Rapid, efficient, targeted degradation of synaptotagmin 1 using knockoff technology
Here we applied knockoff technology to synaptotagmin 1. Knockoff is a new method developed in the Chapman lab to acutely disrupt membrane proteins. Briefly, a small, unique, substrate cleavage site is inserted just above a transmembrane domain; at the same time the hepatitis NS3-4A protease is appended to the carboxy terminus. Following the application or withdrawal of pharmaceutical grade NS3-4A protease inhibitors, cleavage and therefor protein level can be acutely controlled. This method excels when applied to type I membrane proteins as the subsequent cleavage products are degraded via the N-end rule. (A) Illustration of the syt1 knockoff protocol. (B) Representative anti-syt1 immunoblot demonstrating acute syt1 disruption in mouse hippocampal neurons. (Ø) denotes a condition in which cultures have never been exposed to inhibitor. (C) Self-cleavage time course of syt1-SELF, upon inhibitor washout. (D) Rates of spontaneous glutamate release (mGT) in control neurons and during syt1 disruption. (E) Percentage of synchronous glutamate release measured in control neurons and during syt1 disruption via iGluSnFR ΔF/F0 peaks occurring within 10 ms following a single stimulus. (F) Inverse correlation between synchronous release (≤10 ms) and spontaneous (mGT) release in control neurons and during syt1 disruption. Groups are color coded as in previous panels.
Spontaneous release recorded from dissociated hippocampal syt1KO neurons, using iGluSnFR
(A) Diagram of fluorescent glutamate sensor (iGluSnFR) and (B) changes in fluorescence upon glutamate interaction, data from Marvin, J.S. et al. Nat Meth. 2013. (C) Example of spontaneous release from a neuron with disrupted syt1 function. (D) Digitally magnified area with arrow pointing to release site. Trace is from ROI drawn around release site. At least 4 spontaneous events are observed.
Marvin, J.S., Borghuis, B.G., Tian, L., Cichon, J., Harnett, M.T., Akerboom, J., Gordus, A., Renninger, S.L., Chen, T.W., Bargmann, C.I. and Orger, M.B., 2013. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nature methods, 10(2), p.162.
The direct and specific egress of SYT1 to axons. a) A schematic depicting the RUSH system¹ used to retain SYT1 in the ER and release it with the addition of biotin. b) Live-cell image of rat hippocampal neurons showing the SYT1 reporter 30 minutes after biotin addition. The axon is identified by the presence of an axon initial segment (anti pan-neurofascin antibody) and denoted by an arrow. Scale bar is 10 microns. Kymographs were generated from the SYT1 channel, as shown by dashed boxes, and drawn from the soma outward direction for 20 microns. Corresponding kymographs from proximal dendrites (c) show the absence of mobile SYT1 reporter, whereas the proximal axons (d) show the presence of mobile SYT1. e) The displacement for all observed SYT1-containing vesicles is plotted in the anterograde (positive) or retrograde (negative) direction with respect to the soma for each compartment. The total time and area observed is equivalent between all compartments. Figure is modified from reference 2.
1. Boncompain G, Divoux S, Gareil N, et al. Synchronization of secretory protein traffic in populations of cells. Nat Methods. 2012;9(5):493-498. doi:10.1038/nmeth.1928
2. Watson ET, Pauers MM, Vevea JD, Chapman ER. Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons. bioRxiv. February 2022:2022.02.08.479521. doi:10.1101/2022.02.08.479521
In hippocampal neurons, SYT7 is localized to both the axonal plasma membrane and LAMP1+ organelles that include active lysosomes.
Synaptotagmin 7 is cleaved by the intramembrane aspartyl protease presenilin.
Synaptic Vesicle Isolation
Method for purification of synaptic vesicles (SVs). Magnetic Dynabeads conjugated to monoclonal antibodies are used to pull down SVs out of complex brain homogenates. One of these antibodies, rho1D4, enables the use of a peptide to elute SVs in native form, but other antibodies against SV proteins also work well to purify vesicles for downstream chemical analysis. The bottom row of panels depicts analytical confirmation of our SVs: these organelles contain the protein synaptophysin and the amino acid neurotransmitter glutamate, are approximately 45 ± 20 nm in diameter, and have the expected appearance when imaged by transmission electron microscopy.