosophila Ncd protein, moves in opposite direction, toward "-" end of the microtubular track

B. One would expect that the heads of "+"- & "-"-directed KLPs would have a different structure, since the heads contain the catalytic core of the motor domain

C. But the heads of the 2 proteins are virtually indistinguishable; instead differences in direction of movement are determined by differences in the adjacent neck regions of the two proteins

1. When the head of a "-" end-directed Ncd molecule is joined to the neck & stalk portions of a kinesin-1 molecule, the hybrid protein moves toward the "+" end of a MT track

2. Even though the hybrid has a catalytic domain that would normally move toward the "-" end of a MT, as long as it is joined to the neck of a "+" end motor, it moves in the "+" direction

V. A third small subfamily (kinesin-13) of kinesin-like proteins is incapable of movement - KLPs of this group bind to either end of a microtubule & bring about its depolymerization rather than moving along its length

VI. Kinesin-mediated organelle transport – when vesicles move between organelles (like from Golgi to lysosomes), routes followed by cytoplasmic vesicles & organelles are largely defined by MTs

A. Members of kinesin superfamily are strongly implicated as force-generating agents that drive movement of membrane-bound cargo

B. In most cells as in axons, MTs are aligned with plus ends pointed away from center of cell

1. Thus, kinesin & kinesin-like proteins tend to move vesicles & organelles (peroxisomes, mitochondria) in outward direction toward cell's plasma membrane

C. Evidence – isolated cell from normal 9.5-day mouse embryo & stained it to reveal location of its MTs & mitochondria

1. Isolated another cell from a 9.5-day mouse embryo lacking both copies of gene encoding KIF5B kinesin heavy chain

2. Mitochondria of KIF5B-deficient cell are absent from peripheral regions of cell, as would be expected if this plus end-directed kinesin is responsible for outward movement of membranous organelles

Microtubules: The Molecular Motors of Microtubules – Cytoplasmic Dynein

I. First MT-associated motor found (1963); it was responsible for moving cilia & flagella & was called dynein; existence of cytoplasmic forms of dynein was suspected immediately

A. It took >20 years before a similar protein was purified & characterized from mammalian brain tissue & called cytoplasmic dynein

1. Thought to be ubiquitous eukaryotic motor protein; related protein found in variety of nonneural cells

2. While cytoplasmic dynein is present throughout the animal kingdom, there is controversy as to whether or not it is present in plants

3. Whereas each of us has many different kinesins (& myosins), each adapted for specific functions, we manage to function with only 2 cytoplasmic dyneins

4. One of these dyneins appears to be responsible for most transport operations

B. Cytoplasmic dynein is a huge protein (molecular mass of ~1.5 million daltons); made of 2 identical heavy chains & a variety of intermediate & light chains

1. Each dynein heavy chain consists of a large globular head with an elongated projection (called stalk); the dynein head is an order of magnitude larger than a kinesin head & acts as a force-generating engine

2. Each stalk contains the all-important MT-binding site situated at its tip

3. The longer projection, known as the stem (or tail), binds the intermediate & light chains, whose functions are not well defined

4. Structural analyses indicate that the dynein motor domain consists of a number of distinct modules organized in the shape of a wheel

5. This makes the dynein motor domain fundamentally different in both architecture & mode of operation from kinesin & myosin

C. In vitro motility assays indicate that cytoplasmic dynein moves processively along MT toward polymer's minus end — opposite the movement of kinesin

II. A body of evidence suggests 2 well-studied roles for cytoplasmic dynein

A. As a force-generating agent in positioning the spindle & moving chromosomes during mitosis

B. As a "-"-end-directed microtubular motor with a role in positioning the centrosome & Golgi complex & moving vesicles, particles & organelles through the cytoplasm

1. In nerve cells, cytoplasmic dynein implicated in retrograde membranous organelle movement (toward cell body & cell center) & anterograde movement of MTs

2. In fibroblasts & other nonneural cells, it may transport membranous organelles (endosomes, lysosomes, ER-derived vesicles heading toward Golgi complex) from peripheral locations toward cell center

3. Dynein-driven cargo also includes the HIV virus, which is transported to the nucleus of infected cell

III. Cytoplasmic dynein does not interact directly with membrane-bounded cargo, but requires an intervening multisubunit adaptor, dynactin

A. Dynactin may also regulate dynein activity & help bind the motor protein to MT, which increases processivity

IV. Present model may be overly simplistic - kinesin & cytoplasmic dynein move similar materials in opposite directions over the same railway network

A. Individual organelles may bind kinesin & dynein simultaneously although studies suggest that only one type of motor is active at any given time; myosin may also be present on some of these organelles

Microtubules: Microtubule-Organizing Centers (MTOCs)

I. Function of MT in living cell depends on its location & orientation, thus it is important to understand why a MT assembles in one place as opposed to another; controlled by MT-organizing centers (MTOCs)

II. Assembly of MTs from ab-dimers occurs in 2 distinct phases

A. Nucleation – slower phase; small portion of MT is initially formed; occurs in vivo in association with a variety of specialized structures, the microtubule-organizing centers (MTOCs); centrosome is best-studied

B. Elongation - more rapid phase

III. Centrosomes – a complex structure that contains 2 barrel-shaped centrioles surrounded by amorphous, electron dense pericentriolar material (PCM)

A. In animal cells, cytoskeleton MTs typically form in association with centrosome

B. Centrioles – cylindrical structures; ~0.2 µm in diameter & typically about twice as long; contain 9 evenly spaced fibrils

1. Each fibril appears in cross section to be composed of band of 3 fused MTs (A tubule, [the only complete MT]; B & C tubules), A is connected to center of centriole by radial spoke

2. 3 MTs of each triplet arranged in a pattern that gives a centriole a characteristic pinwheel appearance

3. Centrioles nearly always in pairs situated at right angles to each other near cell center just outside nucleus

C. Extraction of isolated centrosomes with 1 M potassium iodide removes ~90% of PCM protein, leaving behind spaghetti-like scaffold of insoluble fibers

D. Series of sections through cell shows that centrosomes are sites where large numbers of MTs converge

IV. Cytoskeleton MT origin in cultured animal cell is best studied by depolymerizing MTs with cold temperature or chemicals (NO, CO) & then following MT reassembly after cells warmed or chemicals removed

A. Disassembly of MTs (by cold or chemicals) & their reassembly can be followed by fixing cells at various times & staining them with fluorescent anti-tubulin antibodies

1. Within a few minutes of removal of inhibition, 1 or 2 bright fluorescent spots seen in cell cytoplasm

2. Within 15 - 30 minutes, number of labeled filaments radiating out of these foci increases dramatically

3. In EM - MTs seen to radiate out from centrosome; MTs don't actually penetrate into centrosome & contact centrioles, but terminate in dense pericentriolar material residing at centrosome periphery

4. PCM apparently initiates MT formation; although centrioles are not directly involved in MT nucleation, they probably play a role in recruiting the surrounding PCM during centrosome assembly

B. Thus, in experiment, centrosomes are shown to be sites of MT nucleation

1. Polarity of MTs is always the same; minus end is associated with centrosome & plus (growing) end is situated at opposite tip

2. Even though MTs are nucleated at MTOC, they are elongated at opposite end of polymer

3. Growing end of MT may contain specific proteins that help attach the MT to a particular target, like an endosome or Golgi cisterna in an interphase cell or a condensed chromosome in a mitotic cell

V. Centrosome is typically situated near the center of cell, just outside the nucleus - in columnar epithelial cells, centrosome migrates from cell center to a site in the apical region of the cell just beneath the cortex

VI. Not all animal cell MTs stay associated with centrosome

A. Many MTs in differentiated epithelial cells are anchored by "-" ends at dispersed sites near the cell's apical end with "+" ends extending toward cell's basal surface

B. Nerve cell axon has large numbers of MTs with no association with centrosome (found in neuron's cell body)

1. They are severed from centrosome, where they are first formed & then moved to axon by motor proteins

C. Some animal cells (mouse oocytes) lack centrosomes entirely, but can still make complex microtubular structures like meiotic spindle

VII. Basal bodies & other MTOCs - centrosomes are not the cell's only MTOCs; outer MTs in cilium or flagellum are generated directly from MTs in a structure called a basal body (resides at base of cilium or flagellum)

A. Basal bodies are identical in structure to centrioles; in fact, the two can give rise to one another

1. Sperm flagellum originates as a basal body derived from sperm centriole that had been part of meiotic spindle of spermatocyte from which the sperm arose

2. Conversely, sperm basal body typically becomes centriole during fertilized egg's first mitotic division

B. Plant MTOC – plant cells lack both centrioles & centrosomes or any other type of obvious MTOC; instead, MTs in plant cell are nucleated around the surface of the nucleus & widely throughout the cortex

VIII. MT nucleation

A. Despite their diverse appearances, all MTOCs play similar roles in all cells; they:

1. Control the number of MTs that form & their polarity

2. Control the number of protofilaments that make up the MT walls

3. Control the time & location of MT assembly

B. All MTOCs share a common protein component, g-tubulin (a type of tubulin discovered in mid-1980s); it is ~0.005% of total cell protein, while a- & b-tubulins are 2.5% of total nonneural cell protein

1. Fluorescent anti-g-tubulin antibodies (ABs) stain all MTOC types, like centrosome PCM; this suggests that it is a critical component in MT assembly & nucleation, which is supported by other studies

2. Microinject anti-g-tubulin ABs into living cell —> blocks MT reassembly after their depolymerization by drugs or cold temperature

C. MT nucleation mechanism revealed by structure & composition studies of PCM at centrosome periphery

1. Insoluble fibers of PCM are thought to serve as attachment sites for ring-shaped structures that have the same diameter as MTs (25 nm) & contain g-tubulin

2. The ring-shaped structures were found when centrosomes were purified & incubated with gold-labeled anti-g-tubulin ABs

3. Gold was clustered in rings or semi-circles at MT minus ends, the ends embedded in the PCM where nucleation occurs

4. Researchers can isolate similar ring-shaped complexes (g-TuRCs) from cell extracts; they nucleate MT assembly in vitro

D. The above studies have suggested the following model – a helical array of g-tubulin subunits forms an open, ring-shaped template on which the first row of ab-tubulin dimers assemble

1. In this model, only the a-tubulin of heterodimer can bind to the ring of g-subunits

2. Thus, the g-TuRC determines the polarity of the entire MT & also forms a cap at its minus end

E. 2 other tubulin isoforms d-tubulin & e-tubulin have also been identified in centrosomes, but their function has not been determined

Microtubules: Dynamic Properties of Microtubules

I. MTs vary markedly in stability even though they are quite similar morphologically & thought to be stabilized by presence of bound MAPs & certain post-translational modifications (e.g., tubulin subunit acetylation)

A. Spindle & cytoskeleton MTs are extremely labile (sensitive to disassembly)

B. Mature neuron MTs are much less labile

C. MTs of centrioles, cilia & flagella are highly stable

1. Ciliary, flagellar & centriolar MTs are stabilized by MAP attachment to wall of structure & by enzymatic modifications of tubulin subunits (e.g. addition of acetyl groups or glutamyl residues)

II. Labile MTs in living cells can be disassembled without disrupting other cell structures by many treatments; these treatments usually interfere with noncovalent bonds holding MTs together

A. Cold temperature

B. Hydrostatic pressure

C. Elevated Ca2+ concentration

D. A variety of chemicals (often used in chemotherapy) like colchicine, vinblastine, vincristine, nocodazole, podophyllotoxin

III. Treatments like the drug taxol disrupt MT dynamic activity by doing the opposite; they inhibit disassembly

A. Taxol binds MT polymer & thus prevents its disassembly; cell cannot assemble new needed MT structures

B. Many of these compounds, including taxol, are used in chemotherapy against cancer since they kill tumor cells preferentially

1. They were once thought effective against tumors since tumor cells were thought to be particularly sensitive to the drugs because of their high rate of cell division, but there is more to the story

2. Normal cells have a mechanism (or checkpoint) that stops mitosis in presence of spindle-altering drugs (vinblastine, taxol, etc.) & the cells usually arrest division activities until drug is eliminated from body

3. Many cancer cells lack this mitotic checkpoint & attempt to complete division even in absence of functional spindle —> usually results in death of tumor cell

IV. Cytoskeletal MT lability reflects fact that they are polymers formed by noncovalent association of dimer building blocks

V. MTs are subject to depolymerization or repolymerization as cell needs change, e.g., plant cells; if one follows a typical plant cell from one mitotic division to the next, 4 distinct arrays of MTs appear, one after another

A. During most of interphase, plant cell MTs are distributed widely throughout the cortex

1. g-tubulin (a nucleation factor, is found to be localized along the lengths of the cortical MTs, suggesting that new MTs might form directly on the surface of existing MTs

2. This is supported by studies of tubulin incorporation in living cells & in in vitro assays that show newly formed MTs branching at an angle off the sides of pre-existing MTs

3. Once formed, the daughter MTs are likely to be severed from the parent MT & incorporated into the parallel bundles that encircle the cell

B. As cell approaches mitosis, MTs disappear from most of cortex, leaving only a single transverse band (the preprophase band) that encircles the cell like a belt & marks the site of the future division plane

C. As cell enters mitosis, preprophase band is lost & MTs reappear in form of mitotic spindle

D. After the chromosomes have been separated, mitotic spindle disappears & is replaced by a MT bundle (phragmoplast) that plays a role in the formation of the cell wall that separates the two daughter cells

VI. These dramatic changes in MT spatial organization may occur by a combination of 2 separate mechanisms:

A. Rearrangement of existing MTs

B. Disassembly of existing MTs & reassembly of new ones in different cell regions

1. MTs making up the preprophase band are formed from same subunits that a few minutes earlier were part of the cortical array or, before that, the phragmoplast

VII. Study of MT dynamics (assembly & disassembly) in vitro gave insight into factors that influence rates of MT growth & shrinkage - suggest that cytoskeleton can rapidly remodel & respond to stimuli

A. Richard Weisenberg (Temple U., 1972) – came up with first successful approach to in vitro MT assembly

1. Reasoned that cell homogenates should possess all the macromolecules needed for MT assembly process

2. Thus, he homogenized brain (all components should be there) & got tubulin polymerization at 37°C by adding Mg2+, GTP & EGTA (binds Ca+2 that is an inhibitor of polymerization)

3. He found he could disassemble & reassemble the MTs repeatedly by lowering & raising temperature

4 Can assemble purified tubulin in vitro, but sometimes get only 11 protofilaments, probably since they lack the 13-subunit template normally provided by g-tubulin ring complexes in vivo

5. Addition of MT seeds (pieces of MTs or structures containing MTs) help by serving as template for the addition of free subunits

6. As occurs in vivo, tubulin subunits are added primarily to the plus end of the existing polymer

B. Early in vitro studies established that GTP is required for MT assembly

1. Tubulin dimer assembly requires that a GTP molecule be bound to b -tubulin subunit; GTP hydrolysis is not needed for actual incorporation of dimer onto end of MT

2. Rather, GTP is hydrolyzed to GDP shortly after the dimer is incorporated into a MT; the resulting GDP stays bound to the assembled polymer

3. After a dimer is released from MT during disassembly & enters the soluble pool, GDP is replaced by a new GTP; this nucleotide exchange thus recharges the dimer so that it can add to polymer again

4. A GTP molecule is also bound to the a-tubulin subunit, but it is not exchangeable & it is not hydrolyzed following subunit incorporation

C. Assembly is not energetically inexpensive, since it includes GTP hydrolysis: why did such a costly pathway of polymerization evolved? – to answer this, must consider effect of GTP hydrolysis on MT structure

1. When MT is growing, plus end is present (seen in EM) as an open sheet to which GTP-dimers are added

2. During rapid growth periods, tubulin dimers are added faster than their GTP can be hydrolyzed

3. The resultant cap of GTP-dimers on MTs at protofilament ends is thought to favor the addition of more subunits & hence MT growth

4. However, MTs with open ends are thought to undergo a spontaneous reaction leading to tube closure

5. In this model, tube closure is accompanied by hydrolysis of bound GTP, which generates subunits that contain GDP-bound tubulin

6. GDP-tubulin subunits have a different conformation from their GTP-tubulin precursors & are less suited to fit into a straight protofilament —> resulting mechanical strain destabilizes MTs

7. Strain energy is released as protofilaments curl outward from tubule & catastrophically depolymerize

D. Answer to above question – GTP hydrolysis is a fundamental component of the dynamic quality of MTs; it allows rapid adjustment of cell cytoskeleton to cell needs

1. GTP hydrolysis makes MTs inherently unstable & - in the absence of other stabilizing factors like MAPs – capable of disassembling soon after their formation

2. MTs can shrink remarkably fast, especially in vivo, allows very rapid cell microtubular cytoskeleton disassembly

VIII. Study of MT dynamics in vivo - dynamic character of microtubular cytoskeleton inside cell is best revealed by microinjecting fluorescently labeled tubulin into a nondividing cultured cell

A. Inject labeled tubulin into nondividing cultured cell —> labeled subunits are rapidly incorporated into preexisting cytoskeleton MTs, even in the absence of any obvious morphological change

B. Watch individual MTs in fluorescence microscope over time —> they grow for a period of time & then unexpectedly shrink

1. Since MTs shrink faster than they grow, in a matter of minutes, most MTs disappear from cell & are replaced by new MTs that grow out from centrosome

IX. Timothy Mitchison & Marc Kirschner (Univ. of Cal. – San Francisco, 1984) – proposed that MT behavior could be explained by dynamic instability

A. Dynamic instability refers to the fact that growing & shrinking MTs can coexist in same cell region & also to the fact that given MT can switch back & forth unpredictably between growing & shortening phases

B. Dynamic instability is a property of the plus end of the MT – subunits are added to the plus end during growth & lost from the plus end during shrinkage

1. Moreover, cells contain a diverse array of proteins that bind to the plus end of MTs & regulate their rates of growth or shrinkage & the frequency of interconversion between the 2 phases

2. Dynamic instability provides a mechanism by which the plus ends of MTs can rapidly explore the cytoplasm for appropriate sites of attachment

3. Attachment temporarily stabilizes MTs & allows the cell to build complex cytoskeletal networks

C. Dynamic instability allows cells to respond rapidly to changing conditions that require remodeling of the microtubular cytoskeleton

1. At mitosis, MTs of cytoskeleton are disassembled & remodeled into a bipolar mitotic spindle

2. This reorganization is associated with a marked change in MT stability; MTs in interphase cells have half-lives that are 5 – 10 times longer than MTs in mitotic cells

3. Unlike MTs of mitotic spindle or cytoskeleton, MTs of organelles (e.g., cilia, flagellae) lack dynamic activity & instead are highly stable

Microtubules: Cilia and Flagella - General Features and Function

I. General features of cilia & flagellae - hairlike, motile organelles projecting from many eukaryotic cell surfaces; prokaryotic flagella are simple filaments with no evolutionary relationship to their eukaryotic counterparts

A. Cilia & flagellae are essentially 2 versions of the same structure

B. Most biologists use one or the other term based on the type of cell from which the organelle projects & its pattern of movement

II. Cilium (like an oar) moves cell in a direction perpendicular to the cilium itself – rigid state in power stroke & pushes against surrounding medium, flexible in recovery stroke offering little resistance to medium

A. Tend to occur in large numbers on cell surface; their beating activity is usually coordinated

1. In unicellular organism, major function is moving the cell possessing them from place to place

2. In multicellular organisms, move fluid & particulate material through various tracts; ciliated epithelium lining human respiratory tract moves mucus & trapped debris away from lungs

B. Not all cilia are motile; many body cells contain a nonmotile cilium (primary cilium) that is thought to have a sensory function in monitoring the properties of extracellular fluids

III. Flagella - fewer on cell surface; usually longer than cilia; exhibit a variety of different beating patterns (waveforms) depending on the cell type

A. Single-celled alga - pulls itself forward (waves 2 flagellae in asymmetric manner like breast stroke of human swimmer); can also push itself through medium by using a symmetric beat like that of sperm

1. Degree of asymmetry in algal beat pattern is regulated by internal Ca2+ ion concentration

B. In sperm, beat is undulatory (>1 wave at a time along flagellum length); generates force pushing cell in direction parallel to flagellum long axis

Microtubules: Cilia and Flagella - Detailed Structure

I. Entire ciliary or flagellar projection is covered by a membrane continuous with the cell plasma membrane

A. Cilium core (axoneme) contains an array of MTs that runs longitudinally through entire organelle

1. Motile ciliary or flagellar axoneme, with rare exceptions, consists of 9 peripheral doublet MTs surrounding a central pair of single MTs; known as 9 + 2 pattern or array

2. This 9 + 2 pattern is seen in axonemes from protists to mammals; another reminder that all living eukaryotes have evolved from a common ancestor

3. All MTs in axoneme array have same polarity ("+" ends at tip of projection, "-" ends at base)

4. Peripheral doublets consist of 1 complete (A tubule; 13 subunits) MT & 1 incomplete (B tubule) MT with 10 or 11 subunits

B. Not all eukaryotes have them; cilia & flagella generally absent among fungi, nematodes & insects

C. Despite high degree of conservation (e.g. 9 + 2 pattern), there are some evolutionary departures:

1. 9 + 1 array in flatworms

2. 9 + 0 array in Asian horseshoe crab, eel, mayfly; some lacking central elements are motile, some not

II. Don Fawcett & Keith Porter (Harvard, 1954) - first described the basic structure of axoneme

A. Improvements in EM resolution revealed less obvious components & fine structure

1. Central MTs enclosed by projections forming central sheath, which is connected to peripheral doublet A MTs by set of radial spokes; doublets connected by interdoublet bridge made of elastic protein, nexin

2. A pair of arms (inner & outer) project from A MT in clockwise direction (when viewed base to tip)

B. MTs are continuous & other elements discontinuous in longitudinal section (cut through axoneme parallel to its long axis); also seen in improved EM

1. Radial spokes typically in groups of three with major repeat of 96 nm

2. Inner & outer dynein arms staggered along A MT length (outer arms spaced every 24 nm; inner arms arranged to match unequal spacing of radial spokes)

C. Cilia & flagellae emerge from basal bodies that are similar in structure to centrioles - 9 peripheral fibers consisting of 3 MTs rather than 2 (A tube complete, B & C tubes incomplete)

1. No central MTs in basal bodies as in centrioles; also similar to centrioles in other ways

2. A & B tubules of basal bodies elongate to form cilia & flagellae doublets

3. If cilium or flagellum is sheared off living cellsurface, new one is regenerated as basal body outgrowth

III. 1993 – researchers observed movement of particles in space between the peripheral doublets & surrounding plasma membrane

A. Subsequent studies revealed process known as intraflagellar transport, which relies on a highly conserved member of the kinesin superfamily (kinesin-II)

B. Intraflagellar transport moves complex arrays of building materials along protofilaments of peripheral doublets to assembly site at tip of growing axoneme

C. Kinesin-II molecules (& recycled axonemal proteins) are transported back toward basal body along the same flagellar MTs by a dynein-powered mechanism

D. Mutations in a number of genes involved in intraflagellar transport can have widespread consequences, like situs invertus, kidney disease, blindness (due to effectrs of nonmotile cilium found in photoreceptor cells

IV. Dynein arms – machinery for ciliary & flagellar locomotion resides within axoneme

A. Isolated sperm tail axoneme without a membrane is capable of normal, sustained beating in presence of added Mg2+ & ATP; the greater the [ATP], the faster the beat frequency of these reactivated organelles

B. Ian Gibbons (Harvard, 1960s) - isolated protein responsible for converting ATP chemical energy into ciliary locomotion mechanical energy; he chemically dissected cilia from the protozoan Tetrahymena

1. He began by dissolving the enclosing cell membrane with the detergent digitonin after shearing flagella off the organism

2. Isolated axonemes of insoluble fraction were then immersed in solution containing EDTA (a compound that binds & removes or chelates divalent cations like Mg2+/Ca2+)

3. When EDTA-treated axonemes were observed in EM, the central MTs were missing as were the arms projecting from the A MTs

4. Simultaneous with the loss of these structures, ATPase activity was lost in the insoluble axonemes & gained in the supernatant (part of huge, up to 2 million dalton protein)

5. Gibbons called it dynein (dyne = force; in = protein); now called ciliary or axonemal dynein to distinguish it from cytoplasmic dynein (related protein involved in organelle transport)

6. Mix insoluble axoneme parts with Mg2+ & soluble ATPase protein —> much ATPase again associated with axoneme as are arms with A MTs —> arms contain ATPase (releases energy for locomotion)

C. Later, treated isolated sperm axoneme with high salt (0.6 M NaCl) —> selectively removes outer arms, leaving inner arms in place

1. Add ATP to axonemes lacking outer arms, get normal wave form beating at about half normal rate of intact axoneme

2. They thus concluded that each arm generates half of the force used in motion; remaining inner arms are able to maintain beat albeit more slowly

D. EM examination of outer arms released by high salt from Tetrahymena showed them to contain:

1. 3 globular heads attached by slender stems to a common base, which is tightly anchored to A MT outer surface in the intact axoneme

2. These heads function as ATP-hydrolyzing cross-bridges projecting toward B MT of neighboring doublet

3. Inner arms extracted with harsher procedures found to consist of a number of distinct types containing either one or two globular heads

V. The mechanism of ciliary & flagellar locomotion

A. Sliding muscle filament model (actin filaments sliding over adjacent myosin filaments) suggested mechanism of ciliary/flagellar movement was sliding of adjacent MT doublets relative to one another

1. Sliding force in muscle is generated by ratchetlike cross-bridges that reside in head of myosin molecule

B. Proposed that ciliary movement happens by sliding of adjacent microtubular doublets relative to one another

1. Dynein arms act as swinging cross-bridges that generate forces needed for ciliary or flagellar movement

2. Dynein arms projecting from one doublet walk along adjacent doublet wall —> the two doublets slide relative to one another

C. Sequence of events in ciliary/flagellar sliding motion

1. Dynein arms anchored along A MT of lower doublet attach to binding sites on B MT of upper doublet

2. Dynein molecules undergo conformational change or power stroke; causes lower doublet to slide toward basal end of upper doublet

3. Dynein arms detach from B tubule of upper doublet

4. Dynein arms reattach to upper doublet so that another cycle can begin

D. Sliding on one side of axoneme alternates with sliding on other side so part of cilium or flagellum bends first one way, then in opposite direction

1. Thus, at given time, arms on one axoneme side are active, while those on other side are inactive

2. Thus, as result of this difference in dynein activity, doublets on inner side of bend extend beyond those on opposite side of axoneme; verified by EM of axoneme tips in different stages of bending cycle

E. Evidence for model has accumulated - MT sliding in beating flagellar axonemes has been directly visualized

1. Tiny gold beads were incubated with & attached to the exposed outer surface of peripheral doublets from isolated flagellar axoneme; beads served as fixed markers of specific sites on doublets

2. Relative bead position was then monitored as axonemes were stimulated to beat by addition of ATP

3. As axonemes bent back & forth, distances between beads on different doublets increased & decreased in alternating manner as expected if neighboring doublets slid up & down over one another

Intermediate Filaments

I. General traits – for many years, solid, smooth-surfaced, unbranched filaments seen in EM, ~10 nm dia (between MFs & MTs); called intermediate filaments (IFs) because of their dimensions relative to MTs & MFs

A. So far, IFs are only identified with certainty in animal cells & radiate through cytoplasm of wide variety of animal cells; often interconnected to other cytoskeletal filaments by thin, wispy cross-bridges

1. In many cells, cross-bridges made of huge, elongated protein (plectin); it can exist in numerous isoforms

2. Each plectin molecule has an IF binding site at one end & depending upon isoform, a binding site for another IF, MT or MF at the other end

B. IFs are chemically heterogeneous group of structures unlike MFs & MTs - >65 different genes encode them in humans

1. IF polypeptide subunits are divided into 5 major classes

2. Classification based on the type of cell in which they are found, as well as biochemical, genetic & immunologic criteria

C. Most, if not all, of these polypeptides have similar arrangement of domains that allows them to form similar looking filaments

1. Each IF polypeptide has central, rod-shaped, fibrous a-helical domain of homologous amino acid sequence & similar length, flanked on each side by globular domains of variable size & sequence

2. 2 such polypeptides spontaneously interact as their a-helical rods wrap around each other to form ropelike dimer (~45 nm long) with N-terminal to C-terminal polarity (aligned parallel)

3. Since the 2 polypeptides are aligned parallel to one another in the same orientation, the dimer has polarity with one end defined by the C-termini of polypeptides & the opposite end by their N-termini

II. Intermediate filament assembly & disassembly

A. Basic unit of IF assembly is thought to be a tetramer formed by 2 dimers that become aligned side-by-side in a staggered fashion with their N- & C-termini pointing in opposite (antiparallel) directions

1. Since dimers point in opposite directions, the tetramer itself lacks polarity

2. Tetramers associate with one another both side-to-side & end-to-end to form poorly described intermediates that assemble into the final filament

3. Since tetrameric building blocks lack polarity so does the assembled filament; again unlike MFs & MTs

B. IFs are highly resistant to tensile (pulling) forces, like ECM's collagen (also made of staggered subunits)

1. Skin outer layer made of dead epidermal cells; made almost entirely of dense, keratin-containing IF mat

2. These filaments help to make skin airtight, watertight, & resistant to bacteria & many chemicals

C. IFs tend to be more resistant to chemical disruption than other cytoskeletal elements & harder to solubilize

1. Due to their insolubility, IFs were first thought to be permanent & unchanging structures, but they behave dynamically in vivo

D. Inject labeled keratin subunits into cultured skin cells —> rapidly incorporate into existing IFs; subunits surprisingly are not incorporated at filament ends as in MTs & MFs, but rather in filament interior

1. Initially, filaments become labeled at scattered sites along their length, but within an hour or so, the entire IF network is labeled

2. This suggests that epidermal cells contain a pool of keratin subunits that (like MT & MF subunits) are in a dynamic equilibrium with the polymerized form; similar results seen in neuron IFs

E. Assembly & disassembly of most types of IFs are controlled by phosphorylation & dephosphorylation of the subunits; example: phosphorylation of vimentin filaments by protein kinase A leads to their disassembly

III. Types of intermediate filaments

A. Keratin filaments constitute primary structural proteins of epithelial cells (epidermal cells, liver hepatocytes, pancreatic acinar cells)

1. Keratin-containing IF bindles form an elaborate, cagelike network around nucleus & radiate through cytoplasm

2. Many of them terminate in the cytoplasmic plaques of desmosomes & hemidesmosomes that attach these cells to other cells & to the underlying basement membrane

B. Neuron cytoplasm contains loosely packed IF bundles whose long axes are oriented parallel to that of axon; these IFs (neurofilaments, NFs) are made of 3 distinct proteins: NF-L, NF-H & NF-M, all of type IV group

1. Unlike other IF polypeptides, NF-H & NF-M have sidearms that project outward from NF; the sidearms are thought to maintain proper spacing between the parallel NFs of the axon

2. In early stages of differentiation when axon is growing toward a target cell, it contains very few NFs, but large numbers of supporting MTs

3. Once the nerve cell has become fully extended, it becomes filled with NFs that provide support as the axon increases dramatically in diameter

4. Aggregation of NFs is seen in several human neurodegenerative disorders, including Parkinson's disease & ALS; such NF aggregates may block axonal transport, leading to death of affected neurons

IV. Recent efforts to probe IF function have relied on genetically engineered mice that fail to make particular IF polypeptide (gene knockout) or make altered IF polypeptide; reveal importance of IFs in particular cell types

A. Mice carrying deletions in gene encoding K14, a type I keratin polypeptide normally synthesized by cells of basal epidermal layer have serious health problems; the mice are very sensitive to mechanical pressure

1. Even mild trauma (passage through birth canal, newborn nursing) —> severe skin or tongue blistering

2. Phenotype similar to epidermolysis bullosa simplex (EBS; rare human skin-blistering disease; caused by defects in gene encoding homologous K14 polypeptide or K5 polypeptide (dimerizes with K14)

3. Studies confirm IF role in imparting mechanical strength to cells situated in epithelial layers

B. Similarly, knockout mice that do not produce desmin polypeptide exhibit serious cardiac & skeletal muscle abnormalities; desmin plays key structural role in alignment of myofibrils of muscle cell

1. Absence of these IFs makes the cells extremely fragile

2. An inherited human disease (desmin-related myopathy) is caused by mutations in gene encoding desmin; symptoms - skeletal muscle weakness, cardiac arrhythmias & eventual congestive heart failure

C. Not all IF polypeptides have such essential functions

1. Mice lacking vimentin gene (expressed in fibroblasts, macrophages, white blood cells) show no obvious abnormalities even though the affected cells lack cytoplasmic IFs

2. Conclude that IFs have tissue-specific functions that are more important in some cells than others

V. Other studies have used transgenic mice whose nerve cells produce 3 - 4 times the normal quantity of NF-L, one of the neurofilament polypeptides

A. Overexpressing this protein leads to gradual accumulation of greater-than-normal NF numbers; impedes normal movement of materials & organelles down axon —> causes nerve cell degeneration, muscle atrophy

B. Accumulation & abnormal assembly of NFs are also seen in motor neurons of patients with certain degenerative neuromuscular diseases, including ALS (Lou Gehrig's disease)

1. Not clear whether these cytoskeletal abnormalities are a cause of or a byproduct of the disease

2. A small fraction of ALS patients has been found to have a mutation in NF-H gene; suggests that disruption of NF structure is one of several primary causes of ALS

Microfilaments: General Features and Structure

I. Many cells are remarkably motile; cells that move around & exhibit other types of motility all need MFs; examples mof MF-dependent processes appear below

A. Neural crest cells in vertebrate embryo leave developing nervous system & migrate across entire width of embryo, forming such diverse products as skin pigment cells, teeth, cartilage of jaws

B. Legions of white blood cells patrol body tissues searching for debris & microorganisms

C. Certain parts of cells can be motile

1. Broad projections of epithelial cells at wound edge act as motile devices that pull the cell sheet over the damaged area, sealing the wound

2. Leading edge of growing axon sends out microscopic processes that survey the substratum & guide the cell toward a synaptic target

D. MFs are also involved in intracellular motile processes like vesicle movement, phagocytosis & cytokinesis

1. Plant cells seem to rely mostly on MFs, rather than MTs for long-distance transport of cytoplasmic vesicles & organelles

2. This bias toward MF-based motility reflects the rather restricted MT distribution in many plant cells

II. MFs (F-actin) - ~8 nm diameter; made of globular actin subunits (G-actin); found in most animal cells, also higher plants; MF, actin filament, & F-actin are synonyms, but F-actin is often used for those formed in vitro

A. In presence of ATP, actin monomers (G-actin) polymerize to form flexible, helical filament

1. As a result of its subunit organization, an actin filament is essentially a two-stranded structure with 2 helical grooves running along its length

2. Each actin subunit has polarity & all actin filament subunits are pointed in same direction, so entire MF has polarity

B. Depending on cell type & activity in which it is engaged, MFs can be organized into highly ordered arrays, loose ill-defined networks, or tightly anchored bundles

III. Actin can be detected in a given cell using a cytochemical test that takes advantage of the fact that actin filaments, regardless of source, will interact in a highly specific manner with the protein myosin

A. Purified myosin (obtained from muscle tissue) is cleaved into fragments using proteolytic enzymes - produce fragments called heavy meromyosin (HMM) or the smaller S1 subfragment

1. S1 binds to actin molecules all along MF, identifying filaments as actin & revealing filament's polarity

2. When S1 fragments are bound, one end of MF is pointed like an arrowhead; other end looks barbed

3. Orientation of arrowheads formed by S1-actin complex provides information as to the direction in which the MFs are likely to be moved by a myosin motor protein

B. Also, can see actin MFs by light miroscopy with fluorescently labeled phalloidin, which binds to actin filaments, or by fluorescently labeled anti-actin antibodies

IV. Actin was identified more than 50 years ago as one of the major contractile proteins of muscle cells

A. Since then, actin has been found to be a major protein in virtually every eukaryotic cell examined

B. Higher plant & animal species possess a number of actin-coding genes whose encoded products are specialized for different types of motile processes

C. Actins have been remarkably conserved during eukaryotic evolution (yeast cell actin & rabbit skeletal actin amino acid sequences are 88% identical); this means that nearly all aminos are crucial to function

1. Actins from diverse sources can copolymerize to form hybrid filaments

Microfilaments: Assembly & Disassembly

I. Before incorporation into a filament, an actin monomer binds a molecule of ATP (like tubulin binds GTP)

A. Actin is an ATPase (like tubulin is GTPase); ATP role in MF assembly is same as GTP in MT assembly; some time after monomer incorporation into growing actin filament, its ATP is hydrolyzed to ADP

B. If filaments are built at a high rate, the filament end has an actin-ATP subunit cap, which hinders filament disassembly & favors continued assembly

1. When MFs are incubated in vitro with a high concentration of labeled actin-ATP subunits, both ends of the MF become labeled but .....

2. One end incorporates monomers at 5 - 10 times higher rate than the other end

C. Decoration of MFs with S1 myosin fragment reveals that barbed (or plus) end of MF is fast-growing end, while the pointed (or minus) end is the slow-growing tip

1. Conversely, minus (pointed) end is preferential depolymerization site so subunits treadmill through MF

2. Actin-ATP subunits add to MF plus end & actin-ADP subunits tend to leave from minus end, so individual monomers move down length of MF in vitro (treadmilling)

3. Studies of live cells containing fluorescently labeled actin subunits support occurrence of treadmilling in vivo, but have also provided evidence that actin filaments exhibit dynamic instability similar to MTs

4. Dynamic instability is characterized by coexistence of randomly growing & shortening filaments

II. Cells maintain a dynamic equilibrium between monomeric & polymeric forms of actin, just as happens with tubulin & MTs – rate of assembly & disassembly can be influenced by a variety of different accessory proteins

A. Changes in local conditions in particular part of cell can push equilibrium either toward assembly or disassembly - allows cell to reorganize its MF cytoskeleton by controlling this equilibrium

1. Need such reorganization for dynamic processes (cell locomotion & cell shape changes, cytokinesis)

B. Can show MFs involved in process by treating with MF inhibitors; treat with inhibitors —> process stops; MF-mediated cell processes rapidly grind to halt when cells are exposed to such compounds (ex.: filopodia)

1. Cytochalasins (group of compounds extracted from mold cells) - promote MF depolymerization; block the plus ends of actin filaments, allowing depolymerization at the minus end

2. Phalloidin (poisonous mushroom extract) - stabilizes MFs so cells can no longer use the structures in its dynamic activities; binds to intact actin filaments & prevents their turnover

3. Latrunculin (obtained from sponge) – binds to free monomers & blocks their incorporation into the polymer

C. Such experiments have shown that actin filaments play a role in nearly all of a cell's motile processes

Microfilaments: Myosin - The Molecular Motor for Actin Microfilaments

I. Myosin's sole known function is as a motor for actin; all motors known to interact with actin are members of myosin superfamily; with major exception of myosin VI, myosins that move toward actin filament plus end

A. First isolated from mammalian skeletal muscle tissue & later from a wide variety of eukaryotic cells: protists, plants, nonmuscle animal cells, vertebrate cardiac & smooth muscle tissues)

B. Structure of myosins – all of them share characteristic motor (head) domain, which has a site that binds the actin filament & one that binds & hydrolyzes ATP to drive the myosin motor

1. While the head domains of myosins are similar, the tail domains are highly divergent

2. Myosins also contain a variety of low molecular weight (light) chains

C. Myosins are generally divided into 2 broad groups – conventional (or type II; first found in muscle tissue) & unconventional myosins

1. Unconventional myosins are subdivided on the basis of amino acid sequence into at least 17 different classes (type I & types III - XVIII)

2. Some of these classes are expressed widely among eukaryotes, whereas others are restricted

3. Myosin X is found only in vertebrates; myosins VIII & XI are present only in plants

4. Humans have ~40 different myosins, each presumed to have its own specialized function(s); type II myosin molecules are best understood; several types of myosins may be present together in same cell

II. Conventional (type II) myosins – myosin II class proteins are the primary motors for muscle contraction but are also found in a variety of nonmuscle cells

A. Nonmuscle activities – they are required for splitting a cell in two during cell division (cytokinesis), for generating tension at focal adhesions & for the turning behavior of growth cones

B. Structure of myosin II molecules – each myosin II composed of 6 polypeptide chains (1 pair of heavy chains, 2 pairs of light chains); organized in way that produces highly asymmetric protein with 3 sections

1. A pair of globular heads that contain the molecule’s catalytic site

2. A pair of necks, each consisting of a single, uninterrupted a-helix & 2 associated light chains

3. A single, long, rod-shaped tail formed by intertwining of long, a-helical sections of the 2 heavy chains

C. In an in vitro assay, researchers immobilized isolated myosin heads (S1 fragments) on glass cover slip —> caused actin filament sliding

1. Thus, a single head domain has all of the machinery needed for motor activity

2. The fibrous tail portion of myosin plays a structural role, allowing the protein to form filaments

3. Myosin molecules assemble so that the tail ends point toward filament center, while the globular heads point away from the center; described as bipolar since polarity reverses at center of filament

D. Skeletal muscle myosin II filaments are highly stable components of the contractile apparatus

1. However, the smaller myosin II filaments that form in most nonmuscle cells often display transient construction (assembling when & where they are needed, then disassembling after they have acted)

III. Unconventional myosins - Thomas Pollard & Edward Korn (NIH, 1973) described a unique myosin-like protein extracted from the protist Acanthamoeba

A. Unlike muscle myosin, this smaller, unconventional myosin (myosin I) had only a single head & was unable to assemble into filaments in vitro

1. Others found in a variety of cell types; classified into many classes based on their amino acid sequences

2. Like myosin IIs, they are able to bind to actin filaments in vitro & move them in presence of ATP

3. Despite considerable study, the precise role of myosin I in cellular activities remains unclear

B. Steps in the (unconventional) myosin V mechanical cycle seen in a series of EM micrographs catching its stages; myosin V is dimeric myosin that, unlike others studied, moves processively along actin MFs

1. Processivity is due to the high affinity of the myosin heads for the actin filament

2. This ensures that each head remains attached to the filament until the second head reattaches

3. Myosin V is also noteworthy for the length of its neck, which at 23 nm is ~3X that of myosin II

4. Because of its long neck, myosin V can take very large steps (>30 nm); very important for motor protein moving processively along actin filament made of helical strands of subunits

5. Actin helix repeats itself ~every 13 subunits (36 nm), close to myosin V step size

6. These & subsequent studies on movements of single myosin V molecules suggest that it walks along an actin filament by a "hand-over-hand" model, similar to that of kinesin

7. To move in this way, each myosin head must swing through a distance of 72 nm, twice the distance between 2 successive binding sites on actin filament

8. Due to its giant strides, it can walk in a straight path along this repeat even though the underlying "roadway" spirals 360°C between its "feet"

IV. Functions of unconventional myosins

A. Some (types I, V, VI) are associated with various types of cytoplasmic vesicles & organelles

1. Some vesicles contain MT-based motors (kinesins and/or cytoplasmic dynein) & MF-based motors (unconventional myosins); in fact, the two kinds of motors may be physically linked to one another

2. Vesicles & other membranous carriers move long distances in animal cells along MTs to their end & then may switch over to MF tracks for local movement through actin-rich cell periphery

B. Cooperation between MTs & MFs has been best studied in pigment cells

1. In mammals, pigment granules (melanosomes) are moved into fine peripheral pigment cell processes by one of the myosin V isoforms (Va)

2. Melanosomes are then transferred to hair follicles where they are incorporated into developing hair

3. Mice lacking myosin Va activity are unable to transfer melanosomes into hair follicle – this causes their coat to have a much lighter color

C. Humans lacking a normal gene encoding myosin Va suffer from a rare disorder (Griscelli syndrome); they exhibit partial albinism (lack of skin coloration)

1. They suffer other symptoms thought to be related to vesicle transport defects (neurologic dysfunction)

2. In 2000, it was discovered that a subset of Griscelli patients had a normal myosin Va gene, but lacked a functional gene encoding a peripheral membrane protein called Rab27a

3. The Rab family of proteins is a group of molecules that tether vesicles to target membranes; Rabs are also involved in the attachment of myosin (& kinesin) motors to membrane surfaces

4. The Rab family contains >60 different members, which offers the potential for different motors to be specifically bound to different membrane compartments

D. Hair cells are particularly good system for studying unconventional myosin functions; they are named for bundle of stiff, hairlike stereocilia projecting from cell's apical surface into fluid-filled inner ear cavity

1. Displacement of stereocilia by mechanical stimuli leads to generation of nerve impulses that we perceive as sound; stereocilia have no relation to the true cilia discussed earlier

2. Instead of containing MTs, each stereocilium is supported by a bundle of parallel actin filaments whose barbed ends are located at the outer tip of the projection & pointed ends at the base

3. Stereocilia have provided some of the most striking images of the actin cytoskeleton's dynamic nature

4. While stereocilia are permanent structures, their actin bundles are in constant flux; actin monomers bind continually to each filament's barbed end, treadmill through its body & dissociate from pointed end

5. Several unconventional myosins (I, V, VI, VII & XV) are localized at various sites within the hair cells of the inner ear; the precise roles of these various motor proteins is unclear

E. Mutations in myosin VIIa are the cause of Usher 1B syndrome, which is characterized by both deafness & blidness; as in humans, mice homozygous for the mutant allele encoding this motor protein are deaf

F. Myosin VI, a processive organelle transporter in many cells' cytoplasm, including hair cells, is distinguished by its movement in "reverse directon", that is toward the pointed (minus) end of an actin filament

1. Myosin VI is thought to be involved in the formation of clathrin-coated vesicles at the plasma membrane & the movement of the uncoated vesicles to the early endosomes

Microfilaments: Muscle Contractility

I. Skeletal muscles derive their name from the fact that most of them are anchored to bones that they move; they are under voluntary control & can be consciously commanded to contract

II. Skeletal muscle cell structure - highly unorthodox; cylindrical cell; typically 10 - 100 µm thick; >100 mm long

A. Skeletal muscle cells are multinucleate (hundreds of nuclei), a product of the embryonic fusion of large numbers of mononucleate myoblasts (premuscle cells)

1. Because of their properties, these cells are more appropriately called muscle fibers

B. Skeletal muscle fiber cells may have the most orderly internal structure of any cell in body

1. Muscle fiber is a cable made up of hundreds of thinner, cylindrical strands (myofibrils); seen in longitudinal section

2. Each myofibril is made up of a repeating linear array of contractile units (sarcomeres)

3. Each sarcomere, in turn, has characteristic banding pattern that gives muscle fiber a striated appearance

4. Myofibrils separated by cytoplasm with intracellular membranes & mitochondria, lipid droplets, glycogen granules

C. Banding pattern seen in EM to be result of partial overlap between the two distinct thick & thin filaments

1. Each sarcomere extends from one Z line to next Z line (~2.5 µm) & contains several dark bands & light zones; there is a pair of lightly staining I bands at outer edges of sarcomere

2. More densely staining A band is between the outer I bands; also lightly staining H zone in A band center

3. Densely staining M line lies in center of H zone

4. I bands – contain only thin filaments; H zone - only thick filaments; the part of the A band on either side of the H zone – represents region of overlap & contains both types of filaments

D. Cross section through overlap region shows thin filaments in hexagonal array around each thick filament with each thin filament situated between 2 thick filaments

1. Longitudinal sections show the presence of projections from thick filaments at regularly spaced intervals

2. The projections represent cross-bridges capable of forming attachments with neighboring thin filaments

III. The Sliding Filament Model of muscle contraction – all skeletal muscles operate by shortening; no other way

A. Contract by shortening at sarcomere level (they are units of shortening); combined decrease in sarcomere length accounts for decrease in length of entire muscle

B. Sarcomere banding patterns shift during contraction, showing that filaments slide & serving as an important clue to mechanism; observed banding patterns at different stages of contractile process

1. As muscle fiber is shortened, the A band in each sarcomere remained essentially constant in length, H zone & I bands decrease in width during contraction & eventually disappear

2. During contraction, Z lines on both ends of sarcomere move inward until contacting A band outer edges

C. Andrew Huxley & Rolf Niedergerke and Hugh Huxley & Jean Hanson - 2 British research groups proposed in 1954 that contraction is not result of filament shortening, but rather of their sliding over each other

1. Thin filaments slide toward sarcomere center; results in increase in overlap between filaments

2. Also explains decreased width of I & H bands; rapidly accepted & evidence is still accumulating

IV. Composition & organization of thick & thin myofilaments - thin filaments mostly actin; thick mostly myosin

A. In addition to actin, skeletal muscle thin filaments also contain 2 other proteins: troponin & tropomyosin

1. Tropomyosin – elongated molecule, ~40 nm long; fits securely into grooves between 2 thin filament actin chains; each rod-shaped tropomyosin interacts with 7 actin subunits linearly along F-actin chain

2. Troponin - globular protein complex made of 3 subunits - each has distinct, important functional role; ~40 nm apart on thin filament, contact both the actin & tropomyosin components of thin filament

B. Actin filaments of each half sarcomere are aligned with their barbed ends linked to Z line by a-actinin

C. Thick filaments are composed of several hundred myosins along with small amounts of other proteins

1. Like filaments formed in vitro, thick filament polarity is reversed at sarcomere center; filament center is composed of opposing tail regions of myosin molecules & is devoid of heads

2. Myosin heads project from each thick filament along remainder of its length due to the staggered positions of the myosin molecules making up the body of the filament

D. Titin (>3.5 x 106 daltons in molecular mass [contains >38,000 amino acids]; 1 µm long) – third most abundant skeletal muscle fiber protein; largest protein yet discovered in any organism

1. The entire titin gene can give rise to isoforms of different length

2. Titin molecules originate at M line in center of each sarcomere & extend along myosin filament, continuing past A band & terminating at the Z line

3. It is highly elastic protein & stretches (like a molecular spring) as certain regions within the molecule become uncoiled

4. It is thought to prevent the sarcomere from being pulled apart during muscle stretching & also maintains myosin filaments in their proper position within sarcomere center during muscle contraction

5. Titin gene mutations have recently been found to be responsible for a condition known as dilated cardiomyopathy, which results in death due to heart failure

V. Molecular basis of contraction - shorten ~65% of relaxed length; ~1 µm/thin filament

A. During muscle contraction, each myosin head extends outward & binds tightly to a thin filament forming cross-bridges between the 2 types of filaments; heads from one myosin filament contact 6 surrounding actins

B. Once bridges form & myosin heads are bound tightly to actin filament, myosin heads change shape (undergo a conformational change), bending toward the sarcomere center (power stroke)

1. Moves thin actin filament over thick filament (~10 nm toward the center of the sarcomere)

2. Since each thin filament contacted by ~100 heads, it exhibits constant motion during cycle

C. Muscle myosin is not a processive motor protein like kinesin since it remains in contact with its track (the thin filament) for only a small fraction (<5%) of the overall cycle

1. However, each thin filament is contacted by a team of ~100 myosin heads that beat out of synchrony with one another; thus the thin filament undergoes continuous motion during each contractile cycle

2. It is estimated that a single muscle cell thin filament can be moved several 100 nm during a period as short as 50 msec; repeats ~50 - 100 mechanical cycles/sec

D. How can single myosin molecule move actin filament ~10 nm in single power stroke? - Ivan Rayment, Hazel Holden, et al. (U. of Wisconsin, 1993) explain myosin mechanism by studying S1 myosin II atomic structure

1. Energy released by ATP hydrolysis induces a small (0.5 nm) conformational change wihin head while it is tightly bound to actin filament

2. The small head movement is amplified ~20-fold by the swinging movement of an adjoining, elongated a-helical neck; the elongated myosin II neck acts as a rigid lever arm

3. Neck causes attached actin filaments to slide much greater distance than would otherwise be possible

4. The two light chains that are wrapped around neck are thought to provide rigidity for lever

E. James Spudich, et al. (Stanford U.) - obtained support for lever-arm proposal

1. Constructed genes encoding altered versions of myosin II molecule with necks of different length

2. Then tested them in in vitro motility assays with actin filaments

3. Apparent myosin molecule power stroke length was directly proportional to neck length as predicted

4. Myosin molecules with shorter necks generated smaller displacements; those with longer necks generated greater displacements

5. Not all studies support correlation between step size & neck length, so lever-arm role is still controversial

VI. Energetics of filament sliding – myosin heads hydrolyze ATP & act as levers for thin filament motion; each myosin cross-bridge mechanical activity cycle (~50 msec) is accompanied by ATPase activity cycle

A. Cycle starts with ATP binding to myosin head, which induces cross-bridge dissociation from actin filament

B. ATP binding is followed by its hydrolysis, which occurs before myosin head contacts the actin filament; ADP & Pi hydrolysis products stay bound at enzyme active site

1. Energy released by hydrolysis is absorbed by myosin as a whole, placing the cross-bridge in an energized state, like a stretched spring capable of spontaneous movement

2. The myosin head is cocked at a new position further along filament

C. Energized myosin attaches to actin & releases its bound Pi

1. Pi release triggers a large conformational change, driven by the stored free energy

2. This conformational change shifts actin filament toward sarcomere center (myosin head power stroke)

D. Bound ADP leaves & new ATP attaches, inducing release of cross-bridge; cycle starts over - in absence of ATP, cross-bridges stay tightly bound to actin filament (causes rigor mortis after death)

VII. Excitation-contraction coupling - steps linking the arrival of nerve impulse at the muscle plasma membrane to the shortening of sarcomeres deep within muscle fiber

A. Muscle fibers are organized into groups called motor units; all fibers of motor unit are jointly innervated by branches of a single motor neuron

1. These fibers contract simultaneously when stimulated by an impulse transmitted along that neuron

2. Point where neuron axon terminus & muscle fiber make contact is called neuromuscular junction

3. Junction is site of nerve impulse transmission from axon across synaptic cleft to muscle fiber

4. Muscle fiber plasma membrane is also excitable & capable of conducting an action potential

B. Unlike a neuron, where an action potential stays at the cell surface, skeletal muscle cell impulse is propagated into cell interior along membranous folds (transverse [T] tubules)

1. T tubules terminate in very close proximity to a cytoplasmic membrane system composing sarcoplasmic reticulum (SR) that forms membranous sleeve around myofibril (specialized form of smooth ER)

2. ~80% of SR membrane integral proteins are Ca2+-ATPases (moves Ca2+ ions out of cytosol into SR lumen where they are stored until they are needed)

C. Sydney Ringer (English physician, 1882) – demonstrated importance of calcium in muscle contraction

1. Found that isolated frog heart would contract in a saline solution made with London tap water, but…

2. The heart would not contract in solution made with distilled water

3. Ringer determined that calcium ions in tap water were an essential factor in muscle contraction

D. In the relaxed state, Ca2+ levels within cytoplasm of muscle fiber are very low (~2 x 10-7 M) & below the threshold concentration required for contraction

1. With arrival of action potential at SR via T tubules, Ca2+ ion channels in SR membrane are opened

2. Ca2+ ions diffuse out of SR compartment & over short distance to myofibrils

3. Intracellular [Ca2+ ion] levels rise from ~2 x 10-7 M to ~5 x 10-5 M – how does this trigger skeletal muscle fiber contraction?

E. In relaxed sarcomere, thin filament tropomyosin molecules block myosin-binding sites on actin molecules

1. Tropomyosin’s position within the filament groove blocking sites is controlled by attached troponin

2. If Ca2+ levels rise, the ions bind troponin C subunit —> causes another troponin subunit to alter its shape (conformation), which moves adjacent tropomyosin ~1.5 nm closer to center of filament’s groove

3. This shift in tropomyosin position exposes myosin-binding sites on adjacent actins, allowing myosin heads to attach to thin filaments & cross-bridges to form, contraction occurs

4. Each troponin molecule controls position of 1 tropomyosin molecule, which, in turn, controls the binding capacity of 7 adjacent actin monomers in thin filament

F. When stimulation from innervating motor nerve fiber stops, SR membrane Ca2+ channels close & excess Ca2+ is pumped from cytosol back into SR by Ca2+-ATPase

1. As [Ca2+ ion] decreases, Ca2+ ions dissociate from troponin binding sites —> tropomyosin molecules drop back to previous position blocking myosin-binding sites & preventing actin-myosin interactions

2. Relaxation process is like competition for Ca2+ between transport protein of SR & troponin

3. Ca2+-ATPase transport protein has greater affinity for Ca2+ ion than troponin, so Ca2+ is preferentially removed from cytosol, leaving troponin molecules without bound Ca2+ ions

Nonmuscle Motility: Background Information

I. Nonmuscle motility study is more difficult since the critical components are present in less ordered, more labile, transient arrangements & they are typically restricted to a thin cortical zone just under plasma membrane

A. Cortical zone is an active cell region that is responsible for a number of activities, all dependent on MF assembly in cortex:

1. Ingestion of extracellular materials

2. Extension of processes during cell movement

3. Constriction of a single animal cell into two cells during cell division (cytokinesis)

B. Nonmuscle contractility & motility depends on actin filaments & sometimes myosin superfamily proteins

II. Actin-binding proteins - purified actin filaments can polymerize in vitro, but such filaments cannot interact with each other or perform useful activities; in EM, look like straw covering barn floor

A. But, in living cells, F-actin forms various types of bundles, thin (2D) networks & complex 3D gels

B. Actin-binding proteins (ABPs; >100 different ones) determine organization & behavior of actin filaments inside cells; they belong to numerous families that have been isolated from different cell types

1. ABPs affect assembly or disassembly of actin filaments, their physical properties, & their interactions with one another & with other cellular organelles

2. ABPs are divided into several categories based on their presumed function in the cell

3. Some of these proteins carry out >1 of the types of activities listed, depending on ABP concentration, state of the protein (phosphorylated or not) & prevailing conditions (concentrations of Ca2+ & H+ ions)

4. Most studies performed in vitro; so it is difficult to extend results to activities occurring in cell

III. Categories of actin-binding proteins

A. Nucleating proteins - slowest step in actin filament formation is the first step, nucleation

1. Nucleation requires that at least 2 or 3 actin subunits come together in the proper orientation to begin formation of the polymer

2. As with MTs, actin filament formation is accelerated by the presence of a preexisting seed or nucleus to which subunits can be added

3. Several proteins have been identified that promote nucleation of actin filaments; the best studied such protein is the Arp2/3 complex, which contains 2 "actin-related proteins"

4. In fact, "Arp" stands for actin-related proteins; they share considerable sequence homology with actins, but are not considered "true" actins

5. Once complex is activated, the 2 Arps are thought to adopt conformation providing a template to which actin monomers can add, analogous to way that g-tubulin is proposed to form MT nucleation template

6. Arp 2/3 complex generates networks of short, branched actin filaments

7. Another nucleating protein (formin) generates unbranched filaments, like those found at focal adhesions & contractile rings of dividing cells

8. Unlike Arp 2/3, which remains at the pointed end of the newly formed filament, formins track with the barbed end even as new subunits are inserted at that site

B. Monomer-sequestering proteins – thymosins, like thymosin b4, are proteins that bind to actin-ATP monomers (often called G-actin) & prevent their polymerization; called actin monomer-sequestering proteins

1. These proteins are thought to account for relatively high [G-actin monomer] without polymerization in nonmuscle cells (50 - 200 µM); they bind G-actin & stabilize the monomer pool

2. Without them, nearly complete polymerization of soluble actin monomers into filaments is favored by conditions within the cytoplasm

3. If their concentration or activity is changed, the cell can shift monomer-polymer equilibrium in a certain region of cell to determine whether polymerization or depolymerization is favored at the time

C. End-blocking (capping) proteins – proteins of this group regulate actin filament length by binding to one or the other end of the filaments, forming a cap that blocks both loss & gain of subunits

1. If cap fast-growing, barbed, "+" end of filament —> depolymerization may occur at opposite end —> filament disassembly; if cap pointed end —> depolymerization is blocked

2. Striated muscle thin filaments are capped at their barbed end by capZ protein at the Z line & at their pointed end by the protein tropomodulin

3. Microinject tropomodulin ABs into muscle cell —> tropomodulin cap disturbed —> thin filaments add more actin subunits at newly exposed pointed end & exhibit dramatic elongation into sarcomere center

D. Monomer-polymerizing proteins - profilin is a protein that binds to actin-ATP monomers

1. For years, profilin was thought to be a monomer-sequestering protein that, like thymosin, inhibited actin polymerization, but its concentration is not high enough to bind enough actin to do this

2. New evidence suggests that it probably promotes actin filament growth by attaching to an actin monomer & catalyzing the dissociation of its bound ADP, which is rapidly replaced with ATP

3. Profilin-ATP-actin monomer can then assemble onto the barbed end of a growing actin filament, which leads to the release of profilin

E. Actin-filament depolymerizing proteins – members of cofilin family of proteins (cofilin, ADF, & depactin) bind to actin-ADP subunits present within the body & at the pointed end of actin filaments

1. Cofilin has 2 apparent activities: it can fragment actin filaments & it can promote their depolymerization into monomers

2. They play a role in rapid turnover of actin filaments at sites of dynamic changes in cytoskeletal structure

3. Essential for cell locomotion, phagocytosis & cytokinesis

F. Cross-linking proteins – these proteins are able to alter the 3D organization of an actin filament population

1. Each has 2 or more actin-binding sites so it can cross-link two or more separate actin filaments

2. Some, like ABP280 & filamin are shaped like long, flexible rods & promote formation of loose networks of filaments interconnected at near right angles to one another

3. Regions of cytoplasm containing such networks have the properties of a 3D elastic gel that resists local mechanical pressures

4. Other cross-linking proteins (villin, fimbrin) - globular shape; cause actin filament bundling into tightly knit parallel arrays in microvilli in some epithelia & stereocilia projecting from inner ear receptor cells

5. Bundling filaments together adds to their rigidity, allowing them to act as supportive internal skeleton for these cytoplasmic projections

G. Filament-severing proteins - bind to side of existing filament & break it in two; gelsolin was first identified

1. Since they reduce actin filament length, they decrease cytoplasmic viscosity

2. Gelsolin was discovered by its ability to liquify (solate) gelated cytoplasmic extracts

3. May also promote actin monomer incorporation by creating additional free barbed ends or they may cap the fragments they generate

H. Membrane-binding proteins – much of contractile machinery of nonmuscle cells lies just beneath the plasma membrane; usually attach MFs to membrane indirectly (via peripheral proteins)

1. The forces generated by contractile proteins act on the plasma membrane, causing it to protrude outward (like during cell locomotion) or invaginate inward (like during phagocytosis & cytokinesis)

2. These activities are generally facilitated by linking actin filaments to the plasma membrane indirectly by attachment to peripheral membrane proteins

3. Examples: inclusion of short actin polymers into membrane skeleton of erythrocytes & attachment of actin MFs to membrane at focal adhesions & adherens junctions

4. Proteins that link membranes to actin: vinculin, ERM family members (ezrin, radixin, moesin), spectrin family members (dystrophin)

IV. Actin filaments, often working with myosin motors, are responsible for a diverse variety of dynamic nonmuscle cell activities, including:

A. Cytokinesis

B. Phagocytosis

C. Cytoplasmic streaming (directed bulk flow of cytoplasm occurring in certain large plant cells)

D. Vesicle trafficking

E. Blood platelet activation

F. Lateral movements of integral proteins within membranes

G. Cell substratum interactions, cell locomotion & axonal outgrowth

H. Changes in cell shape

Nonmuscle Motility: Actin Polymerization as a Force-Generating Mechanism

I. Some types of cell motility occur solely as result of actin polymerization & do not involve myosin activity

A. Listeria monocytogenes - bacterium that can infect macrophages; causes encephalitis or food poisoning

1. Listeria is propelled like a rocket through infected cell's cytoplasm by polymerization of actin monomers just behind the bacterium

2. Listeria contains a surface protein called ActA that is present at only one end of bacterium

3. When ActA is exposed within host cytoplasm, it recruits & activates a number of host proteins (including Arp 2/3 complex) that work together to direct the actin polymerization process

B. Listeria propulsion process was reconstituted in vitro, proving conclusively that myosin is not involved & that actin polymerization by itself (without myosin motors) can provide force required for motility

II. Recent studies suggest that the same events that occur during Listeria propulsion are used for normal cell activities, ranging from propulsion of cytoplasmic vesicles to movement of cells themselves

Nonmuscle Motility: Cell Locomotion

I. Cell locomotion - flagellae, cilia, crawling over substrate, walking (all display repetitive series of activities)

II. Cell locomotion needed for many higher vertebrate activities: tissue/organ development, formation of blood vessels, axon development, wound healing, infection protection; also contributes to cancerous tumor spread

A. Hard to see cell locomotion in body since migratory cells cannot usually be distinguished from opaque milieu in vivo so researchers usually study single cells in vitro as they move over bottom of culture dish

III. Series of activities in cell locomotion is much like walking; cells may assume very different shapes as they crawl over substratum

A. Movement is initiated by protrusion of part of cell surface in direction in which cell is to move

B. A portion of lower surface of protrusion attaches to substratum, forming temporary anchorage sites

C. Bulk of cell is pulled forward over adhesive contacts that will eventually become part of rear of cell

D. Cell breaks its rear contacts with substratum, causing retraction of trailing edge or "tail"

Nonmuscle Motility: Cells That Crawl Over the Substratum

I. Examples of cells that crawl over the substratum - amoeba & mammalian fibroblasts (FBs)

II. Place small piece of living tissue (skin, liver) in culture dish in appropriate culture medium —> individual cells migrate out of specimen & onto culture dish surface

A. These cells are typically fibroblasts, the predominant cells present in connective tissue

B. As it moves, fibroblast flattens itself close to substratum & becomes fan-shaped, with broadened frontal end & a narrow tail; its movement is erratic & jerky, sometimes advancing & other times withdrawing

C. On a good day, fibroblast may move a distance of ~1 mm

III. Key to fibroblast locomotion is seen by examining its leading edge

A. Leading edge extends out from cell as a broad, flattened, veil-like protrusion (lamellipodium)

B. Lamellipodia are typically devoid of particulate structures & the outer edge often exhibits an undulating motion, giving it a ruffled appearance

C. As lamellipodium is extended from cell, it adheres to underlying substratum at specific points, providing temporary anchorage sites for the cell to pull itself forward

IV. Actin monomer polymerization can provide the force that propels Listeria bacterium through the cytoplasm with movement accomplished without the involvement of molecular motors

A. A similar actin-polymerization mechanism is thought to provide motile force required for protrusion of the leading lamellipodium edge

B. Such nonmuscle motility also demonstrates the importance of actin-binding proteins in orchestrating assembly & disassenmbly of actin-filament networks at particular site within cell at a particular time

V. Example: rounded white blood cell receives chemical signal coming from one particular direction where body has been wounded; this is the type of stimulus that will cause the cell to move in that direction

A. Once stimulus is received at plasma membrane, it triggers localized actin polymerization, which leads to the polarization of the cell & its movement toward the source of the stimulus

1. Like Listeria (its ActA protein activates actin polymerization at bacterial surface), mammal cells have a diverse protein family (WASP) that activates Arp2/3 complex at stimulation site near plasma membrane

B. WASP was discovered as a product of the gene responsible for Wiskott-Aldrich syndrome; its patients have a crippled immune system because their white blood cells lack a functional WASP protein

1. Consequently, their white blood cells fail to respond to chemotactic signals

VI. Model for major steps in the formation of a lamellipodium that would guide a cell in a particular direction

A. Stimulus is received at one end of cell, which leads to activation of Arp2/3 protein complexes by activated WASP proteins

B. Activated Arp2/3 complexes serve as nucleating sites for formation of new actin filaments

1. Polymerization of ATP-bound actin monomers onto free barbed ends of growing filaments is promoted by profilin molecules

C. Once actin filaments have formed, Arp2/3 complexes bind to sides of these filaments & nucleate the formation of additional actin filaments that form as branches

1. The Arp2/3 complexes remain at the pointed ends, which are situated at branch points; meanwhile, growth of the barbed ends of older filaments is blocked by the addition of capping protein

2. In contrast, addition of actin monomers to barbed ends of more recently formed filaments of network pushes lamellipodium membrane outward in direction of the attractive stimulus

D. As newer filaments are growing by addition of monomers to their barbed ends, the older capped filaments undergo disassembly from their pointed ends

1. Disassembly is promoted by cofilin, which binds to actin-ADP subunits at pointed end of filaments

2. Actin-ADP monomers released from disassembling filaments are recharged by conversion into profilin-actin-ATP monomers, which can be reutilized in actin filametnt assembly at the leading edge

VII. Major structural features of cell locomotion

A. Branched, cross-linked nature of filamentous actin network resides just beneath plasma membrane of advancing lamellipodium

B. There is a succession of short actin-filament branches with Arp2/3 complexes attached (seen in EM by immunogold labeling)

C. The Arp2/3 complexes reside at the Y-shaped junctions where the newly polymerized filaments have branched off of preexisting filaments

VIII. Lamellipodial movement is a dynamic process

A. As actin filament polymerization & branching continue at very front edge of lamellipodium, actin filaments are depolymerizing toward the rear of the lamellipodium

B. Thus, the entire actin-filament array undergoes a type of treadmilling in which actin monomers are added to barbed ends of array at its front end & lost from pointed ends of array toward rear

IX. Protrusion of the leading edge is followed by the movement of the bulk of the cell

A. The major forces involved in cell locomotion are those generated at sites of adhesion that are required to pull or tow the main body of the cell forward

1. They are often called traction forces since they occur at sites where the cell grips the substrate

B. When cells are allowed to migrate over a thin sheet of elastic material, movements of the cell are accompanied by deformation of the underlying substratum

1. The magnitude of the traction forces exerted at various locations within a live migrating cell can be calculated from the dynamic patterns of substrate deformation

2. In migrating fibroblasts, the greatest traction forces are exerted just behind the cell's leading edge where the cell adheres strongly to the underlying substratum

C. The cell body adheres less strongly to substrate, which allows it to be dragged forward like a contained mass of cargo

X. While actin polymerization apparently extends the cell's leading edge, myosin along with actin filaments generates the contractile (traction) forces that pull the rest of the cell forward - example: fish keratocytes

A. Fish keratocytes are cells derived from epidermis covering fish's scales; they are a favorite for studying locomotion since their rapid gliding movement depends on the formation of very broad, thin lamellipodium

B. Fix moving keratocyte & stain it for actin & myosin II —> advancing lamellipodium edge is filled with actin; myosin, on other hand, is concentrated in a band where rear of lamellipodium joins rest of cell

1. EM of this region shows clusters of small, bipolar myosin II filaments bound to actin network

2. Forces generated by these myosins presumably pull bulk of cell along behind advancing lamellipodium; myosin I may also generate forces for cell locomotion in some organisms

Nonmuscle Motility: Axonal Outgrowth

I. Ross Harrison (Yale Univ., 1907) – classic experiment; removed small piece of tissue from developing frog embryo nervous system & grew tissue from it in tiny drop of lymphatic fluid & watched it for a few days

A. Cells stayed healthy & sprouted processes out into surrounding medium (first cells kept alive in culture)

B. Results showed strong evidence that axons develop by process of active outgrowth & elongation

II. Elongating axon tip (growth cone) is quite different from the rest of the cell; it resembles a highly motile, crawling fibroblast; bulk of axon shows little outward evidence of motile activity

A. Close examination of living growth cone shows several types of locomotor protrusions:

1. Broad, flattened lamellipodium that creeps outward over substratum

2. Short, stiff microspikes that point outward toward lamellipodium edge

3. Highly elongated filopodia that extend & retract in continuous display of motile activity

B. Fluorescence microscopy - all of the above structures are filled with actin filaments; MTs fill up core of axon leading to tip, although a number of individual MTs penetrate into actin-rich peripheral domain

1. Thus, in neurons like other cells, MFs mostly function in motility; MTs primarily do support & transport

III. Function of highly motile growth cone is to explore environment & elongate axon along defined paths

A. In embryo, axons of developing neurons grow along defined paths, following certain topographical features of substratum or responding to presence of certain chemicals that diffuse into their path

B. Lamellipodia & filopodia of growth cone respond to presence of these physical & chemical stimuli, causing pathfinding axons to turn toward attractive environmental & away from repulsive factors

C. The turning behavior of the growth cone, in turn, is dependent on dynamic changes in the organization of the actin cytoskeleton

D. Advancing tip in cultured neurons will make dramatic turns in response to a diffusible factor that causes the disruption of the growth cone actin network

IV. Ultimately, correct wiring of entire nervous system depends on uncanny ability of embryonic growth cones to make the proper steering decisions that lead them to the target organ they must innervate

A. Neurons grow out of embryonic retina toward specific region of the brain; they encounter semaphorins, a family of proteins that acts primarily to repel advancing growth cones

B. The axons may grow along a path that directs them away from this repulsive guidance molecule

Nonmuscle Motility: Changes in Cell Shape During Embryonic Development

I. Each part of body has characteristic shape & internal architecture that arises during embryonic development:

A. The spinal cord is basically a hollow tube

B. The kidney consists of microscopic tubules

C. Each lung is composed of microscopic air spaces

II. Many cell activities are needed for characteristic organ morphology development, including programmed cell shape changes occurring largely by changes in cytoskeletal element orientation within cells

III. Development of the nervous system is one of the best examples

A. Toward the end of gastrulation in vertebrates, the outer (ectodermal) cells situated along the embryo’s dorsal surface elongate & form a tall epithelial layer (neural plate)

1. Neural plate cells elongate as MTs become oriented with their long axes parallel to that of cell

2. After elongation, neural epithelium cells become constricted at one end & thus become wedge shaped so that the entire layer of cells curves inward

B. This curving of the cell layer occurs after a MF band assembles in cell cortical region just beneath apical cell membrane; the MF band then contracts narrowing that end of the cell

C. Eventually, neural tube curvature causes the outer edges to contact one another, forming a cylindrical, hollow tube that will give rise to the animal's entire nervous system

The Human Perspective: The Role of Cilia in Development and Disease

I. Outwardly, humans seem to be relatively symmetrical (left half essentially mirror image of right); internally, humans are strikingly asymmetrical (stomach, heart & spleen shifted to body's left side; liver on right side

A. Sometimes a patient will have the right-left asymmetry of the visceral organs reversed (situs inversus)

1. Situs inversus is seen in persons with Kartagener syndrome, which is also characterized by recurrent sinus & respiratory infections & infertility in males

B. First clues to the underlying cause of this disorder came in 1970s when it was found that immotile sperm from these individuals had an abnormal axonemal structure

1. Depending on patient, axoneme may be missing outer or inner dynein arms, central MTs or radial spoke structures

2. Later studies showed that mutations in a number of genes, including those encoding dynein heavy & intermediate chains, can cause this syndrome

3. Understandable that such patients would have respiratory infections (depend on debris & bacteria clearance by respiratory tract cilia) & male infertility – why left-right asymmetry in half of them?

II. Basic mammal body plan is laid down during gastrulation in association with structure called the embryonic node; each cell making up the embryonic node has a single cilium

A. These cilia have unusual properties

1. They are missing the 2 central MTs (they have a 9 + 0 axonemal structure)

2. They also exhibit an unusual rotary motion

B. If motility of these cilia is impaired, as in mice harboring flagellar dynein gene mutations, roughly half the animals developed reversed asymmetry; suggests left-right asymmetry in mutants is determined by chance

1. Rotation of nodal cilia moves surrounding fluid to left side of embryonic midline as determined by tracking movement of microscopic fluorescent beads

2. It was proposed that extracellular fluid moved by the nodal cilia contains morphogenetic substances (substances that direct embryonic development) that become concentrated on the embryo's left side

3. This leads to the eventual formation of different organs on different sides of the midline

C. This is strongly supported by experimental studies in which mouse embryos were raised in miniaturized chambers in which artificial flow of fluid across the node could be imposed

1. When embryos were subjected to a flow of fluid in a direction opposite to that occurring during normal development, the embryos developed with reversed left-right asymmetry

D. Alternate hypothesis holds that embryonic node contains 2 different types of cilia, a population of motile cilia located in the center of the node & nonmotile primary cilia distributed around the node periphery

1. According to hypothesis, motile cilia generate the leftward flow & the nonmotile cilia act as sensory structures that detect the movement & transmit signals that lead to asymmetry

III. Many body cells have single, nonmotile cilium; ignored by researchers for years, but recent studies suggest an important function as "antennae" (sense chemical/mechanical properties of fluids into which they project)

A. Primary cilia on epithelial cells lining lumen of microscopic kidney tubules where urine formation occurs

1. Their importance was revealed when it was discovered that a pair of membrane proteins called polycystins are located on the surface of these kidney cilia where they form a Ca2+ ion channel

2. Mutations in PKD1 & PKD2, the genes that encode the polycystins, lead to polycystic kidney disease (PKD), in which the kidney develops multiple cysts that destroy kidney function

B. PKD is thought to be a condition resulting from a breakdown in cell division regulation, because the cysts are a result of an abnormally high level of proliferation of epithelial cells that line parts of kidney tubules

1. Mutations in the polycystins are thought to alter the response of the primary cilia to fluid flow

2. This leads to a disturbance in calcium flux across the ciliary membrane, which in turn impairs the transmission of signals to the body of the cell & the nucleus, resulting in abnormal cell proliferation

IV. Cilia importance in human development has gotten even more evident by recent revelation that Bardet-Biedl syndrome (BBS) is caused by mutations in any 1 of a number of genes affecting basal body & cilia assembly

A. Persons afflicted with BBS exhibit a remarkable range of abnormalities, including:

1. Polydactyly (extra fingers & toes)

2. Situs inversus

3. Obesity

4. Kidney disease

5. Heart defects

6. Mental retardation

7. Genital abnormalities

8. Retinal degeneration

9. Decreased olfactory discrimination

10. Diabetes

11. High blood pressure

B. The fact that all of these dysfunctions can be traced to abnormalities in basal bodies & cilia illustrates the widespread importance of these structures in organ development & function

C. Many of the genes responsible for these various cilia-based disorders ("ciliopathies") were first identified in model organisms like Chlamydomonas or C. elegans

1. This provides another example of the importance of basic research on non-vertebrate organisms in furthering our understanding of human disease

The Human Perspective: The Molecular Basis of Muscular Dystrophy

I. Muscular dystrophies comprise a collection of inherited neuromuscular diseases characterized by muscle-fiber degeneration & resulting muscle weakness

A. Duchenne muscular dystrophy (DMD) – named for person who described it (1861); a common, debilitating disorder striking ~1 in 3,300 males; the disease most often associated with the name muscular dystrophy

B. There are several other rare types of muscular dystrophy that have revealed interesting insights into the relationship between the cytoskeleton, plasma membrane & the extracellular matrix

II. The gene responsible for DMD (dystrophin), is the largest gene so far identified in the mammalian genome

A. It stretches for >2.3 million bases along the X chromosome, a length that requires 16 hours to be transcibed into a single mRNA

1. Only ~0.6% of gene actually codes for amino acids; the remaining 99.4% consists of noncoding introns

B. The protein encoded by the dystrophin gene resides in the membrane skeleton of striated muscle cells

1. Like the related spectrin molecules of the erythrocyte cytoskeleton, dystrophin molecules are rod-shaped dimers that lie just beneath the plasma membrane

2. On their cytoplasmic side, dystrophin molecules are attached to actin filaments

3. On their membrane side, they are attached to a cluster of dystrophin-associated proteins (DAPs) that are part of the plasma membrane

4. The DAPs, in turn, are linked on their extracellular surface to components of the basement membrane that surrounds these contractile cells

5. Together these proteins form a functional pathway linking the internal cytoskeleton to the extracellular matrix, providing structural support for the plasma membrane as the muscle fiber contracts & relaxes

6. In the absence of dystrophin, the entire protein complex is lost

III. DMD is a severe form of muscular dystrophy, which is characterized at the cellular level by the virtual absence of dystrophin molecules from both skeletal & cardiac muscle cells

A. A less severe form (Becker muscular dystrophy) also occurs, in which the protein is present but is abnormal and/or greatly reduced in amount

B. The symptoms of DMD usually appear in childhood when the patient has difficulty performing certain motor tasks (climbing stairs, rising from a prone position)

1. The disease progresses with increasing muscular weakness & debilitation

2. It finally takes the patient's life as a result of respiratory or cardiac failure

C. Patients with DMD show dramatic abnormalities in both cardiac & skeletal muscle tissue

1. At the light microscopic level, some of the muscle fibers are seen to be degenerating (necrotic) & often infiltrated by macrophages of the immune system

2. At the EM level, segments of the plasma membrane are seen to be missing from the cell surface so that the extracellular basement membrane remains as the muscle fiber's primary encasement

3. Plasma membrane destruction is accompanied by marked internal cell changes (sarcoplasmic reticulum dilation, mitochondrial swelling, increased proteolytic cell material digestion & myofibril rupture)

4. Plasma membrane damage is apparently caused by mechanical stress exerted on it as muscle contracts; the greatest damage occurs to those muscles in body that are subjected to the greatest mechanical stress

IV. Dystrophin is not the only gene that, when mutated, can cause muscular dystrophy

A. Congenital muscular dystrophy (CMD) – a rare form of the disease that develops in early infancy & strikes boys & girls with equal frequency

1. It has been traced to a deficiency in one of the subunits of the laminin molecule that forms a key component of the muscle cell basement membrane

B. Severe childhood autosomal recessive muscular dystrophy (SCARMD) – results from absence of one of the subunits of the DAP complex

C. Patients with DMD, CMD & SCARMD all exhibit severe forms of the disease; indicates that all 3 components of the molecular chain (dystrophin, DAP, laminin) are required for muscle function

V. Presently, there is no treatment to stem the degenerative changes that occur with any of the MDs; several MD animal models have been developed, including the mdx mouse, which lacks a functional dystrophin gene

A. Unlike its human counterparts, these dystrophin-deficient mice show only mild symptoms but are still useful models for testing new molecular-based therapies & screening drugs that might have therapeutic value

B. Studies of mdx mice have turned up one drug, the antibiotic gentamicin that causes the animals to synthesize dystrophin & protects them from muscle damage

1. mdx mice fail to produce dystrophin because their dystrophin genes contain a mutation that leads to premature termination during synthesis of the protein

2. Gentamicin reverses the effects of this mutation by allowing the ribosome to read through the defect on the mRNA & produce a full-sized dystrophin molecule

3. It is estimated that ~15% of DMD patients fail to synthesize dystrophin because of a similar type of genetic defect

4. Several clinical trials of gentamicin on members of this population are under way; in first preliminary report, researchers failed to detect dystrophin production in 4 patients treated with the drug

5. A number of other compounds that improve muscle strength in the mdx mouse are in clinical trials

C. Another approach that has been tried in clinical trials on human patients, without measureable success, is cell transplantation therapy

1. Normal, undifferentiated muscle cells (myoblasts) were isolated from muscle tissue of close relatives & injected into particular muscles of the patient

2. It was hoped that the donor myoblasts would fuse with the genetically deficient cells of the patient & provide genetic messages for the production of normal dystrophin throughout the entire muscle fiber

3. The survival rate of the injected cells was very low & little or no evidence of the production of dystrophin was observed

D. Recently, a new approach in cell transplantation therapy has begun using stem cells rather than myoblasts

1. Studies have shown that adult stem cells, whether from bone marrow or other sources, can be obtained in larger numbers than myoblasts & are more likely to survive & differentiate after transplantation

2. A recent experiment – stem cells were isolated from knee joints of human donors; they were allowed to proliferate in culture & injected directly into mdx mouse leg nuscles

3. The mice had been treated with drugs to prevent immune rejection of the human cells

4. Muscle tissue examination showed that the fibers made human dystrophin & a particular mouse protein (normally absent from mdx mouse muscle) that serves as marker of muscle contractility restoration

E. It is hoped that eventually stem cells can be removed from DMD patient, provided with normal copy of the dystrophin gene in vitro & then reintroduced into the patient to give rise to normal muscle tissue

1. Alternatively, DMD patients might receive dystrophin-producing stem cells from a healthy, compatible donor