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IMOTO LAB
 

Dissecting the synaptic membrane fission and the trafficking system at milliseconds and nanometer resolution

Research

Cells reproduce by division. This process involves pinching off a part of their plasma membrane and its compartment, organelles. Core proteins for membrane fission are hypothesized to have originated from a single progenitor that likely mediated the endocytosis of endosymbiotic ancestor or cytokinesis in early eukaryotes. It then evolved into various proteins for membrane fission of mitochondria, peroxisomes, endosomes, lysosomes and for receptor mediated endocytosis. One of the most recent innovations of cells is synaptic vesicle recycling. Unlike the general membrane fission events listed above (takes ~10 seconds to tens of minutes), the speed of membrane fission can become at least 100 times faster during synaptic vesicle recycling. How could a protein with such slow kinetics adapt to the speed of rapid synaptic vesicle scission (less than 100 milliseconds)? How can membrane fission machinery recognize different types of organelles within the small presynapses (~ 500 – 1000 nm)? Despite extensive studies over decades, several difficulties prevent a comprehensive understanding of these questions. Such difficulties include: 1) membrane fission events occur randomly and on a rapid time scale. Thus, it is difficult to capture their sequential events. 2) Conventional microscopes cannot distinguish localization or dynamics of molecules between different organelles within such tiny compartments. 3) There are more than 50 endocytic proteins with multiple isoforms / splice variants. As such, making it challenging to distinguish individual functions or possible functional redundancies within synapses. Our long-term scientific goal is to answer these questions and understand the molecular mechanisms underlying membrane fission at synapses.

1. Synaptic vesicle recycling

Neurons can maintain high-frequency synaptic transmission without depleting synaptic vesicles. This feature relies on the efficient endocytic recycling of synaptic vesicle membranes and proteins locally at synapses. Several modes of synaptic vesicle endocytosis have been reported, including clathrin-mediated endocytosis, activity-dependent bulk endocytosis, and ultrafast endocytosis. Many endocytic proteins are shared among these pathways, although the timescale of these endocytic pathways ranges from ~100 milliseconds to tens of seconds or minutes. Thus, it is not clear how a set of endocytic proteins can regulate a wide range of vesicle scission events. Recently, we have identified a specific splice variant of the GTPase dynamin protein that can mediate ultrafast endocytosis. Through multivalent interactions with other endocytic proteins, this specific splice variant constitutively forms a nano-domain at the endocytic zone. The presence of this endocytic nano-domain accelerates the kinetics of membrane fission ~100 times faster than clathrin-mediated endocytosis. Our lab uses our strength in time-resolved electron microscopy and super-resolution imaging to uncover:

1) The molecular mechanisms by which the endocytic nano-domain mediates synaptic vesicle endocytosis during a wide range of neuronal stimuli.
2) The molecular organization around the endocytic nano-domain during synaptogenesis and in matured presynapses.  

2. Sorting of synaptic vesicle proteins

Proteolytic degradation of synaptic vesicle proteins is essential to sustain synaptic function. Old or damaged proteins accumulate on vesicles as a result of prolonged or intense synaptic activity. These abnormal proteins must be sorted out of the recycling pathway. After synaptic vesicle endocytosis, normal membrane proteins are delivered to synaptic endosomes, followed by clathrin-dependent vesicle budding. In contrast, abnormal proteins need to be delivered to multivesicular body (MVBs) and eventually delivered to the cell body for degradation. MVBs play a pivotal role as the initial organelles for proteolysis at synapses. The generation of MVBs involves the acidification of endosomes and the inward bending of membranes to form intraluminal vesicles, a process mediated by ESCRT proteins. These processes require ~5 minutes, which is considerably slower than the rate of synaptic vesicle recycling. We aim to:

1) Capture membrane dynamics during synaptic endosomal sorting.
2) Identify the molecular mechanisms how abnormal proteins are sorted out during the rapid synaptic vesicle recycling pathway.

3. Emergence of modern synaptic system

The development of intelligence among Metazoans correlates with an increased variety of synaptic proteins and neurotransmitters. Synaptic cell adhesion molecules and post-synaptic compartment genes are present from a common ancestor of bilaterians (eg. mammals, fish, octopus, worms, or insects) and a cnidarian specie, as well as sponges. Exocytic SNARE complex proteins are identified in non-bilaterians including placozoans and sponges, but took diversification by increasing isoform numbers or adding extra domain for more complex release properties in Bilaterians. In addition, Bilaterians have established monoaminergic (serotonin, dopamine, norepinephrine, etc.) systems. These increased diversities allow for the development of complex behaviors and cognitive functions including, learning and memory formation. Such properties are maintained by synapse formation and the coupling of exo-endocytosis during neuronal plasticity. Unlike synaptic adhesion molecules or exocytic SNARE complexes, several important synaptic endocytic proteins suddenly expanded their variety during the transition from Cnidaria to Bilateria. This variation is achieved by either generating alternative splice variants or increasing the repetition of functional amino acid motifs. We have identified that such bilaterian-specific modification facilitates synaptic vesicle endocytosis in mammals. Our lab is trying to understand:

1) How bilaterian neurons regulates and have established evolutionally conserved alternative splicing for the synaptic vesicle endocytosis.
2) The original mechanisms of exo-endocytosis coupling required for bilaterian centralized nerve system.

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