Biology subjects

Macha, A.

Publications and source records attributed to Macha, A..

4 recordsLinked to original sources

bioRxiv2025

Phosphoinositide- and Collybistin-Dependent Synaptic Clustering of Gephyrin

Gephyrin is the main scaffolding protein at inhibitory synapses clustering glycine and GABA type A receptors. At specific GABAergic synapses, the nucleotide exchange factor collybistin recruits gephyrin to the postsynaptic membrane via interaction with phosphoinositides. However, the molecular mechanisms underlying the formation, maintenance and regulation of collybistin-dependent gephyrin clusters remain poorly understood. This study sheds light on the molecular mechanism of gephyrin cluster formation based on gephyrin self-oligomerization induced by collybistin, leading to the formation of a high-molecular weight (>5 MDa) gephyrin-collybistin complex, which is regulated in two ways: First, plasma-membrane phosphoinositides promote complex formation demonstrating their critical role in membrane targeting and stabilization of gephyrin-collybistin clusters at postsynaptic sites. Second, gephyrin phosphorylation at Ser325 abolishes complex formation with collybistin thus impairing collybistin-dependent gephyrin clustering at GABAergic synapses. Collectively, our data demonstrates a molecular mechanism for synaptic clustering of gephyrin which involves collybistin- and phosphoinositide-dependent formation of high-molecular gephyrin oligomers.

biochemistry↗

bioRxiv2024

A versatile protocol for purifying recombinant proteins from Nicotiana benthamiana for structural studies

Structural biology is an essential tool for understanding the molecular basis of biological processes. Although predicting protein structures by fold recognition algorithms has become increasingly powerful, especially with the integration of deep-learning approaches, experimentally resolved structures are indispensable for guiding structure-function studies and for improving modelling. However, experimental structural studies of protein complexes are still challenging, owing to, for example, the necessity for high protein concentrations and purity for downstream analyses such as cryogenic electron microscopy (cryo-EM). The use of Nicotiana benthamiana leaves as a transient expression system for recombinant proteins has become an increasingly attractive approach as the plant is inexpensive to cultivate, grows rapidly, allows fast experimental turnaround and is easily scalable compared to other established systems such as insect cell cultures. Using N. benthamiana as an expression system, we present here a robust and versatile protocol for the purification of five heterocomplexes with sizes ranging from [~]140 kDa to [~]660 kDa consisting of immunoreceptors and their associated pathogen effectors, followed by electron microscopy. The plant-based protocol was applied to verify the structure of the insect cell-derived wheat Sr35 resistosome and to co-purify and co-resolve a [~]140 kDa homodimer of the AvrSr35 effector from the fungus Puccinia graminis f sp tritici (Pgt). In several cases, only a single epitope tag is needed for complex purification, reducing complications that come with multiple epitope tags and two-step affinity purifications. We identify codon usage, signal peptide fusion, epitope tag choice and detergents as critical factors for expression and purification of recombinant protein from N. benthamiana leaves.

biochemistry↗

bioRxiv2024

Revisiting Sodium Phosphotungstate and Ammonium Molybdate as non-radioactive negative staining agents for single particle analysis

This study reports the successful replacement of uranyl-based stains by either sodium phosphotungstate or ammonium molybdate for negative staining electron microscopy. Using apoferritin as a test specimen, it is demonstrated that in combination with a facile on-grid fixation step both stains yield comparable images to uranyl formate. Subsequently, using {beta}-galactosidase, it is shown that both stains can also successfully be employed for single particle analysis, yielding virtually indistinguishable results from uranyl formate. As both replacement stains are non-radioactive, they are not subjected to the same handling restrictions as uranyl-based stains. Therefore they are not only cheaper to use, but also make decentralized sample grid preparation - thus directly after purification - accessible to a broader range of scientists.

biochemistry↗

bioRxiv2022

Pentameric assembly of glycine receptor intracellular domains provides insights into gephyrin clustering

Pentameric ligand-gated ion channels represent a large family of receptors comprising an extracellular domain, four transmembrane helices and a cytosolic intracellular domain (ICD). ICDs play important roles in receptor localization and trafficking, thus regulating synaptic activity and plasticity. Glycine and GABA type A receptor ICDs bind to the scaffolding protein gephyrin, a master regulator of inhibitory synapses. Here we report the use of yeast lumazine synthase as soluble pentameric protein scaffold for the study of receptor ICDs derived from GlyR 1- and {beta}-subunits. We were able to create ICDs assemblies in a homo- (LS-{beta}ICD) and hetero-pentameric state (LS-{beta}ICD) and provide first-in-class structural insights on their high structural flexibility using small angle X-ray scattering. We report a high-affinity interaction between the LS-{beta}ICD and gephyrin leading to the in vitro formation of high-molecular mega-Dalton complexes composed of three gephyrin trimers and three pentamers as basic building block. Depending on the stoichiometric ratios between gephyrin and LS-ICDs the formed complexes grow or shrink in size. In cells, LS-ICDs efficiently recruited gephyrin and were able to accumulate gephyrin at GABAergic synapses in neurons. Our findings collectively propose a new, potentially general, mechanistic concept for a gephyrin-dependent bridging of GlyRs at the inhibitory synapse.

biochemistry↗