לוח שנה משולב

Munc13 proteins are key determinants of synaptic vesicle priming and absolutely essential for the completion of the synaptic vesicle cycle at presynaptic active zones. Munc13 function is regulated by three different Ca2+-dependent pathways, and elevations of the presynaptic Ca2+ concentration during neuronal activity leads to a Munc13-dependent increase in the synaptic vesicle priming rate, and consequently to dynamic changes in the efficacy of neurotransmission. I will describe how the Ca2+-dependent regulation of Munc13s affects synaptic signaling in intact circuits, and present the first known synaptopathy caused by a mutation in Munc13-1, which affects a recently discovered interplay between Munc13s, voltage-gated Ca2+ channels, and synaptic vesicle fusogenicity.

Subdiffusion is a hallmark of protein dynamics. Several models predict subdiffusive dynamics, generally either due to some fractional noise or due to trapping dynamics in the underlying energy landscape. However the connection remains unclear, particularly to the underlying energy landscape. We reassess the assumption of a constant single subdiffusive exponent, identifying plateaus in the mean-squared displacement of molecular dynamics trajectories. We associate these with kinetic traps on a minimal model we develop of a hierarchical multibarrier energy landscape, exhibiting a separation of scales in both energy and space. We employ the model to extract the characteristic free energy barriers and length scales associated with the kinetic traps which lead to arrested dynamics. Moreover we identify a second subdiffusive mechanism inherent to the underlying fluctuating process. Lastly we find a relationship between the characteristic energy barriers and the rate of their appea! rance, allowing to reconstruct the effective subdiffusive mean-squared displacement.

Multiplexed ion beam imaging (MIBI) is a novel approach to immunohistochemistry (IHC) that uses secondary ion mass spectrometry (SIMS) and antibodies labeled with elemental mass tags to visualize dozens of proteins simultaneously in a single tissue section. MIBI is compatible with standard formalin-fixed, paraffin-embedded (FFPE) tissue specimens, the most common sample type in clinical repositories worldwide, and can achieve single molecule sensitivity across a five log dynamic range at resolutions equivalent to brightfield microscopy. In recent work, MIBI was validated for imaging breast tumor tissue sections stained with clinically relevant metal-conjugated antibodies via side-by-side comparison with an FDA-approved quantitative image analysis platform. Since that time, my lab has validated over sixty antibodies and has constructed a 45-plex MIBI panel for characterizing phenotypic and epigenetic features of epithelial, stromal, and infiltrating immune cells in clinical breast tumor biopsies. Analogous approaches with metal conjugated oligonucleotides have also been used for multiplexed DNA and RNA ISH. Finally, to permit broader use of this method, we have designed and constructed novel instrumentation optimized for MIBI that is capable of super resolution imaging and one hundred fold faster sample throughput. Taken together, these tools are being used by lab to comprehensively enumerate immune cell populations in normal and neoplastic solid tissues, to develop clinical classifiers for predicting disease progression in pre-invasive cancer lesions, and to discover epigenetic drivers of epithelial to mesenchymal transition.