Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Tuesday Speaker Abstracts

Molecular Choreography of Polarity Regulation in Cell Migration Tobias Meyer . Stanford University, Stanford, USA.

We are investigating molecular mechanisms of polarization both in neutrophils and endothelial cells using fluorescent reporters and chemical and genetic perturbations. I will be presenting work on the order of events by which neutrophils rapidly transition from an unpolarized state to a polarized migrating state in a series of steps involving the small GTPases Cdc42, RhoA, Rac as well as Ras and PI3K. Once neutrophils are persistently migrating they steer their front towards chemotactic sources using primarily local Cdc42 and RhoA activities. In contrast to neutrophils, endothelial cells show two types of polarization, one operating in leader cells using a combination of small GTPases, Ca2+ and diacylglycerol signals and one operating in follower cells based on small GTPases that are locally directed by curved VE-cadherin-based membrane invaginations that we termed "Cadherin fingers". I will be presenting results on how curved membranes generated by these cadherin fingers help orient follower cells. Synaptic Function Illuminated by a Hybrid-Type Fluorescent Glutamate Probe Kenzo Hirose . UTokyo, Tokyo, Japan. To facilitate our understanding of the basic features of synaptic transmission, we have been developing a series of fluorescent glutamate probes named EOS. EOS is a hybrid type fluorescent probe consisting of a fluorescent dye and a glutamate binding domain of AMPA-type glutamate receptor. eEOS, our most recent version of EOS, was obtained by a combinatorial screening. eEOS has good photostability and a large dynamic range. Using eEOS, we successfully imaged glutamate release from single presynaptic terminals of cultured hippocampal neurons. The amounts of glutamate release under various conditions were analyzed to evaluate synaptic parameters that govern the quantal nature of neurotransmitter release. Results show that there exist multiple release sites at each synaptic terminal. There are large varieties in the number of the release sites as well as in release probability. Intriguingly the two synaptic parameters were not correlated. Thus each synapse has its own 'personality' characterized by these independent parameters. We then asked how nanoscale architectures formed by presynaptic proteins control neurotransmitter release. We undertook STORM microscopy for nanoscale visualization of presynaptic proteins. Notably, Mun13-1 molecules were found to assemble as small clusters; there were multiple Mun13-1 clusters per active zone. By combining the glutamate imaging technique and STORM microscopy, we directly counted and compared the number of the release sites and the number of Mun13-1 clusters. We found that these numbers were highly correlated. We also found that Muc13-1 clusters recruited syntaxin-1. These data indicate that Munc13-1 clusters are molecular entities for functional release. Reconstitution experiments in non-neuronal cells reveal that the formation of nanoclusters relies on self- organizing properties of Munc13-1 molecules sites. The self-organizing property of presynaptic molecule underlies presynaptic weights for synaptic computation.

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