Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
VIB-KU Leuven Center for Brain and Disease Research, Campus Gasthuisberg, Leuven, Belgium.
Nat Commun. 2021 May 24;12(1):3057. doi: 10.1038/s41467-021-23116-w.
The active zone of a presynaptic nerve terminal defines sites for neurotransmitter release. Its protein machinery may be organized through liquid-liquid phase separation, a mechanism for the formation of membrane-less subcellular compartments. Here, we show that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation in transfected HEK293T cells. Condensate formation is triggered by Liprin-α3 PKC-phosphorylation at serine-760, and RIM and Munc13 are co-recruited into membrane-attached condensates. Phospho-specific antibodies establish phosphorylation of Liprin-α3 serine-760 in transfected cells and mouse brain tissue. In primary hippocampal neurons of newly generated Liprin-α2/α3 double knockout mice, synaptic levels of RIM and Munc13 are reduced and the pool of releasable vesicles is decreased. Re-expression of Liprin-α3 restored these presynaptic defects, while mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented this rescue. Finally, PKC activation in these neurons acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. Our findings indicate that PKC-mediated phosphorylation of Liprin-α3 triggers its phase separation and modulates active zone structure and function.
神经递质释放的部位位于突触前神经末梢的活性区。其蛋白机制可能通过液-液相分离来组织，这是形成无膜亚细胞隔室的一种机制。在这里，我们表明，转染的 HEK293T 细胞中的 Liprin-α3 可快速且可逆地发生相分离。凝聚物的形成是由 Liprin-α3 PKC 在丝氨酸-760 上的磷酸化触发的，并且 RIM 和 Munc13 被共同募集到膜附着的凝聚物中。磷酸特异性抗体确定了转染细胞和小鼠脑组织中 Liprin-α3 丝氨酸-760 的磷酸化。在新生成的 Liprin-α2/α3 双敲除小鼠的原代海马神经元中，RIM 和 Munc13 的突触水平降低，可释放囊泡的池减少。Liprin-α3 的重新表达恢复了这些突触前缺陷，而突变 Liprin-α3 的磷酸化位点以阻止相凝聚则阻止了这种挽救。最后，这些神经元中 PKC 的激活可急性增加 RIM、Munc13 和神经递质释放，这取决于可磷酸化的 Liprin-α3 的存在。我们的研究结果表明，PKC 介导的 Liprin-α3 磷酸化触发其相分离，并调节活性区的结构和功能。
