Munc13 Clustering on Lipid Membranes

We adapted the protocol described previously (36 (link), 44 (link)) to assess the formation of Munc13 clusters on lipid membrane surface in the absence or presence of DAG/DHG. Liposomes were prepared with following lipid composition (71% DOPC, 25% DOPS, 2% PIP2, ±2% DAG) using extrusion method with HEPES buffer (50 mM HEPES, 140 mM KCl, 1 mM TCEP, pH 7.4). Lipid bilayers were created by Mg²⁺ (5 mM) induced bursting liposomes in ibidi glass-bottom chambers (ibidi GmbH, Germany) and extensively washed with the HEPES buffer supplemented with EDTA (6 mM). Munc13-Halo-Alexa488 (10 nM) was added to the prewashed bilayer and incubated for 60 mins. The samples were imaged on a TIRF (Nikon) microscope with a 63× oil objective. For DHG experiments, we added 500 nM DHG is solution along with Munc13.