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Nature Communications Sep 2023Small ubiquitin-like modifier (SUMO) typically conjugates to target proteins through isopeptide linkage to the ε-amino group of lysine residues. This posttranslational...
Small ubiquitin-like modifier (SUMO) typically conjugates to target proteins through isopeptide linkage to the ε-amino group of lysine residues. This posttranslational modification (PTM) plays pivotal roles in modulating protein function. Cofilins are key regulators of actin cytoskeleton dynamics and are well-known to undergo several different PTMs. Here, we show that cofilin-1 is conjugated by SUMO1 both in vitro and in vivo. Using mass spectrometry and biochemical and genetic approaches, we identify the N-terminal α-amino group as the SUMO-conjugation site of cofilin-1. Common to conventional SUMOylation is that the N-α-SUMOylation of cofilin-1 is also mediated by SUMO activating (E1), conjugating (E2), and ligating (E3) enzymes and reversed by the SUMO deconjugating enzyme, SENP1. Specific to the N-α-SUMOylation is the physical association of the E1 enzyme to the substrate, cofilin-1. Using F-actin co-sedimentation and actin depolymerization assays in vitro and fluorescence staining of actin filaments in cells, we show that the N-α-SUMOylation promotes cofilin-1 binding to F-actin and cofilin-induced actin depolymerization. This covalent conjugation by SUMO at the N-α amino group of cofilin-1, rather than at an internal lysine(s), serves as an essential PTM to tune cofilin-1 function during regulation of actin dynamics.
Topics: Actins; Sumoylation; Lysine; Actin Depolymerizing Factors; Ubiquitin
PubMed: 37709794
DOI: 10.1038/s41467-023-41520-2 -
ELife Nov 2023The MRTF-SRF pathway has been extensively studied for its crucial role in driving the expression of a large number of genes involved in actin cytoskeleton of various...
The MRTF-SRF pathway has been extensively studied for its crucial role in driving the expression of a large number of genes involved in actin cytoskeleton of various cell types. However, the specific contribution of MRTF-SRF in hair cells remains unknown. In this study, we showed that hair cell-specific deletion of or , but not a, leads to similar defects in the development of stereocilia dimensions and the maintenance of cuticular plate integrity. We used fluorescence-activated cell sorting-based hair cell RNA-Seq analysis to investigate the mechanistic underpinnings of the changes observed in and mutants, respectively. Interestingly, the transcriptome analysis revealed distinct profiles of genes regulated by and , suggesting different transcriptional regulation mechanisms of actin cytoskeleton activities mediated by and . Exogenous delivery of calponin 2 using Adeno-associated virus transduction in mutants partially rescued the impairments of stereocilia dimensions and the F-actin intensity of cuticular plate, suggesting the involvement of , as an downstream target, in regulating the hair bundle morphology and cuticular plate actin cytoskeleton organization. Our study uncovers, for the first time, the unexpected differential transcriptional regulation of actin cytoskeleton mediated by and in hair cells, and also demonstrates the critical role of SRF-CNN2 in modulating actin dynamics of the stereocilia and cuticular plate, providing new insights into the molecular mechanism underlying hair cell development and maintenance.
Topics: Hair Cells, Auditory; Actin Cytoskeleton; Stereocilia; Actins; Gene Expression Regulation
PubMed: 37982489
DOI: 10.7554/eLife.90155 -
Nucleus (Austin, Tex.) Dec 2024The view on nuclear filaments formed by non-skeletal β-actin has significantly changed over the decades. Initially, filamentous actin was observed in amphibian oocyte... (Review)
Review
The view on nuclear filaments formed by non-skeletal β-actin has significantly changed over the decades. Initially, filamentous actin was observed in amphibian oocyte nuclei and only under specific cell stress conditions in mammalian cell nuclei. Improved labeling and imaging technologies have permitted insights into a transient but microscopically apparent filament network that is relevant for chromatin organization, biomechanics of the mammalian cell nucleus, gene expression, and DNA damage repair. Here, we will provide a historical perspective on the developing insight into nuclear actin filaments.
Topics: Animals; Cell Nucleus; Actin Cytoskeleton; Actins; Cytoskeleton; Oocytes; Mammals
PubMed: 38384139
DOI: 10.1080/19491034.2024.2320656 -
The Journal of Cell Biology Dec 2023Understanding how numerous actin-binding proteins (ABPs) work in concert to control the assembly, organization, and turnover of the actin cytoskeleton requires...
Understanding how numerous actin-binding proteins (ABPs) work in concert to control the assembly, organization, and turnover of the actin cytoskeleton requires quantitative information about the levels of each component. Here, we measured the cellular concentrations of actin and the majority of the conserved ABPs in Saccharomyces cerevisiae, as well as the free (cytosolic) fractions of each ABP. The cellular concentration of actin is estimated to be 13.2 µM, with approximately two-thirds in the F-actin form and one-third in the G-actin form. Cellular concentrations of ABPs range from 12.4 to 0.85 µM (Tpm1> Pfy1> Cof1> Abp1> Srv2> Abp140> Tpm2> Aip1> Cap1/2> Crn1> Sac6> Twf1> Arp2/3> Scp1). The cytosolic fractions of all ABPs are unexpectedly high (0.6-0.9) and remain so throughout the cell cycle. Based on these numbers, we speculate that F-actin binding sites are limited in vivo, which leads to high cytosolic levels of ABPs, and in turn helps drive the rapid assembly and turnover of cellular F-actin structures.
Topics: Actin Cytoskeleton; Actins; Microfilament Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Cytosol
PubMed: 37801069
DOI: 10.1083/jcb.202306036 -
Molecular Biology of the Cell Nov 2023Most eukaryotic cells utilize clathrin-mediated endocytosis as well as multiple clathrin-independent pathways to internalize proteins and membranes. Although...
Most eukaryotic cells utilize clathrin-mediated endocytosis as well as multiple clathrin-independent pathways to internalize proteins and membranes. Although clathrin-mediated endocytosis has been studied extensively and many machinery proteins have been identified, clathrin-independent pathways remain poorly characterized by comparison. We previously identified the first known yeast clathrin-independent endocytic pathway, which relies on the actin-modulating GTPase Rho1, the formin Bni1 and unbranched actin filaments, but does not require the clathrin coat or core clathrin machinery proteins. In this study, we sought to better understand clathrin-independent endocytosis in yeast by exploring the role of myosins as actin-based motors, because actin is required for endocytosis in yeast. We find that Myo2, which transports secretory vesicles, organelles and microtubules along actin cables to sites of polarized growth, participates in clathrin-independent endocytosis. Unexpectedly, the ability of Myo2 to transport microtubule plus ends to the cell cortex appears to be required for its role in clathrin-independent endocytosis. In addition, dynein, dynactin, and proteins involved in cortical microtubule capture are also required. Thus, our results suggest that interplay between actin and microtubules contributes to clathrin-independent internalization in yeast.
Topics: Saccharomyces cerevisiae; Actins; Clathrin; Microtubules; Endocytosis; Actin Cytoskeleton; Microfilament Proteins; Saccharomyces cerevisiae Proteins
PubMed: 37647159
DOI: 10.1091/mbc.E23-05-0164 -
Communications Biology Sep 2023Actin, an important component of eukaryotic cell cytoskeleton, regulates cell shape and transport. The morphology and biochemical properties of actin filaments are...
Actin, an important component of eukaryotic cell cytoskeleton, regulates cell shape and transport. The morphology and biochemical properties of actin filaments are determined by their structure and protein-protein contacts. Crowded environments can organize filaments into bundles, but less is known about how they affect F-actin structure. This study used 2D IR spectroscopy and spectral calculations to examine how crowding and bundling impact the secondary structure and local environments in filaments and weakly or strongly bundled networks. The results reveal that bundling induces changes in actin's secondary structure, leading to a decrease in β-sheet and an increase in loop conformations. Strongly bundled networks exhibit a decrease in backbone solvent exposure, with less perturbed α-helices and nearly "locked" β-sheets. Similarly, the loops become less hydrated but maintain a dynamic environment. These findings highlight the role of loop structure in actin network morphology and stability under morphology control by PEG.
Topics: Actins; Actin Cytoskeleton; Protein Structure, Secondary; Cytoskeleton; Cell Shape
PubMed: 37660224
DOI: 10.1038/s42003-023-05274-3 -
Transcriptional profiling of rare acantholytic disorders suggests common mechanisms of pathogenesis.JCI Insight Aug 2023Darier, Hailey-Hailey, and Grover diseases are rare acantholytic skin diseases. While these diseases have different underlying causes, they share defects in cell-cell...
Darier, Hailey-Hailey, and Grover diseases are rare acantholytic skin diseases. While these diseases have different underlying causes, they share defects in cell-cell adhesion in the epidermis and desmosome organization. To better understand the underlying mechanisms leading to disease in these conditions, we performed RNA-seq on lesional skin samples from patients. The transcriptomic profiles of Darier, Hailey-Hailey, and Grover diseases were found to share a remarkable overlap, which did not extend to other common inflammatory skin diseases. Analysis of enriched pathways showed a shared increase in keratinocyte differentiation, and a decrease in cell adhesion and actin organization pathways in Darier, Hailey-Hailey, and Grover diseases. Direct comparison to atopic dermatitis and psoriasis showed that the downregulation in actin organization pathways was a unique feature in the acantholytic skin diseases. Furthermore, upstream regulator analysis suggested that a decrease in SRF/MRTF activity was responsible for the downregulation of actin organization pathways. Staining for MRTFA in lesional skin samples showed a decrease in nuclear MRTFA in patient skin compared with normal skin. These findings highlight the significant level of similarity in the transcriptome of Darier, Hailey-Hailey, and Grover diseases, and identify decreases in actin organization pathways as a unique signature present in these conditions.
Topics: Humans; Actins; Skin; Acantholysis; Skin Diseases
PubMed: 37471166
DOI: 10.1172/jci.insight.168955 -
Cell Reports Dec 2023Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to...
Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions.
Topics: Humans; Protein Serine-Threonine Kinases; Cell Cycle Proteins; Ataxia Telangiectasia; Tumor Suppressor Proteins; Ataxia Telangiectasia Mutated Proteins; Chromatin; Reactive Oxygen Species; Proteomics; DNA-Binding Proteins; Phosphorylation; DNA Damage; Cytoskeleton
PubMed: 38088930
DOI: 10.1016/j.celrep.2023.113555 -
PLoS Pathogens Jul 2023L. pneumophila propagates in eukaryotic cells within a specialized niche, the Legionella-containing vacuole (LCV). The infection process is controlled by over 330...
L. pneumophila propagates in eukaryotic cells within a specialized niche, the Legionella-containing vacuole (LCV). The infection process is controlled by over 330 effector proteins delivered through the type IV secretion system. In this study, we report that the Legionella MavH effector localizes to endosomes and remodels host actin cytoskeleton in a phosphatidylinositol 3-phosphate (PI(3)P) dependent manner when ectopically expressed. We show that MavH recruits host actin capping protein (CP) and actin to the endosome via its CP-interacting (CPI) motif and WH2-like actin-binding domain, respectively. In vitro assays revealed that MavH stimulates actin assembly on PI(3)P-containing liposomes causing their tubulation. In addition, the recruitment of CP by MavH negatively regulates F-actin density at the membrane. We further show that, in L. pneumophila-infected cells, MavH appears around the LCV at the very early stage of infection and facilitates bacterium entry into the host. Together, our results reveal a novel mechanism of membrane tubulation induced by membrane-dependent actin polymerization catalyzed by MavH that contributes to the early stage of L. pneumophila infection by regulating host actin dynamics.
Topics: Legionella pneumophila; Actins; Polymerization; Phosphatidylinositol Phosphates; Vacuoles; Bacterial Proteins
PubMed: 37463171
DOI: 10.1371/journal.ppat.1011512 -
The Journal of Cell Biology Jul 2023Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular...
Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.
Topics: Actins; Endosomal Sorting Complexes Required for Transport; Integrins; Protein Transport; Phospholipids; Cell Membrane; Macrophages; Dendritic Cells; Fibroblasts; Humans; Protein Conformation
PubMed: 37200023
DOI: 10.1083/jcb.202205130