1.  Prior to the restriction point, mammalian cells require the presence of growth factors in their culture medium, if they are to progress in cell cycle

2.  After they have passed the restriction point, these same cells will continue through the remainder of the cell cycle without external stimulation

    IV.  Commitment process for mitosis (M phase) entry involves cyclic availability of cyclins different from those needed to enter S, but same Cdk (cdc2) – 2nd transition point (just before G2 end)

A.  Requires cdc2 activation by mitotic cyclins (different from those at START); Cdks containing a mitotic cyclin (e.g., MPF) phosphorylate substrates needed for cell to enter mitosis

B.  G2-activated, Cdk-phosphorylated cytoplasmic proteins start dynamic changes in organization of both chromosomes & cytoskeleton that characterize the shift from interphase to mitosis like those below:

1.  Nuclear proteins (histone H1) - phosphorylation may help compact chromosomes

2.  Nuclear lamins - phosphorylation leads to disassembly of nuclear envelope

V.  Cells make a 3rd commitment during the middle of mitosis, which determines whether they will complete cell division & reenter G1 of the next cycle

A.  Exit from mitosis & entry into G1 depends on rapid decrease in Cdk activity that results from a plunge in mitotic cyclin concentration

    VI.  Cyclin-dependent kinases, which are described as the engines that drive the cell through its various stages, are regulated by a number of factors that operate in combination to brake or accelerate a process, including:

A.  Cyclin concentration

B.  Cdk phosphorylation state

C.  Cdk inhibitors

D.  Controlled proteolysis

E.  Subcellular localization

Control of the Cell Cycle: Factors That Regulate Cyclin-Dependent Kinases (Cdks)

I.  Cyclin concentration – Cdks are activated by association with a regulatory subunit or cyclin; the presence in the cell of a particular cyclin follows the activation of transcription of the gene encoding that cyclin

A.  Different cyclin genes are transcribed at different stages during the cell cycle

B.  When a cyclin is present in the cell, it binds to the Cdk catalytic subunit, causing a major change in that catalytic subunit's conformation

C.  X-ray crystallography - cyclin binding causes a flexible loop of the Cdk polypeptide chain to move away from the opening to enzyme's active site; allows Cdk to phosphorylate its protein substrates

II.  Cdk phosphorylation state – many events in cell are regulated by addition & removal of phosphate groups from protein; same is true for events leading to mitosis onset

A.  Level of mitotic cyclins rises through S & G2; mitotic cyclins present in yeast cell during this period bind to Cdk to form a cyclin-Cdk complex, but the complex shows little evidence of kinase activity

1.  Then, late in G2, cyclin-Cdk complex becomes activated & mitosis is triggered

2.  How does Cdk activity change? – must look at activity of 3 other regulatory enzymes (2 kinases &  phosphatase whose activity was revealed through a combination of genetic & biochemical analyses)

B.  One of kinases, CAK (Cdk-activating kinase), phosphorylates a critical threonine residue (Thr 161 of the Cdk [cdc2]); phosphorylation of this residue is necessary, but not sufficient, for Cdk to be active

C.  A second protein kinase, Wee1, phosphorylates a key tyrosine residue in the ATP-binding pocket of the enzyme (Tyr 15 of cdc2) & inhibits the kinase's activity

1.  If this residue is phosphorylated, the enzyme is inactive, regardless of the phosphorylation state of any other residue

2.  Thus, the effect of Wee1 overrides the effect of CAK, keeping the Cdk in an inactive state

3.  If the wee1 gene is mutated, mutants cannot maintain the Cdk in an inactive state & they divide at an early stage of the cell cycle producing smaller cells, hence the name "wee"

4.  In normal cells, Wee1 keeps the Cdk inactive until the end of G2

D.  Then, at the end of G2, the inhibitory phosphate at Tyr 15 is removed by the third enzyme, a phosphatase named Cdc25

1.  Removal of the phosphate switches the stored cyclin-Cdk molecules into the active state, allowing it to phosphorylate key substrates & drive the yeast cell into mitosis

2.  Cells with a mutant cdc25 gene cannot remove the inhibitory phosphate from the Cdk & cannot enter mitosis

E.  The balance between Wee1 kinase & Cdc25 phosphatase activities, which normally determines whether the cell will remain in G2 or progress into mitosis, is regulated by still other kinases & phosphatases

F.  These pathways can stop the cell from entering mitosis under conditions that might lead to an abnormal cell division

III.  Cdk inhibitors – Cdk activity can be blocked by a variety of inhibitors

A.  In budding yeast, a protein (Sic1) acts as Cdk inhibitor during G1

B.  If Sic1 is degraded —> cyclin-Cdk present in the cell initiates DNA replication

IV.  Controlled proteolysis – cyclin concentrations oscillate during each cell cycle leading to changes in Cdk activity; cells regulate cyclin concentrations & other key cell cycle proteins

A.  They do this by adjusting both the synthesis rate & destruction rate of the molecule at different points in the cell cycle (degradation is accomplished via the ubiquitin-proteasome pathway)

1.  Unlike other mechanisms that control Cdk activity, degradation is an irreversible event that helps drive the cell cycle in a single direction

B.  Cell cycle regulation requires 2 classes of multisubunit complexes (SCF & APC complexes) that function as ubiquitin ligases

1.  They recognize proteins that have been targeted for degradation & link them to polyubiquitin chain —> ensures their destruction in a proteasome

C.  SCF complexes are active from late G1 through early mitosis & mediate the destruction of G1 cyclins, Cdk inhibitors & other cell cycle proteins

1.  These proteins become targets for SCF after they are phosphorylated by the protein kinases (i.e., the Cdks) that regulate the cell cycle

2.  Mutations that inhibit SCFs from mediating proteolysis of key proteins, like G1 cyclins or the Sic inhibitor, can prevent cells from entering S phase & replicating their DNA

D.  The APC complex acts in mitosis & degrades a number of key mitotic proteins, including the mitotic cyclins; destruction of these cyclins allows a cell to exit mitosis & enter a new cell cycle

V.  Subcellular localization – cells contain a number of different compartments in which regulatory molecules can either be united with or separated from the proteins with which they interact

A.  Subcellular localization is a dynamic phenomenon characterized by movement of cell cycle regulators into different compartments at different stages

B.  Example: cyclin B1 (a major animal cell mitotic cyclin) shuttles between the nucleus & cytoplasm until G2, when it accumulates in the nucleus just prior to the onset of mitosis

1.  Nuclear accumulation of cyclin B1 is facilitated by phosphorylation of a cluster or serine residues that reside in its nuclear export signal (NES)

2.  Phosphorylation at this site presumably blocks subsequent export of cyclin back to cytoplasm

3.  If nuclear accumulation of cyclin is blocked, cells fail to initiate cell division

Control of the Cell Cycle: Control of the Cell Cycle in Mammalian Cells

I.  As in yeast, successive waves of synthesis & degradation of different cyclins play key role in driving mammal cells from one stage to next, however……..

A.  Unlike yeast cells (with only 1 Cdk), mammalian cells make several different versions of this protein kinase

1.  Different cyclin-Cdk complexes target different groups of substrates at different points within cell cycle

B.  The pairing between individual cyclins & Cdks is specific; only certain combinations are found

1.  Cyclin E-Cdk2 complex drives the cell into S phase

2.  Cyclin B1-Cdk1 complex drives the cell into mitosis

C.  Cdks do not always stimulate activities, but can also inhibit inappropriate events – cyclin B1-Cdk1 activity during G2 prevents the inappropriate replication of DNA

II.  Studies aimed at identifying the roles of various cyclins & Cdks in mammalian cells have used genetically engineered (knockout) mice that lack a functional gene for that particular protein

A.  The phenotypes of these mice depend on the gene that has been eliminated

1.  Mice lacking Cdk1 or cyclin B1 die as early embryos, suggesting that these genes are essential for a normal cell cycle

2.  In contrast, mice lacking gene encoding one of the other Cdks or cyclin subunits develop surprisingly well; suggests that other members of gene family can take over functions of those that are lacking

B.  Despite this built-in genetic redundancy, the absence of any of these cell cycle regulatory proteins produces distinct abnormalities

1.  Mice lacking a gene for cyclin D1 are smaller than control animals, which stems from a reduction in the level of cell division throughout the body

2.  In addition, cyclin D1-deficient animals display a particular lack of cell proliferation during development of the retina

3.  Mice lacking Cdk2 appear to develop normally but exhibit specific defects during meiosis, which reinforces the important differences in the regulation of mitotic & meiotic divisions

Control of the Cell Cycle: Checkpoints, Kinase Inhibitors and Cellular Responses

I. Ataxia-telangiectasia (AT) – rare, recessive genetic disorder; irradiated cells go into mitosis despite damage

A.  Characterized by a host of diverse symptoms, including a greatly increased risk of certain types of cancer; the basis of the first 2 symptoms below have yet to be determined

1.  Unsteady posture (ataxia) resulting from degeneration of nerve cells in cerebellum

2.  Permanently dilated blood vessels (telangiectasia) in face & elsewhere

3.  Susceptibility to infection

4.  Cells with an abnormally high number of chromosome aberrations

B.  Late 1960s - AT patients died during radiation therapy; they were extremely sensitive to ionizing radiation; patients' cells are sensitive as well, lacking a crucial protective response seen in normal cells

1.  When normal cells are subjected to treatments that damage DNA (ionizing radiation or DNA-altering drugs), their progress through cell cycle stops while the damage is repaired

2.  If normal cell is irradiated during cell cycle G1 phase, it delays progression into S phase; if cells are irradiated in S phase, further DNA synthesis is delayed; cells irradiated in G2 delay entry into mitosis

II.  Leland Hartwell & Ted Weinert (1988) – worked in yeast & proposed that cells possess checkpoints as part of their cell cycle

A.  Checkpoints are surveillance mechanisms that halt progress of cell cycle if:

1.  Any of the chromosomal DNA is damaged or

2.  Certain critical processes, such as DNA replication during S phase or chromosome alignment during M phase, have not been properly completed

B.  Checkpoints ensure that each of the various events that make up the cell cycle occurs accurately & in the proper order

1.  Mostly, the proteins of checkpoint machinery have no role in normal cell cycle events & are only called into action when an abnormality appears

2.  Genes encoding checkpoint proteins were first found in screens for mutant yeast cells that kept going through cell cycle, despite suffering DNA damage or other abnormalities that caused serious defects

III.  Checkpoints are activated throughout the cell cycle by a poorly understood system of sensors that recognize DNA damage or cellular abnormalities

A.  If a sensor detects the presence of a defect, it triggers a response that temporarily arrests further cell cycle process

B.  The cell can then use the delay to repair the damage or correct the defect rather than continuing on to the next stage

1.  This is especially important because mammalian cells that undergo division with genetic damage run the risk of becoming transformed into a cancer cell

2.  If the DNA is damaged beyond repair, the checkpoint mechanisms can transmit a signal that leads to the death of the potentially hazardous cell

C.  The gene responsible for AT (ATM gene) encodes a protein kinase that is activated by certain DNA lesions, particularly double-stranded breaks  

1.  Remarkably, the presence of a single break in one of the cell's DNA molecules is sufficient to cause rapid, large-scale activation of ATM molecules, causing cell cycle arrest

2.  A related protein kinase (ATR) is activated by other types of lesions, including those resulting from incompletely replicated DNA or UV irradiation

3.  Both ATM & ATR are part of multiprotein complexes that can bind to chromatin that contains damaged DNA

4.  Once bound, ATM & ATR can phosphorylate a large variety of proteins that participate in cell cycle checkpoints & DNA repair

IV.  How does a cell stop its progress from one stage of the cell cycle to the next? - 2 pathways are available to mammalian cells to arrest cell cycle

A.  Arrest pathway #1 – if a cell preparing to enter mitosis is subjected to UV irradiation, ATR kinase is activated & the cell arrests in G2

1.  ATR kinase molecules are thought to be recruited to sites of protein-coated, single-stranded DNA, like those present as UV-damaged DNA is repaired

2.  ATR phosphorylates & activates a checkpoint kinase (Chk1)

3.  Chk1, in turn, phosphorylates Cdc25 on a particular serine residue, making the Cdc25 molecule a target for a special adaptor protein that binds to the phosphatase in the cytoplasm

4.  This interaction inhibits Cdc25's phosphatase activity & prevents it from being reimported into the nucleus

5.  Cdc25 normally plays a key role in the G2/M transition by removing inhibitory phosphates from Cdk1; thus, Cdc25 absence from the nucleus leaves Cdk in an inactive state & the cell arrested in G2

B.  Arrest pathway #2 - damage to DNA also leads to synthesis of proteins that directly inhibit the cyclin-Cdk complex that drives the cell cycle; ATM is involved in this checkpoint mechanism

1.  Cells exposed to ionizing radiation in G1 make a protein (p21; 21 kD molecular mass) that inhibits the G1 Cdk kinase activity, preventing cells from phosphorylating key substrates & entering S phase

2.  ATM phosphorylates & activates another checkpoint kinase (Chk2) that phosphorylates transcription factor (p53), which leads to transcription & translation of p21 gene & subsequent inhibition of Cdk

a.  ~50% of all human tumors show evidence of mutations in the gene that encodes p53 gene, which reflects its importance in the control of cell growth

3.  p21 (see above) is only 1 of at least 7 known Cdk inhibitors - a related Cdk inhibitor (p27) & another cyclin-Cdk complex interact as well

a.  The p27 molecule drapes itself across both subunits of the cyclin A-Cdk2 complex, changing the conformation of the catalytic subunit & inhibiting its kinase activity

b.  In many cells, p27 must be phosphorylated & then degraded before progression to S phase can occur

V.  Cdk inhibitors, like p21 & p27, are also active in cell differentiation; just before they differentiate, cells of all types (muscle, liver, blood, etc) typically withdraw from the cell cycle & stop dividing

A.  Cdk inhibitors are thought to either allow or directly induce cell cycle withdrawal

B.  Study Cdk inhibitors with genetically engineered knockout mice unable to make one of them

1.  p27 gene knockout mice – have distinctive phenotype; larger than normal mice; certain organs (thymus gland, spleen) contain significantly greater numbers of cells than those of normal animal

2.  In normal mice, thymus & spleen cells make relatively high levels of p27; it is thought that absence of p27 in p27-deficient animals allows cells to divide several more times before differentiation

C.  Certain Cdk inhibitors seem to prevent uncontrolled growth that can lead to cancer development - most apparent when Cdk inhibitor is not made so that checkpoint control is disturbed – ex.: Cdk inhibitor p16

1.  Gene encoding Cdk inhibitor p16 is often deleted in a variety of human tumors

2.  Knockout mice lacking p16 Cdk inhibitor gene exhibit greatly increased incidence of cancer

3.  Mimicking effects of such Cdk inhibitory proteins could lead to synthesis of new anticancer drugs that block uncontrolled cell growth

M Phase: Mitosis and Cytokinesis

I.  Mitosis - from Greek mitos, for thread; named in 1882 by German biologist Walther Flemming to describe threadlike chromosomes that mysteriously appeared just before a cell divided in two

A.  What we know about M phase is largely based on years of animal & plant observations & research

B.  Mitosis is process of nuclear division in which replicated DNA molecules of each chromosome are faithfully segregated into 2 nuclei

1.  Mitosis is usually accompanied by cytokinesis – dividing cell splits in two, partitioning cytoplasm into 2 cellular packages

2.  The 2 daughter cells are genetically identical to each other & mother cell after mitosis & cytokinesis

C.  It maintains chromosome number & generates new cells for organism growth, maintenance & repair

D.  Happens in diploid or haploid (fungi, plant gametophytes, a few animals like male bees [drones]) cells

E.  Most metabolic activities (transcription, translation) are curtailed; cell is unresponsive to external stimuli; virtually all cell energy is devoted to one activity - chromosome segregation

II.  Our understanding of events that occur during mitosis has been greatly aided by the use of extracts prepared from frog eggs; extracts contain all materials needed to support mitosis (histones, tubulins, etc.)

A.  May use original frog egg nucleus present in egg or a foreign nucleus added to egg extract

1.  Add foreign chromatin (or DNA) to egg extract —> chromatin compacted into mitotic chromosomes, which are segregated by a mitotic spindle that assembles spontaneously in cell-free mixture

B.  Can study role of particular protein in mitosis by removing that protein from egg extract by addition of antibody against it (immunodepletion) & determining whether process can continue in its absence

III.  Usually divided into 5 distinct stages - prophase, prometaphase, metaphase, anaphase, telophase 

A.  Each is characterized by a particular series of events

B.  Each stage represents a segment of a continuous process - division into arbitrary phases is done only for the sake of discussion & experimentation

The Stages of Mitosis: Prophase

I.  During prophase, duplicated chromosomes are prepared for segregation & mitotic machinery is assembled

A.  Formation of mitotic chromosomes

B.  Formation of mitotic spindle

C.  Dissolution of nuclear envelope and fragmentation of cytoplasmic organelles

II.  Mitotic chromosome formation

A.  Interphase cell nucleus contains tremendous lengths of chromatin fibers; this extended state is ideal for transcription & replication, but not for segregation into daughter cells

1.  Chromosomes are converted into much shorter, thicker structures by chromosome compaction or condensation that occurs during early prophase

2.  Interphase chromatin is organized into fibers ~30 nm in diameter; mitotic chromosomes are made of similar types of fibers as seen in EM of whole chromosomes isolated from mitotic cells

B.  Thus, chromosome compaction does not alter the nature of a chromatin fiber, but rather its packaging

1.  Treat mitotic chromosomes with solutions to solubilize histones & most nonhistone proteins

2.  After treatment, EMs of the treated chromosomes reveal a structural framework or scaffold that retains the basic shape of the mitotic chromosome

3.  DNA loops attach at their base to the nonhistone proteins that form chromosome scaffolding

4.  During interphase, scaffold proteins are dispersed within nucleus, probably as part of nuclear matrix

C.  In recent years, research on chromosome compaction has focused on an abundant multiprotein complex called condensin

1.  Its proteins were found by incubating nuclei in frog egg extracts & identifying the proteins that associated with chromosomes as they underwent compaction

2.  Removal of condensin from the frog egg extracts prevented normal chromosome compaction

D.  How does condensin induce compaction? – supercoiled DNA fills a much smaller volume than relaxed DNA; supercoiling may play role in compacting chromatin into tiny volume of mitotic chromosome

1.  In presence of topoisomerase & ATP, condensin can bind to DNA in vitro & curl it into positively supercoiled loops

2.  This fits with observation that chromosome compaction at prophase in vivo requires topoisomerase II, which along with condensin is present as part of mitotic chromosome scaffold

3.  Condensin is activated at mitosis onset by phosphorylation of several of its subunits by the Cdk-cyclin responsible for driving cells from G2 into mitosis

4. Presumably, condensin is one of targets through which Cdks trigger cell cycle activities

E.  Due to compaction, mitotic chromosomes appear as distinct, rodlike structures; each one is made of 2 mirror-image sister chromatids formed during replication in previous interphase

1.  Before replication, DNA of each interphase chromosome becomes associated at sites along its length with a multiprotein complex called cohesin

2.  After replication, cohesin functions as a physical bridge that holds the 2 sister chromatids together through G2 & into mitosis, when they are ultimately separated

3.  Condensin & cohesin have similar structural organization – EMs suggest cohesin adopts circular configuration with latch at one end that can open & close in response to ATP binding/hydrolysis

4.  Recent experiments support the hypothesis that the cohesin ring encircles 2 sister DNA molecules

F.  In vertebrates, cohesin is released from the chromosomes in 2 distinct stages

1.  Most of the cohesin dissociates from the arms of the chromosomes as they become compacted during prophase

a.  Dissociation is induced by phosphorylation of cohesin subunits by 2 important mitotic enzymes (Polo-like kinase & Aurora B kinase)

2.  After phosphorylation, the chromatids of each mitotic chromosome are held relatively loosely along their extended arms, but much more tightly at their centromeres, where cohesin stays bound

a.  Cohesin is thought to remain at centromeres due to the presence there of a phosphatase that removes any phosphate groups added to the protein

b.  Release of cohesin from centromeres is normally delayed until anaphase

c.  If the phosphatase is experimentally inactivated, sister chromatids separate from one another prematurely prior to anaphase

III.  Centromeres and kinetochores – the most notable mitotic chromosome landmark is the primary constriction, an indentation that marks the position of the centromere

A.  Centromere is residence of highly repeated DNA sequences that are binding sites for specific proteins

B.  At outer surface of centromere of each chromatid is proteinaceous, buttonlike structure (kinetochore) – seen in sections through a mitotic chromosome

1.  The kinetochore assembles on the centromere during prophase

2.  It serves as chromosome attachment site for dynamic MTs of mitotic spindle, as the residence of several MT-based motor proteins & as a key component of an important mitotic checkpoint

IV.  Mitotic spindle formation – as cells move from G2 to mitosis, G2 MTs undergo sweeping disassembly, then reassemble forming mitotic spindle with focus at centrosome, a special animal cell MT-organizing structure

A.  Rapid disassembly of interphase cytoskeleton is thought to be accomplished by inactivation of proteins that stabilize MTs (MT-associated proteins or MAPs) & activation of proteins that destabilize MTs

B.  Centrosome cycle as it progresses in concert with cell cycle - animal cells after mitosis have 1 centrosome with 2 centrioles situated at right angles to one another

1.  Each centriole of centrosome starts its replication in the cytoplasm at the onset of S phase as DNA replication begins in nucleus; process starts as centrioles move apart within centrosome

2.  Soon, a small daughter centriole appears next to each preexisting (maternal) centriole oriented at right angles to it; later, MTs of new centriole elongate, bringing the centriole to full length

3.  At the start of mitosis, the centrosome splits into 2 adjacent centrosomes, each containing a pair of mother-daughter centrioles

4.  The start of centrosome duplication at the G1-S transition is triggered by phosphorylation of a centrosomal protein by cyclin E-Cdk2, the same agent that triggers the onset of DNA replication

5.  Centrosome duplication errors can lead to abnormal cell division & may help cancer develop

C.  First stage of spindle formation in typical animal cell – MTs appear in sunburst arrangement (aster) around each centrosome during early prophase

1.  Phosphorylation of key proteins by newly active mitotic Cdk increases centrosome MT-nucleating activity at mitosis; the mitotic Cdk controls G2 to M progression

2.  Aster formation is followed by centrosomes separating from each other & their subsequent movement around the nucleus to the opposite ends of the cell

3.  Centrosome separation is driven by motor proteins associated with the adjacent MTs

4.  As centrosomes separate, MTs stretching between them increase in number & elongate

5.  Eventually, they reach points opposite one another & establish 2 poles of bipolar mitotic spindle

6.  After mitosis, one centrosome is distributed to each daughter cell  

D.  Centrosomes are not essential components in formation of bipolar mitotic spindle in all cells

1.  Some animal cells (early mouse embryo) & most higher plant cells lack centrosomes, yet all of these cells undergo relatively typical mitosis

2.  Functional mitotic spindles even form in mutant Drosophila cells lacking centrosomes or in mammalian cells in which centrosome was experimentally removed

3.  In all of these cases, the MTs of the mitotic spindle are nucleated near the chromosomes rather than at the poles where centrosomes would normally reside

4.  Once they have polymerized, the minus ends of MTs are brought together (focused) at each spindle pole through the activity of motor proteins

E.  These types of experiments suggested that cells possess 2 fundamentally different mechanisms — one centrosome-dependent & the other centrosome-independent — to achieve the same end result

1.  Recent studies have indicated that both pathways to spindle formation operate simultaneously in the same cell

2.  These studies also show that even cells with functional centrosomes nucleate a significant fraction of their spindle MTs at the chromosomes

V.  Nuclear envelope dissolution & cytoplasmic organelle partitioning

A.  Nuclear envelope breakdown occurs at prophase end; it allows spindle-chromosome interaction since spindle forms in the cytoplasm & chromosomes compact in the nucleus

1.  Classical view of nuclear envelope breakdown – nuclear envelope is fragmented into a population of small vesicles that disperse throughout mitotic cell

B.  This view has been challenged in recent years by mammalian cell studies that suggest that the nuclear envelope is torn apart mechanically by forces exerted through MTs & molecular motors

1.  Events begin with the association of cytoplasmic dynein with the outer surface of nuclear envelope

2.  Dynein molecules then move along MTs toward their minus ends (toward centrosome) pulling on attached nuclear envelope, which forms deep invaginations in the vicinity of centrosomes

3.  These invaginations containing a nearby centrosome have been seen for years in mitotic cell EMs

4.  Nuclear envelope invagination on one side of nucleus is proposed to produce a stretching force (tension) at the opposite side of the nucleus, which tears open nucleus

5.  The opening in the membrane spreads & eventually produces fragments that are transported along MTs away from chromosomes & toward centrosomes

6.  The disintegration of nuclear envelope provides access to the chromosomes by MTs of the spindle

C.  Some membranous cytoplasmic organelles remain relatively intact through mitosis - mitochondria, lysosomes, peroxisomes, plant chloroplasts

D.  Golgi complex partitioning mechanism during mitosis has been controversial in recent years – 3 views of what happens; ultimately, we may learn that different cell & organism types use different mechanisms

1.  Forward (anterograde) membrane/material movement from ER to Golgi stops during prophase, but vesicle movement in opposite (retrograde) direction continues

a.  Thus Golgi complex contents are incorporated into ER & Golgi ceases to exist briefly as a distinct organelle

2.  In alternate view, Golgi membranes are fragmented to form a distinct population of small vesicles that are partitioned between daughter cells

3.  Third view based primarily on algae & protist studies – entire Golgi complex splits in two, with each daughter cell receiving half of original structure

E.  Ideas about fate of ER have changed & are somewhat controversial as well – recent studies on living, cultured mammalian cells suggest that the ER network remains relatively intact during mitosis

1.  This view challenges earlier studies performed largely on eggs & embryos that suggested that ER undergoes fragmentation during prophase

The Stages of Mitosis: Prometaphase

I.  Prometaphase - starts with dissolution of the nuclear envelope

A.  During this stage, mitotic spindle assembly is completed

B.  Chromosomes are moved into position at center of cell

II.  At prometaphase start, compacted chromosomes are scattered throughout space that was nuclear region

A.  As spindle MTs penetrate central cell region, MT free ends grow & shrink in dynamic fashion as if they are searching for chromosome

1.  It is not certain whether searching is entirely random, as evidence suggests that MTs may grow preferentially towards a site containing chromatin

2.  Those MTs that contact a kinetochore are captured & stabilized

3.  Lateral surface (side-wall) of MT makes initial contact with kinetochore, not its free end

4.  Once initial contact is made, some chromosomes then move actively along MT wall powered by motor proteins located in the kinetochore

5.  Soon, kinetochore tends to become stably associated with plus end of ≥1 spindle MTs from one of poles; then unattached sister chromatid kinetochore captures its own MTs from opposite spindle pole

6.  The 2 sister chromatids become connected by kinetochores to MTs extending from opposite poles

B.  Chromosomes not moved directly to spindle center; they oscillate back & forth in both a poleward & anti-poleward direction; ultimately moved by process (congression) to spindle center midway between poles

1.  Forces needed are generated by motor proteins associated with both kinetochores & chromosome arms

2.  As chromosomes congress to mitotic spindle center, longer MTs attached to one kinetochore are shortened, while shorter ones attached to sister kinetochore are elongated

3.  These changes in MT length are thought to be governed by differences in pulling force (tension) on the 2 sister kinetochores

4.  MT elongation & shortening is the result of a gain or loss of subunits at MT (+) end; remarkably, this dynamic activity occurs while each MT (+) end remains attached to kinetochore

5.  Eventually, each chromosome moves into position along a plane at spindle center so that MTs from each pole are equivalent in length

The Stages of Mitosis: Metaphase

I.  Starts with chromosomes aligned at spindle equator in a plane (metaphase plate); one chromatid attached by its kinetochore to spindle fiber from one pole, other chromatid attached to fiber from opposite pole

II.  Metaphase MTs are highly organized array that is ideally suited for the task of separating duplicated chromatids & the MTs of animal cell are divided into 3 groups of fibers, all of which have same polarity

A.  Astral MTs - radiate outward from centrosome into region outside the body of the spindle; they help position the spindle apparatus in the cell & determine the plane of cytokinesis

B.  Chromosomal (kinetochore) MTs - extend between centrosome & chromosome kinetochores; exert a pulling force on kinetochores during metaphase

1.  In mammalian cells, each kinetochore is attached to a 20 – 30 MT bundle, forming a spindle fiber

2.  Maintain chromosomes in equatorial plane by tug-of-war between balanced pulling forces exerted by chromosomal spindle fibers from opposite poles

3.  During anaphase, chromosomal MTs are needed for movement of chromosomes toward the poles

C.  Polar (interpolar) MTs - extend from centrosome past chromosomes; they form a structural basket that maintains spindle mechanical integrity

1.  Polar MTs from one centrosome overlap with their counterparts from the opposite centrosome

III.  Metaphase appears to be a stage during which the cell pauses for a brief period as if all the mitotic activities suddenly come to a halt, however, analysis shows that important events occur during this time

IV.  Microtubule flux in the metaphase spindle – even though chromosomal MTs do not appear to change in length while chromosomes are aligned at metaphase plate, studies suggest they are in highly dynamic state

A.  When they use fluorescently labeled tubulin to follow MT's state, subunits are rapidly lost & added from the plus ends of chromosomal MTs, even though these ends are presumably attached to kinetochore

1.  Thus, kinetochore does not act like cap at end of MT, blocking entry or exit of terminal subunits

2.  Instead, it is the site of dynamic activity

B.  Since more subunits are added than lost at plus end, there is a net addition of subunits at the kinetochore; meanwhile, the minus end of the MTs experiences a net loss

1.  Thus, subunits are thought to move along chromosomal MTs from kinetochore to pole

2.  Loss of tubulin subunits at the poles is likely aided by a member of the kinesin-13 family of motor proteins whose function is to promotes MT depolymerization rather than movement

The Stages of Mitosis: Anaphase

I.  Starts as sister chromatids of each chromosome split apart & begin movement toward opposite poles – the control of anaphase & its initiation mechanism have been revealed by genetic & biochemical approaches

A.  2 distinct multiprotein complexes (SCF & APC) act at different stages of the cell cycle to ubiquinate proteins & target them for destruction by a proteasome

1.  SCF acts primarily during interphase

2.  In contrast, the Anaphase promoting complex (APC) could be described as the mitotic terminator; it plays a key role in regulating the events that occur during mitosis

B.  APC contains ~12 subunits, one of which plays a key role in determining which proteins will serve as APC substrate; this "substrate-targeting" subunit is represented by members of a family of proteins

1.  2 members of the family in budding yeast cells (Cdc20 & Cdh1) play an important role in substrate selection during mitosis

2.  APC complexes having one or the other of these subunits are called APCCdc20 or APCCdh1

C.  APCCdc20 is activated at the metaphase/anaphase transition & ubiquinates a major anaphase inhibitor (securin) since it secures the attachment between sister chromatids

1.  Ubiquitination & destruction of securin at end of metaphase releases an active protease (separase)

2.  Separase then cleaves a key subunit of the cohesin molecules that hold sister chromatids together

3.  Cleavage of cohesin triggers the separation of the sister chromatids to mark the onset of anaphase

D.  Near the end of mitosis in budding yeast cells, the Cdc20 subunit of APC is replaced by the other substrate-targeting subunit, Cdh1

1.  When Cdh1 is attached to APC, the enzyme completes the ubiquitination of mitotic cyclins

2.  Destruction of these cyclins leads to a precipitous drop in activity of the mitotic Cdk & the progression of the cell out of mitosis & into the G1 phase of the next cell cycle

II.  Events of anaphase – all metaphase plate chromosomes split synchronously at anaphase onset; the chromatids are now referred to as chromosomes, since they are no longer attached to their duplicates

A.  The chromosomes then begin to migrate poleward - as chromosome moves during anaphase, its centromere is seen at its leading edge with the arms of the chromosome trailing behind

B.  Chromosomes move at ~1 µm/min (very slow compared to other types of cell movements); completed in anywhere from 2 to 60 min; equivalent to a trip from North Carolina to Italy in ~14 million years

1.  Slow rate of movement may ensure that chromosomes segregate accurately without entanglement

III.  Anaphase chromosomes exhibit 2 types of movement – anaphase A & anaphase B

A.  Anaphase A – movement of chromosomes toward poles

B.  Anaphase B – a separate but simultaneous movement; the 2 spindle poles move farther apart

1.  Polar MTs from opposite poles overlap in the region of the spindle equator

2.  Separation of poles during anaphase B is accomplished by the sliding of overlapping MTs from opposite poles over one another in opposite directions

3.  The elongation of the mitotic spindle during anaphase B is accompanied by the net addition of tubulin subunits to the plus ends of the polar MTs

4.  Thus, subunits can be preferentially added to polar MTs & removed from chromosomal MTs at the same time in different regions of the same mitotic spindle

IV.  Forces required for chromosome movements at anaphase

A.  Over the past two or three decades, two broad proposals to explain the forces required for chromosome segregation during anaphase have been debated – both factors likely contribute to force generation

1.  Some think that motor proteins provide the necessary forces

2.  Others argue that microtubule dynamics is responsible

B.  Molecular motors – cytoplasmic dynein & at least 2 kinesin-related proteins have been identified at the kinetochores of mitotic chromosomes

1.  Thus, the chromosomes are endowed with all of the motor equipment needed to move themselves from one place to another along a MT

2.  Fluorescently labeled antibodies against dynein show that during interphase, the protein is localized in cytoplasm; as cell enters mitosis, the motor protein is found at both spindle poles & kinetochores

3.  Cytoplasmic dynein moves along MT surface toward its minus end & thus would tend to move an attached chromosome toward the poles

4.  Inhibition of dynein function at anaphase greatly slows chromosome movement, suggesting that this motor protein at least contributes to poleward chromosome migration

C.  Poleward movement of chromosomes is accompanied by the obvious shortening of chromosomal MTs - several processes can contribute to this phenomenon

1.  It has long been appreciated that tubulin subunits are lost from the plus (kinetochore-based) end of chromosomal MTs during anaphase A

2.  The loss of tubulin subunits from the plus end may be aided by the presence at the kinetochore of a type of kinesin that promotes MT depolymerization rather than movement

3.  Subunits are also lost from the minus end of these MTs as a result of the continued poleward flux of tubulin subunits that occurs during prometaphase & metaphase

4.  The primary difference in MT dynamics between metaphase & anaphase is that subunits are added to MT plus ends during metaphase, whereas subunits are lost from the plus ends during anaphase

5.  Addition of subunits to plus ends at metaphase keeps the length of chromosomal fibers constant, whereas the loss of subunits at plus ends during anaphase results in chromosomal fiber shortening

6.  This change in behavior at the MT plus ends is thought to be triggered by a change in tension on the kinetochores following separation of the sister chromatids

7.  The basis for this change in MT dynamics at the kinetochore is discussed below

D.  Shinya Inoué (early 1960s; Marine Biological Lab, Woods Hole) said that chromosomal MT disassembly during anaphase was not simply a consequence of chromosome movement, but the cause of it

1.  He suggested that depolymerization of MTs that comprise a spindle fiber could generate sufficient mechanical force to pull a chromosome forward

E.  Early experimental support for the disassembly-force model came from studies in which chromosomes underwent considerable movement as the result of the depolymerization of attached MTs

1.  Movement of a MT-bound chromosome occurs in vitro after dilution of the medium

2.  Dilution reduces the concentration of soluble tubulin, which, in turn, promotes MT depolymerization

3.  Such experiments indicate that MT depolymerization alone can generate enough force to pull chromosomes considerable distances, but don't address question whether mechanism is used by cells

V.  Events proposed to occur during chromosomal movement at anaphase

A.  MTs that comprise chromosomal spindle fibers undergo depolymerization at both their minus & plus ends during anaphase; these combined activities lead to movement of chromosomes toward the pole

1.  Depolymerization at MT minus ends serves to transport MTs toward the poles due to poleward flux (like person on moving walkway at airport)

2.  In contrast, depolymerization at MT plus ends serves to "chew up" the fiber that is towing the chromosomes

3.  Some cells rely more on poleward flux, others more on plus-end depolymerization

B.  Studies of animal cells in anaphase have shown that both the plus & minus ends of chromosomal fibers are sites where depolymerizing kinesins (members of kinesin-13 family) are localized

1.  If either of these MT "depolymerases" are specifically inhibited, chromosome segregation during anaphase is at least partially disrupted

2.  Such findings suggest ATP-dependent, kinesin-mediated depolymerization (as opposed to type of depolymerization that typifies dynamic instability) forms basis for mitotic chromosome segregation

C.  Other recent studies on yeast cells have revealed the apparent mechanism by which the depolymerizing plus ends of these MTs are physically linked to the chromosome kinetochores

1.  Each yeast chromosome kinetochore contains a ring-shaped protein complex  (Dam1) whose inner diameter of 32 nm is large enough to comfortably surround a MT

2.  In the presence of MTs, Dam1 assembles to form rings & helices that encircle the MTs

3.  If these encircled MTs are induced to depolymerize in vitro, the rings are seen to slide along the MTs for distances of several micrometers, just behind the depolymerizing tip

4.  According to this model, the energy released by the protofilaments as they peel away from the MT is utilized to push the Dam1 ring toward the opposite end of the MT

5.  Because the ring is attached to the kinetochore of the anaphase chromosome, the entire chromosome is moved toward the spindle pole

6.  Although vertebrates do not possess close homologues of the Dam1 proteins, it is very likely that a protein complex with a similar structure & function is present in all eukaryotic cells

7.  Even if such rings are not present, molecular motors present at kinetochores could play a critical role in anchoring chromosomes to the plus ends of chromosomal MTs as they lose subunits

VI.  The spindle checkpoint – operates at transition between metaphase & anaphase; best revealed when one or more chromosomes fails to align properly at metaphase plate

A.  When this happens, the checkpoint mechanism delays onset of anaphase until the misplaced chromosome has assumed its proper position along the spindle equator

B.  If a cell were not able to postpone chromosome segregation, it would greatly elevate the risk of daughter cells receiving an abnormal number of chromosomes (aneuploidy)

1.  This expectation has received confirmation with the identification of a number of children with inherited deficiencies in one of the spindle checkpoint proteins

2.  These individuals exhibit a disorder (named MVA), which is characterized by a high percentage of aneuploid cells & a greatly increased risk of developing cancer

VII.  How do cells know if chromosomes have aligned themselves properly at metaphase plate? - cells monitor at least 2 distinct properties of mitotic spindle

A.  If metaphase cell has chromosome connected by MTs to only one spindle pole, cell delays anaphase onset until chromosome becomes attached to spindles from both poles & aligned at the equator

1.  Unattached kinetochores contain a complex of proteins (best-studied one is Mad2) that mediate the spindle checkpoint

2.  The presence of these proteins at an unattached kinetochore sends a "wait" signal to the cell-cycle machinery that prevents the cell from continuing into anaphase

3.  Once the wayward chromosome is attached to spindle fibers from both spindle poles & becomes properly aligned at the metaphase plate, the signaling complex leaves the kinetochore

4.  This turns off the "wait" signal & allows the cell to progress into anaphase

B.  Evidence for above – in mitotic spindle of cell arrested prior to metaphase due to a single unaligned chromosome, the unaligned chromosome kinetochore is the only one that still contains Mad2 protein

1.  As long as cell contains unaligned chromosomes, Mad2 molecules can inhibit cell cycle progress

2.  Inhibition is achieved through direct interaction between Mad2 & the APC activator Cdc20

3.  While Cdc20 is bound to Mad2, APC complexes cannot ubiquinate securin (the anaphase inhibitor), keeping all of the sister chromatids attached to one another by their cohesin "glue"

C.  What trait of an unattached kinetochore makes it a binding site for checkpoint proteins like Mad2? – 2 interdependent properties distinguish unattached kinetochore; lack of either can arrest cell in metaphase

1.  A lack of physical interaction with microtubules and

2.  A lack of tension that is normally exerted at the kinetochore by attached microtubules

D.  Another abnormality that can delay anaphase is the occasional attachment of both chromatids of a chromosome to MTs from the same spindle pole; this leaves the chromosome lacking bipolar tension

1.  Kinetochores possess a corrective mechanism that is mediated by an enzyme called Aurora B kinase & thought to respond to a lack of tension

2.  According to favored model, Aurora B kinase molecules of the incorrectly oriented chromosome phosphorylate an unidentified substrate, which destabilizes MT attachment to both kinetochores

3.  Once freed of their bonds, the kinetochores of each sister chromatid have a fresh opportunity to become attached to MTs from opposite spindle poles

4.  Aurora B kinase inhibition in cells or extracts leads to chromosome misalignment & missegregation

The Stages of Mitosis: Telophase

I.  Telophase, the final stage of mitosis, start is marked by chromosomes collecting in mass as they near their respective poles

II.  During telophase, daughter cells return to their interphase condition

A.  Mitotic spindle disassembles

B.  Nuclear envelope reforms - membranous vesicles attach to chromosome surfaces & fuse laterally with one another forming increasingly larger double membrane envelope

C.  Chromosomes become more & more dispersed until they disappear from view under microscope

D.  Vesicles that were once part of ER fuse & begin to reform cell's membranous cytoplasmic network

E.  Partitioning of the cytoplasm into 2 separate daughter cells occurs by cytokinesis

Forces Required for Mitotic Movements

I.  Mitosis is characterized by extensive movements of cellular structures

A.  Prophase – spindle poles move to opposite ends of cells

B.  Prometaphase – chromosomes move to the spindle equator

C.  Anaphase A – chromosomes move from spindle equator to its poles

D.  Anaphase B – elongation of the spindle

II.  Molecular motors are responsible for movements during mitosis

A.  A number of different molecular motors have been identified in different locations in mitotic cells of widely diverse species; the motors involved in mitotic movements are primarily MT motors

1.  A number of different kinesin-related proteins & cytoplasmic dynein have been shown to be involved in mitosis

2.  Some motors move toward microtubule plus ends, others toward minus ends; one group of kinesins doesn't move anywhere, but promotes microtubule depolymerization

B. Motor proteins found at spindle poles, along spindle fibers & within kinetochores & chromosome arms

III.  Studies that reveal roles of specific motor proteins

A.  Analysis of phenotypes of cells lacking motor because of mutation in gene coding for part of motor

B.  Injection of antibodies against motor or inhibitors of motor into cells at various stages of mitosis

C.  Depletion of the motor protein from cell extracts in which mitotic spindles are formed

IV.  Tentative interpretations & conclusions about functions of various motor proteins:

A.  Motor proteins located along polar MTs probably contribute by keeping the poles apart

B.  Motors on chromosomes are probably important in chromosome movements during prometaphase, in maintaining chromosomes at metaphase plate & in separating them during anaphase

C.  Motor proteins sited along overlapping polar MTs in spindle equator region are likely responsible for cross-linking antiparallel MTs & sliding them over one another; elongates spindle during anaphase B

Cytokinesis

I.  Cytokinesis – process during which cell is divided into 2 daughter cells; usually coordinated with mitosis

II.  First hint of cytokinesis – occurs in late anaphase with cell surface indentation in a narrow band around the cell; in some cells like the ctenophore egg, the furrow initiates from only one side

A.  As time progresses, indentation deepens & forms furrow completely encircling cell

1.  Furrow plane lies in same plane previously occupied by metaphase plate chromosomes (perpendicular to spindle long axis); ensures that chromosome sets are partitioned into 2 different cells

2.  As one cell becomes two, additional plasma membrane is delivered to cell surface via cytoplasmic vesicles that fuse with the advancing cleavage furrow

3.  Furrow keeps deepening as it passes through tightly packed remnants of the central portion of the mitotic spindle, which forms cytoplasmic bridge between daughter cells called midbody

4.  Opposing surfaces ultimately fuse with one another in the center of cell, thus splitting the cell in 2

B.  Contractile ring theory (Douglas Marsland, late 1950s) – the force required to cleave cell is generated in a thin band of contractile cytoplasm found in cortex just under plasma membrane of furrow

1.  Large numbers of actin filaments are found in the cortex under the furrow of a cleaving cell aligned in an array parallel to cleavage furrow, seen in microscopic examination

2.  A smaller number of short, bipolar myosin II filaments is interspersed among the actin filaments (identified by their ability to bind anti-myosin II antibodies)

C.  Importance of myosin II in cytokinesis is evident from following experiments:

1.  Inject anti-myosin II antibodies into dividing cell —> rapid cessation of cytokinesis

2.  If cells lack functional myosin II gene —> cells cannot divide normally into daughter cells, but they do carry out nuclear division by mitosis

D.  The assembly of the actin-myosin contractile machinery in the plane of the future cleavage furrow is orchestrated by a G protein called RhoA

1.  In its GTP-bound state, RhoA triggers a cascade of events that lead to both the assembly of actin filaments & the activation of myosin II's motor activity

E.  The force-generating mechanism operating during cytokinesis is thought to be similar to the actin- & myosin-based contraction of muscle cells

1.  Sliding of actin filaments in muscle shortens muscle fiber; in contractile ring, sliding filaments pull cortex & attached plasma membrane into center of cell

2.  Contractile ring constricts equatorial region of cell much like a purse string narrows a purse opening

        F.  It is generally agreed that the position of the cleavage furrow is determined by the anaphase mitotic spindle, but there has been considerable debate as to how this occurs at the molecular level

1.  Early marine invertebrate egg study (Ray Rappaport, Union College, NY) - contractile ring forms in plane midway between spindle poles, even if one pole is displaced by microneedle inserted in cell

2.  Suggests that the actin-filament assembly site & thus the cytokinesis plane is determined by signal emanating from spindle poles (thought to travel from spindle poles to cell cortex along astral MTs)

3.  When one modifies the distance between the poles & the cortex experimentally, the timing of cytokinesis can be dramatically altered

4.  In contrast, studies of smaller mammalian cells show evidence that cleavage furrow formation site is defined by stimulus originating in the central part of the mitotic spindle rather than its poles

5.  Researchers have struggled to reconcile these opposing findings; reflected in title of recent series of articles in Trends in Cell Biology: "Cytokinesis: The Great Divide"

6.  Simplest explanations are that (1) different cell types utilize different mechanisms or (2) that both mechanisms operate in the same cell

III.  Cytokinesis in plant cells: formation of the cell plate – plant cells do cytokinesis by a very different mechanism because they are enclosed by a relatively inextensible cell wall

A.  Plant cells must construct an extracellular wall inside a living cell

1.  Wall formation starts in plane at the center of cell & grows outward to meet the existing lateral walls

2.  The formation of new cell wall starts with the construction of a simpler precursor, the cell plate

B.  The plane in which the cell plate forms is perpendicular to the mitotic spindle axis, but unlike animals, the plane is not determined by the spindle position

1.  The orientation of the mitotic spindle & cell plate are determined by a belt of cortical MTs, the preprophase band; it forms in late G2

2.  Even though the preprophase band has disassembled by prometaphase, it leaves an invisible imprint that determines the future division site

C.  The first sign of cell plate formation is seen in late anaphase with appearance of phragmoplast in the center of the dividing cell - steps in process below

1.  Phragmoplast is made of clusters of interdigitating MTs oriented perpendicular to future plate along with actin filaments, membranous vesicles  & electron-dense material

2.  The MTs of the phragmoplast, which arise from mitotic spindle remnants, serve as tracks for the movement of small Golgi-derived secretory vesicles into the region

3.  The vesicles become aligned along a plane between the daughter nuclei

D.  How do Golgi-derived vesicles become reorganized into the cell plate? - EMs of rapidly frozen tobacco cells; steps in the process below:

1.  Vesicles send out fingerlike tubules that contact & fuse with neighboring vesicles to form an interwoven tubular network in center of cell

2.  Additional vesicles are then directed along MTs to lateral edges of network; these newly arrived vesicles continue tubule formation & fusion process, which extends network in an outward direction

3.  Eventually, the leading edge of growing network contacts parent plasma membrane at cell boundary

4.  Ultimately, tubular network loses its cytoplasmic gaps & matures into a continuous, flattened partition; tubular network membranes become plasma membranes of the 2 adjacent daughter cells

5.  Secretory products that had been carried within the vesicles contribute to the intervening cell plate

6.  Once cell plate is finished, cellulose & other materials are added over time to make mature cell wall

Meiosis: Background and Overview

I.  Meiosis - process during which chromosome number is reduced; the cells made only have 1 member of each homologous pair; sexual reproduction includes the union of 2 cells each with full haploid chromosome set

A.  Why reduction via meiosis needed? - without it, chromosome number would double with each generation, and sexual reproduction would be impossible

1.  Doubling of chromosome number at fertilization is compensated for by an equivalent reduction in chromosome number at a stage prior to gamete formation

2.  The reduction is accomplished by meiosis (coined in 1905 from Greek word meaning "reduction")

B.  Meiosis ensures production of haploid phase in the life cycle; fertilization ensures a diploid phase

II.  Prior to both mitosis & meiosis, diploid G2 cells contain pairs of homologous chromosomes, with each chromosome consisting of 2 chromatids

A.  During mitosis, each chromosome's chromatids are split apart & separated into 2 daughter nuclei in a single division —> cells have homologous chromosome pairs & are genetically identical to their parents

1.  But G1 cell chromosomes no longer contain 2 chromatids

B.  Mitosis events contrast with those of meiosis, in which the 4 chromatids of a pair of replicated homologous chromosomes are distributed among 4 daughter nuclei

1.  This is accomplished with 2 sequential divisions without an intervening round of DNA replication

2.  In first meiotic division, each chromosome (with its 2 chromatids) is separated from its homologue; each cell now contains one member of each pair of homologous chromosomes

3.  To ensure that each daughter nucleus formed by meiosis has one member of each homologous pair, chromosomes are paired during prophase I by an elaborate process with no mitotic counterpart

4.  During pairing, homologous chromosomes engage in genetic recombination that produces chromosomes with new combinations of maternal & paternal alleles; increases variability

5.  Variability important for evolution; species better responds to adverse environmental changes

6.  In second meiotic division, the 2 chromatids of each chromosome are separated from one another

III.  Eukaryotic life cycle stage at which meiosis occurs varies as does duration of haploid phase - 3 groups

A.  Gametic or terminal meiosis – includes all multicellular animals & many protists

1.  Meiotic divisions are closely linked to gamete formation; for example, meiosis occurs just prior to the differentiation of the spermatozoa

2.  Spermatogonia (mitosis) —> primary spermatocytes (undergo 2 meiotic divisions) —> 4 spermatids differentiate into 4 spermatozoa; male vertebrates - meiosis occurs just prior to sperm differentiation

3.  Oogonia (mitosis) —> primary oocytes (undergo greatly extended meiotic prophase I; oocyte grows, fills with yolk/other materials)

4.  When differentiation is complete (oocyte has reached essentially the same state as when it is fertilized) —> meiotic divisions then finish

5.  Vertebrate eggs are typically fertilized at a stage before meiosis completion (usually at metaphase II); meiosis is completed after fertilization while the sperm still resides in the egg cytoplasm

B.  Zygotic or initial meiosis – includes only protists & fungi

1.  Meiotic divisions occur just after fertilization, so all cells haploid; process produces haploid spores

2.  Spores divide by mitosis to produce haploid adult generation

3.  Life cycle diploid stage is restricted to brief period after fertilization when individual is still a zygote

C.  Sporic or intermediate meiosis – includes all plants & some algae

1.  Meiosis occurs at a stage unrelated to either gamete formation or fertilization

2.  Life cycle begins with union of male gamete (pollen grain) & female gamete (the egg)

3.  Diploid zygote thus formed undergoes mitosis & develops into a diploid sporophyte

4.  Sometime during sporophyte development, sporogenesis (including meiosis) occurs

5.  Spores produced by sporogenesis germinate directly into haploid gametophyte (either an independent stage or a tiny structure kept in ovules, as in seed plants)

6.  In either case, the gametes are produced from a haploid gametophyte by mitosis

IV.  The prelude to meiosis includes DNA replication - premeiotic S takes several times longer than premitotic S

The Stages of Meiosis: Prophase I

I.  Prophase I - typically lengthened extraordinarily compared to prophase of mitosis; in human female - oocytes initiate prophase I prior to birth & then enters a period of prolonged arrest

A.  Oocytes resume meiosis just prior to the time they are ovulated, which occurs every 28 days or so after an individual reaches puberty

B.  Thus, many human oocytes remain arrested in same approximate stage of prophase for several decades

1.  According to a report in Nature (2004), mouse ovaries contain germline stem cells that produce new oocytes throughout adult life

2.  If this is true in humans, it would overthrow the longstanding belief that all oocytes produced during a woman's lifetime are generated in the fetus & enter meiosis prior to birth

C.  The first meiotic prophase is also very complex & is customarily divided into several (5) stages that are similar in all sexually reproducing eukaryotes

1.  Leptotene

2.  Zygotene

3.  Pachytene

4.  Diplotene

5.  Diakinesis

II.  Leptotene – first prophase I stage; chromosomes become gradually visible in light scope; lasts a few hours

A.  There is no indication that there are 2 chromatids in light microscope (but there are since the chromosomes replicated at an earlier stage), can be seen in EM

B.  Compaction of chromosomes continues through leptotene until homologues can be seen to associate with one another; this process of chromosome pairing is called synapsis & is first event in next stage

III.  Zygotene – the second prophase I stage; synapsis (process by which homologues joined) occurs; lasts a few hours

A.  A number of questions about synapsis remain unanswered

1.  On what basis do the homologues recognize one another?

2.  How does the pair become so perfectly aligned?

3.  When does recognition between homologues first occur?

B.  Homologous DNA regions of homologous chromosomes are already associated with one another during yeast cell leptotene - Nancy Kleckner et al. (Harvard)

1.  It was originally assumed for years that interaction between homologous chromosomes first began as chromosomes initiate synapsis in zygotene, not leptotene

2.  Chromosome compaction & synapsis during zygotene simply make this arrangement visible under the microscope

3.  First step in genetic recombination is the deliberate introduction of double-stranded breaks in aligned DNA molecules

4.  Yeast & mouse studies suggest DNA breaks occur in leptotene, well before chromosomes are visibly paired

C.  The above findings are supported by studies aimed at locating particular DNA sequences within the nuclei of premeiotic & meiotic cells

1.  Individual chromosomes are thought to occupy discrete regions within nuclei rather than being randomly dispersed throughout the nuclear space

2.  Recent studies in yeast show that cells just entering meiotic prophase have each pair of homologues sharing a joint territory, distinct from those shared by other homologous pairs

3.  Suggests that homologous chromosomes are paired to some extent before meiotic prophase begins

D.  In maize, homologues are paired very early in meiotic prophase, but not as early as in yeast - maize leptotene chromosome telomeres (terminal segments) are distributed throughout nucleus

1.  Near leptotene end, telomeres are localized at nuclear envelope inner surface at one side of nucleus

2.  This telomere clustering occurs in wide variety of eukaryotic cells & causes chromosomes to look like flower bouquet (bouquet stage); may facilitate chromosome alignment in preparation for synapsis

3.  In most organisms, synapsis begins at one end of each homologous pair & progresses along the length of the chromosomes during zygotene

E.  Synapsis accompanied by synaptonemal complex (SC) formation, a complex, ladderlike structure made of 3 parallel bars with transverse protein filaments connecting central element with 2 lateral elements

1.  Chromatin of each homologue is organized into loops extending from one of the SC lateral elements, which are made primarily of cohesin (presumably binds together the chromatin of sister chromatids)

2.  For years, SC was thought to hold each homologous pair in proper position to start genetic recombination between homologous DNA strands, but it is now evident that it is not required

3.  SC forms after genetic recombination has been initiated; mutant yeast cells unable to form SC still engage in genetic recombination between homologues

4.  It is currently thought that the SC functions primarily as a scaffold to allow interacting chromatids to complete crossover activities

5.  Complex formed by a pair of synapsed homologous chromosomes is called bivalent (referring to 2 homologues) or tetrad (referring to 4 chromatids)

F.  The end of zygotene is marked by the end of synapsis

IV.  Pachytene - starts when synapsis ends; often inordinately long (extended for a period of days or weeks unlike leptotene & zygotene which generally last a few hours)

A.  Homologues are held closely together along their length by SC; characterized by a fully formed SC - DNA of sister chromatids is extended into parallel loops

B.  Within SC center, a number of electron-dense bodies (~100 nm dia) are seen in EM at irregular intervals

1.  These bodies called recombination nodules; they contain some of the enzymatic machinery that facilitates genetic recombination, which is completed by the end of pachytene

2.  They correspond to the sites where crossing over is taking place as evidenced by the associated synthesis of DNA that occurs during intermediate steps of recombination

V.  Diplotene – its beginning is usually recognized by SC dissolution & the tendency of homologous chromosomes of the bivalents to pull away somewhat from each other

A. As they separate, homologous chromosomes are seen to remain attached to each other at specific points by X-shaped structures (chiasmata; chiasma is the singular) at crossover sites

1.  They are located at sites on chromosomes where crossing-over between DNA molecules from the two chromosomes had previously occurred

2.  Chiasmata are formed by covalent junctions between a chromatid from one homologue & a non-sister chromatid from the other homologue; shows extent of genetic recombination

B.  Diplotene is an extremely extended phase of oogenesis, during which the bulk of oocyte growth occurs

1.  In many spermatocytes & oocytes, diplotene chromosomes become dispersed into a particular configuration not seen at any other time during the organism's life cycle, lampbrush chromosomes

2.  Lampbrush chromosomes have an axial backbone from which pairs of loops extend out in opposite directions; they arise in pairs since each chromosome consists of a pair of duplicated chromatids

3.  Each loop is a projection from a single chromatid, and the two homologues still remain attached to each other at the chiasmata

4.  DNA situated between loops is tightly compacted & transcriptionally inactive

5.  In contrast, loop DNA is highly extended & the site of intense transcriptional activity; RNAs made are used in protein synthesis during both oogenesis & early embryo development after fertilization

VI.  Diakinesis - meiotic spindle assembled & chromosomes are prepared for separation; final stage of meiotic prophase I

A.  In those species in which chromosomes become highly dispersed during diplotene, the chromosomes become recompacted during diakinesis

B.  Diakinesis ends with the disappearance of the nucleolus, nuclear envelope breakdown & tetrad movement to the metaphase plate

1.  In vertebrate oocytes, these events are triggered by an increase in the protein kinase activity of MPF (maturation-promoting factor)

2.  MPF was first identified by its ability to initiate these events, which represent oocyte maturation

The Stages of Meiosis: Metaphase I

I.  In most eukaryotes, homologous chromosomes at meiosis I metaphase plate often contain visible chiasmata

A.  In fact, chiasmata hold homologues together as a bivalent during this stage

1.  In humans & other vertebrates, every homologous pair typically contains ≥1 chiasma; longer chromosomes tend to have 2 or 3 of them

2.  It is thought that some mechanism exists to ensure that even smallest chromosomes have at least 1

3.  If there is no chiasma between homologues, they tend to separate after SC dissolution

4.  This premature homologue separation can result in nondisjunction & an abnormal chromosome number in nucleus; chiasmata are probably a way of preventing abnormal chromosome segregation

B.  A mechanism also exists to prevent formation of >2 or 3 chiasmata regardless of chromosome length; inhibition of excess chiasmata is called crossover interference & is thought to be mediated by SC

C.  At metaphase I, the 2 homologous chromosomes of each bivalent are connected to the spindle fibers from opposite poles

1.  In contrast, sister chromatids are connected to MTs from the same spindle pole, which is made possible by the side-by-side arrangement of their kinetochores

2.  The orientation of maternal & paternal chromosomes of each bivalent on metaphase I plate is random; the maternal member of a particular bivalent has an equal likelihood of facing either pole

3.  Thus, when homologous chromosomes separate during anaphase I, each pole receives a random assortment of maternal & paternal chromosomes

4.  Thus, anaphase I is the cytological event corresponding to Mendel's law of independent assortment; due to independent assortment, organisms can generate a nearly unlimited variety of gametes

D.  Abnormalities in formation of metaphase I spindle trigger the arrest of meiosis by a checkpoint mechanism similar to that in mitosis

The Stages of Meiosis: Anaphase I, Telophase I and Interkinesis

I.  Anaphase I – cohesion between chromosome arms is lost; homologous chromosomes of each bivalent separate, while cohesion of joined centromeres of sister chromatids stays strong & they stay together

A.  Separation of homologous chromosomes at anaphase I requires the dissolution of the chiasmata that hold the bivalent together

1.  Chiasmata are maintained by cohesion between sister chromatids in regions that flank these sites of recombination

2.  Chiasmata disappear at the metaphase I – anaphase I transition, as the arms of each bivalent's chromatids lose cohesion

B.  Loss of cohesion between the arms is accomplished by proteolytic cleavage of the cohesin molecules in those regions of the chromosome

1.  Cohesion between the joined centromeres of sister chromatids remains strong, because the cohesin situated there is protected from proteolytic attack

2.  Thus, sister chromatids stay firmly attached to each other as they move toward a spindle pole during anaphase I; each chromosome made of 2 chromatids attached at centromere throughout stage

II.  Telophase I – less dramatic changes than mitotic telophase; chromosomes usually disperse a bit, but they do not reach the extremely extended state of interphase nucleus; nuclear envelope may or may not reform

III.  Interkinesis - stage between the 2 meiotic divisions; generally short-lived

A.  Animal cells in this fleeting stage are called secondary spermatocytes or secondary oocytes

B.  These cells have a haploid number of chromosomes (one member of each homologous pair), but they have diploid amount of nuclear DNA, since each chromosome still consists of 2 chromatids

1.  Secondary spermatocytes are said to have a 2C amount of DNA, half as much as a primary spermatocyte with a 4 C amount of DNA & twice as much as a sperm cell with a 1C DNA content

The Stages of Meiosis: Meiosis II

I.  Meiosis II - very similar to mitotic division, except haploid number of chromosomes involved

II.  Prophase II – much simpler than prophase I; chromosomes become recompacted & line up at metaphase plate; if nuclear envelope had reformed in telophase I, it is broken down again

III.  Metaphase II – unlike metaphase I, kinetochores of sister chromatids of metaphase II face opposite poles & become attached to opposing sets of chromosomal spindle fibers

A.  Oocytes of vertebrates halt here & await fertilization; progression through meiosis stops here

B.  Metaphase II arrest is brought about by factors that inhibit APCCdc20 activation, thereby preventing cyclin B degradation

1.  As long as cyclin B levels remain high within the oocyte, Cdk activity is maintained & the cells cannot progress to the next meiotic stage

2.  Metaphase II arrest is only released when the oocyte (now called egg) is fertilized

3.  Fertilization leads to rapid influx of Ca2+ ions, the activation of APCCdc20 & cyclin B destruction

4.  Fertilized egg responds to these changes by completing meiosis II; only happens if egg is fertilized

IV.  Anaphase II - begins with the synchronous splitting of the centromeres, which hold the sister chromatids together; allows chromosomes to move toward opposite poles of the cell

V.  Telophase II – ends meiosis II; chromosomes are once again enclosed by a nuclear envelope; cells have haploid amount (1C) of nuclear DNA (half that in normal G1 cell) & haploid chromosome number

Genetic Recombination During Meiosis

I.  Meiosis reduces chromosome number as required by sexual reproduction; also increases genetic variability in population of organisms from one generation to next - how is this genetic variability introduced?

A.  Independent assortment allows maternal & paternal chromosomes to be shuffled as gametes form

B.  Genetic recombination (crossing over) allows maternal & paternal alleles on a given chromosome to be shuffled as well

1.  Without genetic recombination, alleles along a particular chromosome would remain tied together from generation to generation

2.  By mixing maternal & paternal alleles between homologous chromosomes, meiosis generates organisms with novel genotypes & phenotypes upon which natural selection can act