Shataakshi Dube O'Neil,1 Bence Rácz,2 Walter Evan Brown,3 Yudong Gao,3 Erik J Soderblom,3,4 Ryohei Yasuda,5 and Scott H Soderling1,3
1Department of Neurobiology, Duke University Medical Center, Durham, United States
2Department of Anatomy and Histology, University of Veterinary Medicine, Budapest, Hungary
3Department of Cell Biology, Duke University Medical Center, Durham, United States
4Proteomics and Metabolomics Shared Resource and Center for Genomic and Computational Biology, Duke University Medical Center, Durham, United States
5Max Planck Florida Institute for Neuroscience, Jupiter, United States
Scott H Soderling: [email protected]
In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments. Strikingly, we find Rac1 and Arp2/3 are closely associated with synaptic vesicle membranes in adult mice. Using three independent approaches to alter presynaptic Rac1 activity (genetic knockout, spatially restricted inhibition, and temporal optogenetic manipulation), we discover that this pathway negatively regulates synaptic vesicle replenishment at both excitatory and inhibitory synapses, bidirectionally sculpting short-term synaptic depression. Finally, we use two-photon fluorescence lifetime imaging to show that presynaptic Rac1 activation is coupled to action potentials by voltage-gated calcium influx. Thus, this study uncovers a previously unrecognized mechanism of actin-regulated short-term presynaptic plasticity that is conserved across excitatory and inhibitory terminals. It also provides a new proteomic framework for better understanding presynaptic physiology, along with a blueprint of experimental strategies to isolate the presynaptic effects of ubiquitously expressed proteins.
Light was delivered through a 20x water-immersion objective using an LED light source (CoolLED pE-300ultra) with 460 nm and 525–660 m excitation peaks and corresponding filter sets, with the shutter controlled by TTL inputs.
Product Associated Features
The broad spectrum pE-300ultra offers TTL triggering, individual channel control and software integration. This makes it ideal for fast pulsing at differing durations and irradiance, such as in these optogenetic protocols.
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