Research Field

Our current research interests are the molecular mechanisms of synaptic function under normal and pathological conditions.

Research Activity

The human brain is composed of more than a trillion nerve cells whose signal-carrying protrusions are interconnected at special points of contact called synapses. Neurotransmitter release at the synapse is a multi-step process that is coordinated by a large number of synaptic proteins and depends on proper protein-protein interactions. Modulation of these processes is believed to underlie the processes of learning and memory. Our main interest is to study the molecular mechanisms of these processes under normal conditions and during neurodegenerative diseases such as Huntington and Alzheimer diseases.

In the last years we have been investigating the function of key synaptic proteins in these processes using molecular biology, electrophysiology, biochemistry, Ca2+ imaging and computer simulation techniques. We took advantages of a unique experimental approach that allows efficient manipulations of the level and composition of specific proteins using overexpression and knock down approach. The approach allows studying structure-function relationships of synaptic proteins and their role in normal and pathological conditions. We perform detail electrophysiological and fluorescent measurements from chromaffin cells and neurons and compare the phenotype cells expressing the different mutated proteins. Specifically, we investigate the function of Munc13, Munc18, tomosyn, PLD and DOC2 in exocytosis. Our long-term goal is to correlate changes in protein expression to synaptic plasticity and learning and memory processes.

Brief Summary

We have found that tomosyn inhibits priming of vesicles and decreases exocytosis in chromaffin cells. In addition we have defined the N-terminal part of tomosyn and its linker as a minimal domain necessary for its inhibitory activity and found that the SNARE motif of tomosyn is involved in modulation of tomosyn activity.We are now investigating the role of tomosyn in synaptic plasticity processes (see below Boaz). In addition, we investigate tomosyn’s dynamic interactions with syntaxin and with other proteins and examine intramolecular interactions between different domains of tomosyn (see below Noa).In different projects we found that phosphorylation of Munc18 potentiates vesicle pool refilling. In addition, we found that PLD1 enhances priming of large dense core vesicles in chromaffin cells.
To elucidate the functions of calcium-dependent proteins in the synapse we investigated the effect of calcium and calmodulin on the priming activity of ubMunc13-2. We  propose that ubMunc13-2 is activated by two
Ca2+-dependent processes: a slow activation mode operating at low Ca2+ concentrations, where ubMunc13-2 acts as a priming switch, and a fast mode at high Ca2+ concentrations, in which ubMunc13-2 is activated in a Ca2+/CaM-dependent manner and accelerates vesicle recruitment and maturation during stimulation. In addition, we investigate the function of DOC2B, a double C2 domain protein in synaptic transmission. We found that the proteins DOC2A and DOC2B translocate to the plasma membrane in presence of [Ca2+]i below 0.5 µM, and we are currently investigating its function in exocytosis (see below Reut).

In a recent project we have started to elucidate the function of the active zone protein, Bassoon in secretion of large dense core vesicles from chromaffin cells (see below Anton, Merav, Reut). To investigate the role of synaptic proteins in synaptic plasticity we are investigating the changes in the expression levels and distribution of synaptic proteins during and following learning (see below Boaz) and the effects of manipulation of synaptic proteins on synaptic plasticity (see below Ayal). We have also several biophysical projects that aim to understand the dynamic of proteins diffusion, assembly and disassembly and signal transduction. We discovered the Ras-related particle, which we called rasosome. Rasosomes are small particles, containing multiple lipid-modified Ras proteins that diffuse randomly in the cytosol and interact with the plasma membrane.
We are now characterizing if rasosome sample the membrane at specific points, or hot spots, and if rasosome can transmit Ras-related signals (see below Merav). In a project related to Huntington disease, we investigate the role of the Huntingtin interacting protein 1 (HIP1) in endocytosis, and we show that HIP1 particles are collocalized with clathrin on the plasma membrane and appear and disappear concomitant with clathrin particles following treatments that influence pits formation (see below Irit).
These data suggest that HIP1 participates in early stages of clathrin-mediated endocytosis. In another project we investigate the kinetics of SNARE complex formation and disassembly by NSF. We found that SANRE complexes are assembled in two phases and that disassembly depends on the concentration of NSF and alpha SNAP. To learn more about the dynamics of the SANRE proteins on the PM, with or without syntaxin domains we are using a Monte Carlo-based 2-dimensional simulation (see below Dana).

In addition we have developed a novel simulation program that describes the process of exocytosis as dynamic interactions between synaptic proteins. This study provides an excellent platform to predict and quantify the effects of protein manipulations on exocytosis (see below Dana).

1. Ashery, U., Betz, A., Xu, T., Brose, N. & Rettig, J. An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system. Eur J Cell Biol 78, 525-532 (1999).

2. Ashery, U. et al. Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells. EMBO J 19, 3586-3596 (2000).

3. Yizhar, O. et al. Tomosyn inhibits priming of large dense-core vesicles in a calcium-dependent manner.
Proc Natl Acad Sci U S A 101, 2578-2583 (2004).

4. Mezer, A., Nachliel, E., Gutman, M. & Ashery, U. A new platform to study the molecular mechanisms of exocytosis. J Neurosci 24, 8838-8846 (2004).

5. Groffen, A. J., Friedrich, R., Brian, E. C., Ashery, U. & Verhage, M. DOC2A and DOC2B are sensors for neuronal activity with unique calcium-dependent and kinetic properties. J Neurochem 97, 818-833 (2006).

6. Ashery, U., Yizhar, O., Rotblat, B. & Kloog, Y. Nonconventional trafficking of Ras associated with Ras signal organization.
Traffic 7, 119-126 (2006).

7. Ashery, U. et al. Spatiotemporal Organization of Ras Signaling: Rasosomes and the Galectin Switch.
Cell Mol Neurobiol 26, 471-495 (2006).

8. Mezer, A. et al. Systematic search for the rate constants that control the exocytotic process from chromaffin cells by a Genetic Algorithm. Biochim Biophys Acta 1763, 345-355 (2006).

9. Nili, U. et al. Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells. Neuroscience 143, 487-500 (2006).

10. Rotblat, B., Yizhar, O., Haklai, R., Ashery, U. & Kloog, Y. Ras and its signals diffuse through the cell on randomly moving nanoparticles. Cancer Res 66, 1974-1981 (2006).

11.Gladycheva, S. E. et al. Receptor-mediated regulation of tomosyn-syntaxin 1A interactions in bovine adrenal chromaffin cells. J Biol Chem 282, 22887-22899 (2007).

12. Singer-Lahat, D. et al. K+ channel facilitation of exocytosis by dynamic interaction with syntaxin.
J Neurosci 27, 1651-1658 (2007).

13. Yizhar, O. et al. Multiple functional domains are involved in tomosyn regulation of exocytosis.
J Neurochem 103, 604-616 (2007).

14. Zeniou-Meyer, M. et al. PLD1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granule at a late stage. J Biol Chem 282, 21746-21757 (2007).

15. Zikich D. Junge H. Brose N. and Ashery U. Calmodulin regulates vesicle refilling via ubMunc13-2.
J. Neurosci (2008) In Press.

Ph.D Students

1. Irit Gottfried

Huntingtin-interacting protein 1 (HIP1) is related to clathrin mediated endocytosis (CME), However, its exact role and the exact stage in CME are still a debate. It was shown to colocalize with several endocytic proteins but its effect on internalization of cargoes and its involvement in early steps of CME are unclear. To study the involvement of HIP1 in early steps of CME we followed fluorescently tagged proteins, using TIRF and confocal microscopy. We show that HIP1 particles are collocalized with clathrin on the plasma membrane and appear and disappear concomitant with clathrin particles following treatments that influence pits formation (such as butanol and sucrose). In addition, similar to clathrin, HIP1 colocalizes with transferrin receptors on the plasma membrane but not often with those in internalized vesicles, suggesting it might detach from the vesicle close to clathrin uncoating. Nevertheless, examination of the two proteins behavior in untreated cells reveals some differences; HIP1 particles stay in the vicinity of the plasma membrane for longer times and are less mobile than clathrin particles suggesting interaction with other components of the endocytotic machinery or with lipids. We also show that a fragment of HIP1 (HIP1(218-604)) is mislocalized and creates large cellular structures that contain clathrin, AP2 and EEA1. These structures are comprised from smaller units that attach and detach from each other dynamically. We found some HIP1(218-604) in smaller clusters close to the plasma membrane. These HIP1(218-604) clusters behave in a way that resembles clathrin. These results suggest that HIP1 is found on coated pits, but not vesicles and therefore plays a role in the early stages of CME.

2. Reut Friedrich

Elevation of the intracellular calcium concentration ([Ca2+]i) to levels below 1 lM alters synaptic transmission and induces short-term plasticity. To identify calcium sensors involved in this signalling, we investigated soluble C2 domain-containing proteins and found that both DOC2A and DOC2B are modulated by submicromolar calcium levels. Fluorescent-tagged DOC2A and DOC2B translocated to plasma membranes after [Ca2+]i elevation. DOC2B translocation preceded DOC2A translocation in cells co-expressing both isoforms. Half-maximal translocation occurred at 450 and 175 nM [Ca2+]i for DOC2A and DOC2B, respectively. This large difference in calcium sensitivity was accompanied by a modest kinetic difference (halftimes, respectively, 2.6 and 2.0 s). The calcium sensitivity of DOC2 isoforms can be explained by predicted topologies of their C2A domains. Consistently, neutralization of aspartates D218 and D220 in DOC2B changed its calcium affinity. In neurones, both DOC2 isoforms were reversibly recruited to the plasma membrane during trains of action potentials. Consistent with its higher calcium sensitivity, DOC2B translocated at lower depolarization frequencies. Styryl dye uptake experiments in hippocampal neurons suggest that the overexpression of mutated DOC2B alters the synaptic activity. We conclude that both DOC2A and DOC2B are regulated by neuronal activity, and hypothesize that their calcium-dependent translocation may regulate synaptic activity (Link, Ref). We are now investigating DOC2B roles in exocytosis.

3. Dana Bar-On

Syntaxin-1A, SNAP-25 and synaptobrevin/VAMP-II form the SNARE complex, which is crucial for vesicle fusion. The formation of the non-productive cis-SNARE complexes between PM-SNAREs counteracts the formation of trans-SNARE complexes between the syntaxin and SNAP-25 located on the plasma membrane (PM) with the vesicular, synaptobrevin. The formation of SNARE complexes depends on the local availability of the SNARE proteins which exist in a dynamics quasi –equilibrium; formed by the interaction of the SNARE proteins and dis-assembled by the continuous action of the ATP dependent NSF and the αSNAP system. Syntaxin-1A is concentrated in cholesterol-rich clusters at PM and generate non-homogenous distribution on the PM. The goal of our research is to examine the SNARE distribution and kinetics on the PM in various molecular, microscopy and computational methods.    

4. Boaz Barak

My aim is to elucidate the molecular mechanisms of synaptic transmission and plasticity, mechanisms, which are the basis of both developmental and pathological learning and memory processes. In particular, I am investigating the role of Tomosyn in Hippocampal neurons. Tomosyn is a synaptic protein that inhibits vesicle priming and exocytosis in chromaffin cells. We have preliminary results that tomosyn is also involved in synaptic plasticity. Discovering Tomosyn’s role in synaptic plasticity is an important step towards understanding and treating many psychiatric and neurological disorders that are characterized by impaired synaptic transmission.

5. Ayal Levenstain  

Within the line of research I intend to focus on the investigation of biological neuron networks. The main purpose is to study how different manipulations affect the behavior of the neuronal network. Two different methods are utilized to implement this approach:

1. Imaging – Different fluorescent cellular indicators assist in the visualization and characterization of the activity of
ex vivo cultured neural networks.

2. Multi-Electrode Array – Electrode-plated dishes are used to measure the membrane potential of up to 60 neurons simultaneously.

Combining the two resources should provide interesting aspects of the interplay between modification in the cellular level and changes in the network level.

6. Noa Lipsthein

Work is done in Prof. Nils Brose lab in the Max Planck Institute of Experimental Medicine, Department of Molecular Neurobiology, Göttingen, Germany:

Changes in the level of synaptic activity at chemical synapses lead to changes in synaptic strength and efficacy, a process termed synaptic plasticity. Short-term synaptic plasticity (STP) is a presynaptically expressed form of synaptic plasticity that takes place milliseconds to minutes after repetitive synaptic stimulation. The significance of STP was demonstrated in diverse brain functions such as motor control, sensory adaptation, and sound localization. STP can be attributed to changes in residual presynaptic Ca2+ levels, which in turn modulate synaptic vesicle depletion and replenishment during continued synaptic activity. The molecular mechanism accounting for this phenomenon is still largely unknown. Munc13 proteins constitute a family of presynaptic proteins that are critical for synaptic vesicle priming and the replenishment of releasable vesicles. Ca2+ ions tightly regulate the priming activity of Munc13 proteins via an evolutionarily conserved regulatory unit composed of a C1 domain, a C2 domain, and a Ca2+-dependent calmodulin-binding domain. In autaptic hippocampal neuron cultures, the calmodulin-binding domain was shown to regulate Munc13-dependent synaptic efficacy, thus shaping STP characteristics. I am studying the significance of the Ca2+-dependent calmodulin binding to Munc13-1 and its implications for short- and long-term synaptic plasticity in vivo. For that purpose I am generating a Munc13-1 knock-in mouse line, in which Calmodulin binding to Munc13-1 is abolished by a point mutation in the Calmodulin binding site (W464R). Using electrophysiology in autaptic hippocampal cultures, slice electrophysiology, and behavioral analysis of the knock-in mice, I hope to be able to determine the role of Munc13 proteins as molecular mediators of STP in vivo.

M.Sc Students

1. Noa Bielopolski

Syntaxin, Synaptobrevin and synaptosome-associated protein of 25 kDa (SNAP-25) form the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which plays an essential role in priming and/or in the fusion reaction itself. Changes in the quantity or availability of SNARE complexes directly affect the number of fusion-competent vesicles and neurotransmitter release. Under resting conditions, the major restriction for the SNARE complex formation is the availability of its different components. Tomosyn, a brain-specific protein, was identified as a binding partner for Syntaxin. The C-terminal domain of Tomosyn is homologous to the SNARE motif of Synaptobrevin and can replace it in creating an unproductive complex with Syntaxin and SNAP-25. It was demonstrated that overexpression of Tomosyn causes a reduction of the exocytotic response.

To learn more about the molecular mechanisms of tomosyn function, we use the Fluorescence Resonance Energy Transfer (FRET) method to investigate intra and inter molecule interactions of tomosyn and its dynamic regulation by its interaction with syntaxin.

2. Merav Kofer

Ras proteins are essential signal transducers that regulate cell growth, differentiation and death and play a key role in malignant transformation. Ras signaling to its downstream effectors includes translocation of Ras from the plasma-membrane (PM) to intracellular compartments. Rasosomes are recently discovered small particles, containing multiple lipid-modified Ras proteins that diffuse randomly in the cytosol and interact with the plasma membrane. Considering this, rasosomes apparently provide a mean by which multiple copies of Ras proteins and their signal information can spread rapidly across cells, thus contributing to Ras signal robustness.

Our previous observations suggested that rasosomes might sample the membrane at specific points, raising the possibility that information and signaling transfer occur at discrete specific areas on the PM. To examine that, we combine TIRF (total internal reflection fluorescence) microscopy and a new imaging analysis algorithm that examine the dynamic behavior of rasosomes near the PM. Applying such an algorithm demonstrate that there are hotspots on the PM in which rasosomes have higher probabilities to arrive. We next examine what determine the hotspot location and what restricts their narrow localization. Co-localization analysis of actin cytoskeleton and hotspots demonstrate that the hotspots are localized in between actin cages. Next, we investigated if hotspots represent areas in which ras signaling is transfer. We therefore use BiFC assay to examine the localization of Ras signaling domains in the context of actin. We found that the areas of activated ras are in between actin cages, similar to the hotspots of the rasosomes, suggesting that rasosomes may transmit signals at hotspots. Finally, we show that ras proteins that are localized on rasosomes, can recruit GFP-RBD which implies that rasosomes are capable of transmitting signals. This knowledge is expected to improve our understanding of signal robustness and its role in the control of fundamental biological processes.

Research Assistant

Dr. Anton Sheinin

Technical Staff

Reli Melamed

Previous Lab Members

Ofer Yizhar: oferyizhar@gmail.com
Aviv Mezer: avivmezer@gmail.com
Uri Nili: niliuri@gmail.com
Naama Zabari: zabarina@gmail.com
Adam Grundland: a_grundland@yahoo.com
Dragoslav Zikich: dzikich@gmail.com
Keren Sirota: kerensirota@gmail.com

1. Introduction to Neurobiology

2. Workshop in Neurophysiology