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.