Biology subjects

Nachmias, D.

Publications and source records attributed to Nachmias, D..

10 recordsLinked to original sources

bioRxiv2025

Loss of CHMP2A implicates an ordered assembly of ESCRT-III proteins during cytokinetic abscission

The ESCRT machinery mediates membrane remodeling in fundamental cellular processes including cytokinesis, endosomal sorting, nuclear envelope reformation, and membrane repair. Membrane constriction and scission is driven by the filament-forming ESCRT-III complex and the AAA-ATPase VPS4. While ESCRT-III-driven membrane scission is generally established, the mechanisms governing the assembly and coordination of its twelve mammalian isoforms in cells remain poorly understood. Here, we examined the spatial organization and interdependence of ESCRT-III subunits during mammalian cytokinetic abscission by depleting CHMP2A, a core ESCRT-III component. Using live cell imaging, structured illumination microscopy (SIM) and correlative light-electron microscopy (CLEM), we show that CHMP2A knockout cells display a significant delay--but not failure--in abscission, accompanied by distinct mislocalization phenotypes across ESCRT-III subunits. While IST1 and CHMP2B were minimally disrupted, CHMP4B, CHMP3, and CHMP1B display progressively severe organization defects at the abscission site. Dual- protein imaging reveals disrupted coordination between ESCRT-III subunits in individual CHMP2A-deficient cells, supporting an ordered assembly of ESCRT-III subunits in cytokinetic abscission. Together, our findings provide the first in vivo evidence for hierarchical assembly of ESCRT-III subunits during ESCRT-mediated membrane remodeling and identify CHMP2A as a key organizer of ESCRT-III architecture essential for timely membrane abscission.

cell biology↗

bioRxiv2024

AuroraB-kinase methylation by SETD6 regulates cytokinesis and protects cells from chromosomal instability

SETD6 is a non-histone lysine methylatransferase, previously shown to participate in several housekeeping signaling pathways such as the NFkB pathway, Wnt signaling pathway, mitosis and more. In the current study we show evidence that SETD6 methylation is involved in the regulation of cytokinesis - the final process that divides cell contents into two daughter cells. SETD6 depleted HeLa cells presented high levels of chromatin bridges and actin patches, which are commonly observed following chromosomal segregation errors. In a proteomic screen we identified Aurora-B as a novel SETD6 substrate. Aurora-B kinase is an essential regulator of cytokinesis, known to actively delay cytokinesis as a response to the presence of chromatin in the midzone. We found that SETD6 binds and methylates Aurora-B on two adjacent lysine residues. Upon replication stress, Aurora-B methylation by SETD6 increases but is abolished when the two lysine methylation targets are substituted. In addition, replication stress led to a high tendency of SETD6 depleted cells to multinucleate, a major chromosomal-instability (CIN) phenotype. We detected a significant reduction in the Aurora-B kinase activity during cytokinesis in SETD6 knockout cells upon replication stress, which could be the mechanism underlying the accumulation of CIN phenotypes in these cells. CIN is a hallmark of cancer and is associated with tumor cell malignancy. Our findings suggest that Aurora-B methylation by SETD6 carries meaningful implications on tumorigenic cellular pathways.

cell biology↗

bioRxiv2023

Differential role for VPS4 isoforms in cytokinetic abscission confers a regulatory function for monomeric VPS4A and VTA1 in mammalian cells

Mutations in the human AAA-ATPase VPS4 isoform, VPS4A, cause severe Neurodevelopmental defects and Congenital Dyserythropoietic Anemia (CDA). VPS4 is a crucial component of the ESCRT system, which drives membrane remodeling in numerous cellular processes, including receptor degradation, cell division, and neural pruning. Notably, while most organisms encode for a single VPS4 gene, human cells have two VPS4 paralogs, namely VPS4A and VPS4B, but the functional differences between these paralogs is mostly unknown. Here, we set out to investigate the role of the human VPS4 paralogs in cytokinetic abscission using a series of knockout cell lines. We found that VPS4A and VPS4B hold both overlapping and distinct roles in abscission. VPS4A depletion resulted in a severe abscission delay, which was fully rescued by VPS4A expression but only partially rescued by VPS4B overexpression. Unexpectedly, expressing a monomeric-locked VPS4A mutant also partially rescued the abscission delay in VPS4A KO cells and bound the abscission checkpoint proteins CHMP4C and ANCHR. Depletion of VTA1, a co-factor of VPS4, disrupted VPS4A- ANCHR interactions and accelerated abscission, indicating a role for VTA1 in the abscission checkpoint. Our findings reveal a dual role for VPS4A in abscission, one that is canonical and can be compensated by VPS4B, and another that is regulatory and is mediated by its monomeric form. These observations provide a potential mechanistic explanation for the neurodevelopmental defects and other related disorders reported in VPS4A-mutated patients with a fully functional VPS4B paralog.

cell biology↗

bioRxiv2022

Asgard ESCRT-IIIs assemble into helical filaments in the presence of DNA and remodel eukaryotic membranes

The ESCRT machinery mediates membrane remodeling in numerous processes in cells including cell division and nuclear membrane reformation. The identification of ESCRT homologs in Asgard archaea, currently considered the closest ancestor of eukaryotes, implies a role for ESCRTs in the membrane remodeling processes that occurred during eukaryogenesis. Yet, the function of these distant ESCRT homologs is mostly unresolved. Here we show that Asgard ESCRT-III proteins of the Lokiarcheota self-assemble into helical filaments, a hallmark of the ESCRT system. We determined the cryo-EM structure of the filaments at 3.6 [A] resolution and found that they share features of bacterial and eukaryotic ESCRT-III assemblies. Markedly, Asgard ESCRT-III filaments bound and deformed eukaryotic-like membrane vesicles. Oligonucleotides facilitated the assembly of ESCRT-III filaments and tuned the extent of membrane remodeling. The ability of Asgard archaeal ESCRTs to remodel eukaryotic-like membranes, which are fundamentally different from archaeal membranes, and the structural properties of these proteins places them at the junction between prokaryotes and eukaryotes, substantiating a role for ESCRTs in eukaryogenesis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/506706v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@114999forg.highwire.dtl.DTLVardef@171a296org.highwire.dtl.DTLVardef@16931e0org.highwire.dtl.DTLVardef@1497c50_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Characterization of individual HIV-1 budding event using ultra-fast atomic force microscopy reveals a multiplexed role for VPS4

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Whereas the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (< 100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wildtype (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 30% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.

biophysics↗

bioRxiv2021

Asgard ESCRT-III and VPS4 reveal evolutionary conserved chromatin binding properties of the ESCRT machinery

The ESCRT machinery drive membrane remodeling in numerous processes in eukaryotes. Genes encoding for ESCRT proteins have been identified in Asgard archaea, a newly discovered superphylum, currently recognized as the ancestor of all eukaryotes. This begs the question of the functional evolutionary origin of this machinery and its conservation across lineages. Here, we find that Asgard-ESCRTs exhibit conserved DNA-binding properties, which is derived from recruitment of specific members. We show that Asgard-ESCRT-III/VPS4 homologs interact with one another inside mammalian cells, associate with chromatin, and recruit their counterparts to organize in discrete foci in the mammalian nucleus. This is congruent with human-ESCRT-III homologs. We find that human- and Asgard-ESCRT-IIIs associate with chromatin via the same N terminal domain, and that human-ESCRT-III can recruit Asgard-VPS4 to the nucleus to form foci. Therefore, ESCRTs possess chromatin binding properties that were preserved through the billion years of evolution that separate Asgard and human cells.

cell biology↗

bioRxiv2020

Cytokinetic abscission is part of the mid-blastula transition switch in early zebrafish embryogenesis

Animal cytokinesis ends with the formation of a thin intercellular membrane bridge connecting the two newly formed sibling cells that is ultimately resolved by abscission. While mitosis is completed within 15 minutes, the intercellular bridge can persist for hours, maintaining a physical connection between sibling cells and allowing exchange of cytosolic components. Although cell-cell communication is fundamental for development, the potential role of intercellular bridges during embryogenesis have not been fully elucidated. Here, we found that in early zebrafish (Danio rerio) embryogenesis, abscission is delayed and cells do not resolve their intercellular bridges until midblastula transition (MBT), giving rise to the formation of small inter-connected cell clusters. Interestingly, abscission commences during the MBT switch, which is manifested by cell cycle elongation, loss of synchronized divisions and genome activation. Moreover, depletion of Chmp4bb which is an essential ESCRT-III component for scission, delayed abscission beyond the MBT switch. Hallmark features of MBT, including transcription onset and cell shape changes, were similar in sibling cells connected by intercellular bridges, proposing a role for intercellular bridges in maintaining cell-cell communication in the embryo. Taken together, our data suggest that abscission is part of the cellular changes that occur during MBT and that cells coordinate their behavior during this critical embryonic phase through persisted intercellular bridges. Significance StatementIn this work we show that the last step of cytokinesis, termed abscission, is inhibited in early zebrafish embryos. As a result, sibling cells remain connected to one another for several cycles and mutually time their developmental progress including transcription onset. Abscission commences at the 10th cell cycle, when embryos enter the midblastula transition (MBT) switch in which embryonic cells become individualized and exhibit the characteristics of mature cells. Our data suggest that abscission is part of the MBT switch and that embryonic sibling cells mutually time their developmental progress by maintaining physical connections between them in the early embryo.

developmental biology↗

bioRxiv2019

A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

In the high-resolution microscopy era, genetic code expansion (GCE)-based bioorthogonal labeling offers an elegant way for direct labeling of proteins in live cells with fluorescent dyes. This labeling approach is currently not broadly used live cell applications, partly because it needs to be adjusted to the specific protein under study. Here, we present a generic, 14-residues long, N-terminal tag for GCE-based labeling of proteins in live mammalian cells. Using this tag, we generated a library of GCE-based organelle markers, demonstrating the applicability of the tag for labeling a plethora of proteins and organelles. Finally, we show that the HA epitope, used as a backbone in our tag, can be substituted with other epitopes and, in some cases, can be completely removed, reducing the tag length to 5 residues. The GCE-tag presented here offers a powerful, easy-to-implement tool for live cell labeling of cellular proteins with small and bright probes.

cell biology↗

bioRxiv2019

Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

ABSRACTTracking the localization and mobility of individual proteins in live cells is key for understanding how they mediate their function. Such information can be obtained from single molecule imaging techniques such as Single Particle Tracking (SPT) and Single Molecule Localization Microscopy (SMLM). Genetic code expansion (GCE) combined with bioorthogonal chemistry offers an elegant approach for direct labeling of proteins with fluorescent dyes, holding great potential for improving protein labeling in single molecule applications. Here we calibrated conditions for performing SPT and live-SMLM of bioorthogonally labeled plasma membrane proteins in live mammalian cells. Using SPT, the diffusion of bioorthogonally labeled EGF receptor and the prototypical Shaker voltage-activated potassium channel (Kv) was measured and characterized. Applying live-SMLM to bioorthogonally labeled Shaker Kv channels enabled visualizing the plasma membrane distribution of the channel over time with ~30 nm accuracy. Finally, by competitive labeling with two Fl-dyes, SPT and live-SMLM were performed in a single cell and both the density and dynamics of the EGF receptor were measured at single molecule resolution in subregions of the cell. We conclude that GCE and bioorthogonal chemistry is a highly suitable, flexible approach for protein labeling in quantitative single molecule applications that outperforms current protein live-cell labeling approaches.

biophysics↗

bioRxiv2017

Reducing Pyrrolysine tRNA Copy Number Improves the Performance of Genetic Code Expansion in Live Cell Imaging of Bioorthogonally Labeled Proteins

Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine synthetase/[Formula] pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNA contributes to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cells images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

synthetic biology↗