RIM C2B Domains Target Presynaptic Active Zone Functions to PIP2-Containing Membranes

Abstract

Rapid and efficient synaptic vesicle fusion requires a pool of primed vesicles, the nearby tethering of Ca channels, and the presence of the phospholipid PIP in the target membrane. Although the presynaptic active zone mediates the first two requirements, it is unclear how fusion is targeted to membranes with high PIP content. Here we find that the CB domain of the active zone scaffold RIM is critical for action potential-triggered fusion. Remarkably, the known RIM functions in vesicle priming and Ca influx do not require RIM CB domains. Instead, biophysical experiments reveal that RIM C domains, which lack Ca binding, specifically bind to PIP. Mutational analyses establish that PIP binding to RIM CB and its tethering to the other RIM domains are crucial for efficient exocytosis. We propose that RIM CB domains are constitutive PIP-binding modules that couple mechanisms for vesicle priming and Ca channel tethering to PIP-containing target membranes.

RIM and RIM-BP Form Presynaptic Active-Zone-like Condensates via Phase Separation

“…The PRM sequence between two C2 domains of RIM can specifically bind to RIM-BP (Wang et al, 2000). The C2B domain forms a weak dimer (Guan et al, 2007) and has direct interaction with liprin and the phospholipid PI(4,5)P 2 (de Jong et al, 2018;Schoch et al, 2002). Knockout of RIMs impairs Ca 2+ -dependent neurotransmitter release and targeting and clustering of Ca 2+ channels to presynaptic terminals.…”

VAP‐SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function

“…All wild-type SCRN1, SCRN1-N (1-293), SCRN1-C (293-414), SCRN2, and SCRN3 constructs were generated using PCR-based cloning strategies and inserted into b-actin (for HA-SCRN1) or GW1 (for all other constructs) vectors. The RIM1a-mCherry construct was obtained by exchanging the HA-tag of the previously reported pAJ14063-pFUGW-RIM1aWT-HA construct (de Jong et al, 2018). SCRN1 FFAT-like mutant constructs were obtained for each predicted FFAT-like motif identified by a previously reported algorithm (Murphy & Levine, 2016).…”

“…Proteolytic dead mutant constructs SCRN1-C9A, SCRN2-C12A, and SCRN3-C6A were generated by replacing the predicted proteolytic cysteine residue, as identified by the online MEROPS database, by a non-catalytic alanine residue. The RIM1a-mCherry construct was obtained by exchanging the HA-tag of the previously reported pAJ14063-pFUGW-RIM1aWT-HA construct (de Jong et al, 2018). The following shRNAs inserted in pSuper vectors were used in this study: VAPA shRNA #1 (5 0 -GCATGCAGAGTGCTGTTTC-3 0 ; Teuling et al, 2007), VAPA shRNA #2 (5 0 -GGAAACTGATGGAAGAGTG-3 0 ; Teuling et al, 2007), and VAPB shRNA #1 (5 0 -GGTGATGGAA GAGTGC-3 0 ; Teuling et al, 2007); and SCRN1 shRNA #1 (5 0 -GATCCTTCCAGGTCCATAT-3 0 ), SCRN1 shRNA #2 (5 0 -GCACTTA CATCTCAATTGA-3 0 ), and SCRN1 shRNA #3 (5 0 -CAGGC TTGGTTTAGAACGA-3 0 ).…”

A PI(4,5)P2‐derived “gasoline engine model” for the sustained B cell receptor activation

“…IP3 is a key regulator of the cytoplasmic Ca 2+ concentration by controlling a Ca 2+ channel IP3 receptor on the endoplasmic reticulum (ER) . In addition to being the precursor of DAG and IP3, PI(4,5)P2 is also a key messenger that regulates the polymerization of the actin cytoskeleton, plasma membrane tension, exocytosis, membrane fusion, activity of ion channels, and cell adhesion, etc . Thus, PI(4,5)P2 is of great importance during receptor signaling and cell activation.…”