10 research outputs found
Neuroligin 2 induzierter allosterischer Übergang in Collybistin liegt der inhibitorischen postsynaptischen Differenzierung zugrunde
Synaptische Inhibition ist essentiell für die Hirnfunktion. GABAerge und glycinerge Synapsen vermitteln die schnelle Inhibition im zentralen Nervensystem von Säugetieren und balancieren die Aktivität in neuronalen Netzwerken aus. An der inhibitorischen Postsynapse akkumulieren GABA und Glycinrezeptoren an einem Proteingerüst, das aus dem Protein Gephyrin besteht. Durch eine Interaktion mit Neuroligin 2, einem Mitglied der Neuroligin-Familie der synapsenorganisierenden Zelladhäsionsmoleküle, wird Gephyrin an Stellen gegenüber von präsynaptischen Endigungen rekrutiert, die GABA oder Glycin freisetzen. Ein weiteres Gephyrin-bindendes Molekül, Collybistin, reguliert diesen Rekrutierungsprozess, indem es als Schalter in Membranbereichen aktiviert wird, in denen Neuroligin 2 akkumuliert wird. Diese Collybistin-Aktivierung führt zum Anlagern des Gephyrin-Gerüstes an der postsynaptischen Membran und erlaubt so die nachfolgende Assemblierung von Rezeptoren. Trotz vieler Studien über die kritische Rolle von Collybistin für die Entwicklung inhibitorischer Synapsen ist der Mechanismus der Collybistin-Funktion weiterhin unbekannt. In der vorliegenden Arbeit wurden die strukturellen Grundlagen der Collybistin-Funktion und die Lipid-Interaktionen von Collybistin untersucht. Die Ergebnisse führten zur Identifikation von zwei entscheidenden molekularen Mechanismen: (1) Eine durch Neuroligin-2-Bindung induzierte Konformationsänderung des Collybistinproteins, und (2) die darauf folgende, durch Protein-Lipid-Interaktionen vermittelte Bindung des Gephyrin-Collybistin-Komplexes an die Plasmamembran. Die vorliegenden Daten führen zu einem kohärenten molekularen Modell zum Aufbau inhibitorischer Synapsen und schließen eine Lücke in unserem bisherigen Verständnis der Entwicklung synaptischer Inhibition innerhalb des zentralen Nervensystems von Säugetieren
Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin
Neurotransmission at synapses is mediated by the fusion and subsequent endocytosis of synaptic vesicle membranes. Actin has been suggested to be required for presynaptic endocytosis but the mechanisms that control actin polymerization and its mode of action within presynaptic nerve terminals remain poorly understood. We combine optical recordings of presynaptic membrane dynamics and ultrastructural analysis with genetic and pharmacological manipulations to demonstrate that presynaptic endocytosis is controlled by actin regulatory diaphanous-related formins mDia1/3 and Rho family GTPase signaling in mouse hippocampal neurons. We show that impaired presynaptic actin assembly in the near absence of mDia1/3 and reduced RhoA activity is partly compensated by hyperactivation of Rac1. Inhibition of Rac1 signaling further aggravates impaired presynaptic endocytosis elicited by loss of mDia1/3. Our data suggest that interdependent mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate synaptic vesicle endocytosis by controlling presynaptic F-actin
Synaptic Vesicle Endocytosis Occurs on Multiple Timescales and Is Mediated by Formin-Dependent Actin Assembly
Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly
A conformational switch in collybistin determines the differentiation of inhibitory postsynapses
The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin‐based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin‐2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans‐synaptic neuroligin‐dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin‐based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses