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

AbstractThe 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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Phosphorylation triggers presynaptic phase separation of Liprin-α3 to control active zone structure

10.1101/2020.10.28.357574 ◽

2020 ◽

Author(s):

Javier Emperador-Melero ◽

Man Yan Wong ◽

Shan Shan H. Wang ◽

Giovanni de Nola ◽

Tom Kirchhausen ◽

...

Keyword(s):

Phase Separation ◽

Active Zone ◽

Neurotransmitter Release ◽

Phosphorylation Site ◽

Double Knockout ◽

Zone Structure ◽

And Function ◽

Pkc Activation ◽

Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation enables the assembly of membrane-less subcellular compartments, but testing its biological functions has been difficult. The presynaptic active zone, protein machinery in nerve terminals that defines sites for neurotransmitter release, may be organized through phase separation. Here, we discover that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation upon phosphorylation by PKC at a single site. RIM and Munc13 are co-recruited to membrane-attached condensates, and phospho-specific antibodies establish Liprin-α3 phosphorylation in vivo. At synapses of newly generated Liprin-α2/α3 double knockout mice, RIM, Munc13 and the pool of releasable vesicles were reduced. Re-expression of Liprin-α3 restored these defects, but mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented rescue. Finally, PKC activation acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. We conclude that Liprin-α3 phosphorylation rapidly triggers presynaptic phase separation to modulate active zone structure and function.

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Liprin-α3 controls vesicle docking and exocytosis at the active zone of hippocampal synapses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1719012115 ◽

2018 ◽

Vol 115 (9) ◽

pp. 2234-2239 ◽

Cited By ~ 22

Author(s):

Man Yan Wong ◽

Changliang Liu ◽

Shan Shan H. Wang ◽

Aram C. F. Roquas ◽

Stephen C. Fowler ◽

...

Keyword(s):

Synaptic Vesicle ◽

Active Zone ◽

Hippocampal Neurons ◽

Stimulated Emission ◽

Loss Of Function ◽

Zone Structure ◽

Vesicle Docking ◽

Synaptic Vesicle Exocytosis ◽

Vesicle Exocytosis ◽

And Function

The presynaptic active zone provides sites for vesicle docking and release at central nervous synapses and is essential for speed and accuracy of synaptic transmission. Liprin-α binds to several active zone proteins, and loss-of-function studies in invertebrates established important roles for Liprin-α in neurodevelopment and active zone assembly. However, Liprin-α localization and functions in vertebrates have remained unclear. We used stimulated emission depletion superresolution microscopy to systematically determine the localization of Liprin-α2 and Liprin-α3, the two predominant Liprin-α proteins in the vertebrate brain, relative to other active-zone proteins. Both proteins were widely distributed in hippocampal nerve terminals, and Liprin-α3, but not Liprin-α2, had a prominent component that colocalized with the active-zone proteins Bassoon, RIM, Munc13, RIM-BP, and ELKS. To assess Liprin-α3 functions, we generated Liprin-α3–KO mice by using CRISPR/Cas9 gene editing. We found reduced synaptic vesicle tethering and docking in hippocampal neurons of Liprin-α3–KO mice, and synaptic vesicle exocytosis was impaired. Liprin-α3 KO also led to mild alterations in active zone structure, accompanied by translocation of Liprin-α2 to active zones. These findings establish important roles for Liprin-α3 in active-zone assembly and function, and suggest that interplay between various Liprin-α proteins controls their active-zone localization.

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Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjab010 ◽

2021 ◽

Author(s):

Jonathon A Ditlev

Keyword(s):

Phase Separation ◽

Cell Biology ◽

Cellular Environment ◽

Condensate Formation ◽

Associated Proteins ◽

Available Technology ◽

And Function ◽

Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

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Low calcium-induced disruption of active zone structure and function at the frog neuromuscular junction

Synapse ◽

10.1002/(sici)1098-2396(199609)24:1<1::aid-syn1>3.0.co;2-a ◽

1996 ◽

Vol 24 (1) ◽

pp. 1-11 ◽

Cited By ~ 7

Author(s):

Stephen D. Meriney ◽

Birgit Wolowske ◽

Elham Ezzati ◽

Alan D. Grinnell

Keyword(s):

Neuromuscular Junction ◽

Active Zone ◽

Structure And Function ◽

Frog Neuromuscular Junction ◽

Zone Structure ◽

Low Calcium ◽

And Function

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Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis

The Journal of Cell Biology ◽

10.1083/jcb.200711167 ◽

2008 ◽

Vol 181 (5) ◽

pp. 831-846 ◽

Cited By ~ 86

Author(s):

Sergio Leal-Ortiz ◽

Clarissa L. Waites ◽

Ryan Terry-Lorenzo ◽

Pedro Zamorano ◽

Eckart D. Gundelfinger ◽

...

Keyword(s):

Synaptic Vesicle ◽

Active Zone ◽

Hippocampal Neurons ◽

Synapse Formation ◽

Zone Formation ◽

Homologous Protein ◽

Synaptic Vesicle Exocytosis ◽

Vesicle Exocytosis ◽

Presynaptic Plasma Membrane ◽

And Function

Active zones are specialized regions of the presynaptic plasma membrane designed for the efficient and repetitive release of neurotransmitter via synaptic vesicle (SV) exocytosis. Piccolo is a high molecular weight component of the active zone that is hypothesized to participate both in active zone formation and the scaffolding of key molecules involved in SV recycling. In this study, we use interference RNAs to eliminate Piccolo expression from cultured hippocampal neurons to assess its involvement in synapse formation and function. Our data show that Piccolo is not required for glutamatergic synapse formation but does influence presynaptic function by negatively regulating SV exocytosis. Mechanistically, this regulation appears to be calmodulin kinase II–dependent and mediated through the modulation of Synapsin1a dynamics. This function is not shared by the highly homologous protein Bassoon, which indicates that Piccolo has a unique role in coupling the mobilization of SVs in the reserve pool to events within the active zone.

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A presynaptic spectrin network controls active zone assembly and neurotransmitter release

10.1101/812032 ◽

2019 ◽

Author(s):

Qi Wang ◽

Lindsey Friend ◽

Rosario Vicidomini ◽

Tae Hee Han ◽

Peter Nguyen ◽

...

Keyword(s):

Active Zone ◽

Neurotransmitter Release ◽

Synaptic Cleft ◽

Structure And Function ◽

Dynamic Changes ◽

Synaptic Structure ◽

Synaptic Boutons ◽

Active Zones ◽

And Function ◽

Spectrin Network

ABSTRACTWe have previously reported that Drosophila Tenectin (Tnc) recruits αPS2/βPS integrin to ensure structural and functional integrity at larval NMJs (Wang et al., 2018). In muscles, Tnc/integrin engages the spectrin network to regulate the size and architecture of synaptic boutons. In neurons, Tnc/integrin controls neurotransmitter release. Here we show that presynaptic Tnc/integrin modulates the synaptic accumulation of key active zone components, including the Ca2+ channel Cac and the active zone scaffold Brp. Presynaptic α-Spectrin appears to be both required and sufficient for the recruitment of Cac and Brp. We visualized the endogenous α-Spectrin and found that Tnc controls spectrin recruitment at synaptic terminals. Thus, Tnc/integrin anchors the presynaptic spectrin network and ensures the proper assembly and function of the active zones. Since pre- and postsynaptic Tnc/integrin limit each other, we hypothesize that this pathway links dynamic changes within the synaptic cleft to changes in synaptic structure and function.

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RIM proteins and their role in synapse function

Biological Chemistry ◽

10.1515/bc.2010.064 ◽

2010 ◽

Vol 391 (6) ◽

Cited By ~ 35

Author(s):

Tobias Mittelstaedt ◽

Elena Alvaréz-Baron ◽

Susanne Schoch

Keyword(s):

Synaptic Vesicle ◽

Active Zone ◽

Neurotransmitter Release ◽

Multidomain Proteins ◽

Short Term ◽

Presynaptic Nerve Terminal ◽

Presynaptic Plasticity ◽

Active Zones ◽

Synaptic Vesicle Release ◽

The Brain

Abstract Active zones are specialized areas of the plasma membrane in the presynaptic nerve terminal that mediate neurotransmitter release and synaptic plasticity. The multidomain proteins RIM1 and RIM2 are integral components of the cytomatrix at the active zone, interacting with most other active zone-enriched proteins as well as synaptic vesicle proteins. In the brain, RIMs are present in multiple isoforms (α, β, γ) diverging in their structural composition, which mediate overlapping and distinct functions. Here, we summarize recent findings about the specific roles of the various RIM isoforms in basic synaptic vesicle release as well as long- and short-term presynaptic plasticity.

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Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

CNS & Neurological Disorders - Drug Targets ◽

10.2174/1871527319666201026165855 ◽

2020 ◽

Vol 19 ◽

Author(s):

Sumei Li ◽

Jifeng Zhang ◽

Jiaqi Zhang ◽

Jiong Li ◽

Longfei Cheng ◽

...

Keyword(s):

Neurite Outgrowth ◽

Hippocampal Neurons ◽

Neuronal Development ◽

Phosphorylation Site ◽

Microtubule Dynamics ◽

Microtubule Assembly ◽

C Terminus ◽

Mediator Protein ◽

Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

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