Research
Interdisciplinary approach between cellular/molecular biology and
soft-matter physics to decipher various cellular membrane processes
Session 1. Synuclein and neuronal disease
Session 1. Synuclein and neuronal disease
- Neurons
- Neurons
Synucleinopathies are neurological diseases that are characterized by the accumulation of aggregates of a cytosolic protein, α-synuclein, at the plasma membrane. Even though the pathological role of the protein is established, the mechanism by which it damages neurons remains unclear due to the difficulty of correctly mimicking the plasma membrane in vitro. Recently, we monitored the α-synuclein dynamics on the in vitro asymmetric plasma membrane. We found α-synuclein i) freezes membrane by inserting itself into the inside of the membrane hydrophobic core, ii) preferably binds to the inner leaflet, iii) reduces membrane thickness, and iv) induces pores and disrupts membrane at the end (publication #18).
Synucleinopathies are neurological diseases that are characterized by the accumulation of aggregates of a cytosolic protein, α-synuclein, at the plasma membrane. Even though the pathological role of the protein is established, the mechanism by which it damages neurons remains unclear due to the difficulty of correctly mimicking the plasma membrane in vitro. Recently, we monitored the α-synuclein dynamics on the in vitro asymmetric plasma membrane. We found α-synuclein i) freezes membrane by inserting itself into the inside of the membrane hydrophobic core, ii) preferably binds to the inner leaflet, iii) reduces membrane thickness, and iv) induces pores and disrupts membrane at the end (publication #18).
Currently, we are elucidating how cytosolic α-synuclein regulates neuronal secretion and neurotransmission, and how they are found in the extracellular compartment of cells. Also, we are applying the same principle to characterize the dynamics of amyloid beta peptide (Aβ) which is abundantly found in Alzheimer's disease.
Currently, we are elucidating how cytosolic α-synuclein regulates neuronal secretion and neurotransmission, and how they are found in the extracellular compartment of cells. Also, we are applying the same principle to characterize the dynamics of amyloid beta peptide (Aβ) which is abundantly found in Alzheimer's disease.
Session 2. Neuronal fusion pores (presynaptic and secretory fusion pores)
Session 2. Neuronal fusion pores (presynaptic and secretory fusion pores)
- Neurons
- Neurons
Membrane fusion is the process of merging two different membranes through the production of a fusion pore which is a nanometric cylinder-like hole that spans two membranes. This process locally and temporarily neutralizes a part or all of membrane asymmetries (see session 1). In neurons, the set of neuronal SNAREs in the synaptic vesicles and the plasma membrane form the coiled-coil SNARE complex in the cytosol and mediate membrane fusion and synaptic fusion pore formation. The synaptic fusion pore kinetics (e.g., successive initial formation and expansion) is an important matter to release neurotransmitters as quickly as possible, less than 1 ms. Recently, we recapitulate the synaptic fusion pore formation in our 3D-printed microfluidic membrane setup that is combined with a patch amplifier and spinning-disk confocal microscope and success to monitor the individual fusion pore dynamics in 0.1 nm and 10 µs resolution, which exactly follows in vivo kinetics (publication #19 and #3, 5, 8, 9, 11, 12, 14, 15).
Membrane fusion is the process of merging two different membranes through the production of a fusion pore which is a nanometric cylinder-like hole that spans two membranes. This process locally and temporarily neutralizes a part or all of membrane asymmetries (see session 1). In neurons, the set of neuronal SNAREs in the synaptic vesicles and the plasma membrane form the coiled-coil SNARE complex in the cytosol and mediate membrane fusion and synaptic fusion pore formation. The synaptic fusion pore kinetics (e.g., successive initial formation and expansion) is an important matter to release neurotransmitters as quickly as possible, less than 1 ms. Recently, we recapitulate the synaptic fusion pore formation in our 3D-printed microfluidic membrane setup that is combined with a patch amplifier and spinning-disk confocal microscope and success to monitor the individual fusion pore dynamics in 0.1 nm and 10 µs resolution, which exactly follows in vivo kinetics (publication #19 and #3, 5, 8, 9, 11, 12, 14, 15).
Currently, we are characterizing the impact of membrane asymmetry in the synaptic fusion pore to provide systemic understandings.
Currently, we are characterizing the impact of membrane asymmetry in the synaptic fusion pore to provide systemic understandings.
What is the role of SNAP25 S-palmitoylation in cellular trafficking?
What is the role of SNAP25 S-palmitoylation in cellular trafficking?
S-palmitoylation is one of membrane conjugation strategies: adult SNAP25, called SNAP25b, contains cysteines at 85C, 88C, 90C, and 92C and they are covalently attached to palmitic acids in the cytosolic leaflet of the plasma membrane. Recent in vivo studies show impaired exocytosis in the non-palmitoylated SNAP25 condition, however, they are seldom recapitulated in vitro.
S-palmitoylation is one of membrane conjugation strategies: adult SNAP25, called SNAP25b, contains cysteines at 85C, 88C, 90C, and 92C and they are covalently attached to palmitic acids in the cytosolic leaflet of the plasma membrane. Recent in vivo studies show impaired exocytosis in the non-palmitoylated SNAP25 condition, however, they are seldom recapitulated in vitro.
Currently, we apply the principles and techniques to understand VAMP7-mediated secretion.
Currently, we apply the principles and techniques to understand VAMP7-mediated secretion.
Session 3. Building and controlling the asymmetry of the cell membranes and surroundings
Session 3. Building and controlling the asymmetry of the cell membranes and surroundings
- Membrane biophysics
- Membrane biophysics
How to build a controllable system of membrane asymmetries?
How to build a controllable system of membrane asymmetries?
Cellular plasma membrane is asymmetric: each leaflet (lipid monolayer) of the bilayer contains significantly different lipid types and distribution and the adjacent cytosol and extracellular fluid contain different ions, chemicals, and various molecules. Because the asymmetry cannot be changed in vivo, this question is seldom addressed. Our 3D-printed microfluidic membrane setup is capable to control such 4 parameters (each leaflet and environment), hence, it is suitable to recapitulate the most accurate biomimetic plasma membrane system (publication #17 in the 'Publication' tab).
Cellular plasma membrane is asymmetric: each leaflet (lipid monolayer) of the bilayer contains significantly different lipid types and distribution and the adjacent cytosol and extracellular fluid contain different ions, chemicals, and various molecules. Because the asymmetry cannot be changed in vivo, this question is seldom addressed. Our 3D-printed microfluidic membrane setup is capable to control such 4 parameters (each leaflet and environment), hence, it is suitable to recapitulate the most accurate biomimetic plasma membrane system (publication #17 in the 'Publication' tab).
What would be outcomes when two leaflets of the membranes are asymmetric?
What would be outcomes when two leaflets of the membranes are asymmetric?
The asymmetric lipid distribution represents the phenotypic difference of each leaflet. For example, the inner leaflet is ~10 times more negatively charged due to mainly PS and more laterally fluid by containing mostly unsaturated lipids (i.e. having one or more double bonds in the acyl chain) and by forming a liquid-disordered (Ld) phase. The extracellular leaflet, by contrast, is less charged and less fluid by containing mostly saturated lipids and forming two phases composed of liquid-ordered (Lo) and Ld phases. A genuine question is then whether the outer leaflet induces phase change in the inner leaflet by interleaflet coupling.
The asymmetric lipid distribution represents the phenotypic difference of each leaflet. For example, the inner leaflet is ~10 times more negatively charged due to mainly PS and more laterally fluid by containing mostly unsaturated lipids (i.e. having one or more double bonds in the acyl chain) and by forming a liquid-disordered (Ld) phase. The extracellular leaflet, by contrast, is less charged and less fluid by containing mostly saturated lipids and forming two phases composed of liquid-ordered (Lo) and Ld phases. A genuine question is then whether the outer leaflet induces phase change in the inner leaflet by interleaflet coupling.
We are currently characterizing the induced lipid domains.
We are currently characterizing the induced lipid domains.
More about one of our tools: Horizontal and free-standing model membrane-on-the-chip
More about one of our tools: Horizontal and free-standing model membrane-on-the-chip
As the boundary between two regions, biological membranes are the active location where many biological processes occur, including molecular reactions and signal exchanges. Recently, we established the most high-end in vitro membrane setup which includes a 3D-printed microfluidic chip, patch-clamp amplifier, and spinning-disc confocal microscope. Any physiological membrane is produced over horizontally flat ~10,000 µm2. The membrane can have asymmetric (i) lipid distribution on each monolayer and (ii) molecular gradient across the membrane, (iii) correctly oriented membrane proteins. Currently, this simultaneous electrical and optical measurements on any of recapitulated membrane processes are successfully used to answer fundamental and difficult questions in membrane biophysics, intracellular trafficking, viral infect, and various neurodegenerative diseases (publication #17).
As the boundary between two regions, biological membranes are the active location where many biological processes occur, including molecular reactions and signal exchanges. Recently, we established the most high-end in vitro membrane setup which includes a 3D-printed microfluidic chip, patch-clamp amplifier, and spinning-disc confocal microscope. Any physiological membrane is produced over horizontally flat ~10,000 µm2. The membrane can have asymmetric (i) lipid distribution on each monolayer and (ii) molecular gradient across the membrane, (iii) correctly oriented membrane proteins. Currently, this simultaneous electrical and optical measurements on any of recapitulated membrane processes are successfully used to answer fundamental and difficult questions in membrane biophysics, intracellular trafficking, viral infect, and various neurodegenerative diseases (publication #17).