Vesicle Tethering on the Surface of Phase-Separated Active Zone Condensates

Wu, Xiandeng; Ganzella, Marcelo; Zhou, Jinchuan; Zhu, Shihan; Jahn, Reinhard; Zhang, Mingjie

doi:10.1016/j.molcel.2020.10.029

Cited by 76 publications

(79 citation statements)

References 60 publications

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“…While many release mechanisms are established, key questions on how these dense scaffolds assemble, and how they remain dynamic to support the high spatiotemporal demands of the synaptic vesicle cycle remain unanswered. Purified RIM1 and RIM-BP2 form liquid condensates in vitro, indicating that active zones may assemble following phase transition principles 6 , 7 . Other synaptic compartments may also be organized through phase separation 8 – 11 .…”

Section: Introductionmentioning

confidence: 99%

PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 99%

PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure

et al. 2021

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“…Tight clustering of proteins participating in secretion is a hallmark of neuronal CAZ, and recent work in worms showed during synapse development the worm homologues of ELKS and liprin undergo LLPS and then solidify (McDonald et al, 2020). Moreover, different mammalian CAZ components can undergo LLPS in vitro or when overexpressed in cells (Emperador-Melero et al, 14 2021, Liang et al, 2021, McDonald et al, 2020, Sala et al, 2019, Wu et al, 2019, Wu et al, 2021. This raised the question of whether LLPS plays a role in organizing secretion machinery in β-cells.…”

Section: Discussionmentioning

confidence: 99%

“…Although the importance of focal adhesion remodeling for insulin secretion has been confirmed in vivo (Cai et al, 2012), little is known about the relationship between focal adhesions and the proteins organizing insulin exocytosis. Furthermore, a flurry of recent studies suggested that in neurons, CAZ may be formed by liquid-liquid phase separation (LLPS) of the key players, such as liprins, ELKS and RIMs 4 (Emperador-Melero et al, 2021, Liang et al, 2021, McDonald et al, 2020, Sala et al, 2019, Wu et al, 2019, Wu et al, 2021; reviewed in (Chen et al, 2020, Hayashi et al, 2021). The idea that the exocytotic machinery in β-cells would form by LLPS is attractive, but it has not been tested.…”

Section: Introductionmentioning

confidence: 99%

Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells

Noordstra

Berg

Boot

et al. 2021

Preprint

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Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including Bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain non-neuronal proteins LL5β and KANK1, which in migrating cells organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. While previous analyses in vitro and in neurons suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

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“…In addition to LLPS at PSDs, phase separation of synapsins has been shown to organize SVs at presynaptic boutons (Milovanovic et al, 2018), and LLPS of Rab3A-interacting molecules (RIMs) and RIM-BPs compartmentalizes voltage-gated Ca 2+ channels to organize the active zones (Wu et al, 2019), both of which ll are essential for the speed, strength, and accuracy of neurotransmission. More recently, negatively charged small lipid vesicles were shown to coat synapsin and RIM/RIM-BP condensates and separate the reserved or tethered pools of SVs in vitro (Wu et al, 2021), illustrating an interesting example in which membraneless and membrane-bound organelles may collectively promote synaptic signaling. Although it is tempting to infer a role for LLPS in neuronal networks, most studies of synaptic LLPS have been limited to model cell lines.…”

Section: Synaptic Signaling Organized By Biomolecular Condensatesmentioning

confidence: 99%

Liquid-liquid phase separation: Orchestrating cell signaling through time and space

Mehta

Zhang

2021

Molecular Cell

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Cell signaling is a complex process. The faithful transduction of information into specific cellular actions depends on the synergistic effects of many regulatory molecules, nurtured by their strict spatiotemporal regulation. Over the years, we have gained copious insights into the subcellular architecture supporting this spatiotemporal control, including the roles of membrane-bound organelles and various signaling nanodomains. Recently, liquid-liquid phase separation (LLPS) has been recognized as another potentially ubiquitous framework for organizing signaling molecules with high specificity and precise spatiotemporal control in cells. Here, we review the pervasive role of LLPS in signal transduction, highlighting several key pathways that intersect with LLPS, including examples in which LLPS is controlled by signaling events. We also examine how LLPS orchestrates signaling by compartmentalizing signaling molecules, amplifying signals non-linearly, and moderating signaling dynamics. We focus on the specific molecules that drive LLPS and highlight the known functional and pathological consequences of LLPS in each pathway.

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Vesicle Tethering on the Surface of Phase-Separated Active Zone Condensates

Cited by 76 publications

References 60 publications

PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure

PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure

Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells

Liquid-liquid phase separation: Orchestrating cell signaling through time and space

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