Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons.

ELife
Jason D Vevea, Edwin R Chapman

Abstract

The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.

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Citations

Feb 24, 2021·Cellular and Molecular Life Sciences : CMLS·Mónica C Quiñones-Frías, J Troy Littleton
Sep 22, 2021·Molecular Neurobiology· Neha S, Pankaj Singh Dholaniya

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Methods Mentioned

BETA
ubiquitination
fluorescence microscopy
PCR

Software Mentioned

Axograph X
ImageJ
Olympus
Fiji
JaCoP
FALI
CALI
Just Another Colocalization Plugin ( JACoP )
Knocksideways
Trim

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