Molecular Biology of the Cell最新文献

{"title":"Imaging-based screen identifies novel natural compounds that perturb cell and chloroplast division in <i>Chlamydomonas reinhardtii</i>.","authors":"Manuella R Clark-Cotton, Sheng-An Chen, Aracely Gomez, Aditya J Mulabagal, Adriana Perry, Varenyam Malhotra, Masayuki Onishi","doi":"10.1091/mbc.E24-09-0425","DOIUrl":"10.1091/mbc.E24-09-0425","url":null,"abstract":"<p><p>Successful cell division requires faithful division and segregation of organelles into daughter cells. The unicellular alga <i>Chlamydomonas reinhardtii</i> has a single, large chloroplast whose division is spatiotemporally coordinated with furrowing. Cytoskeletal structures form in the same plane at the midzone of the dividing chloroplast (FtsZ) and the cell (microtubules), but how these structures are coordinated is not understood. Previous work showed that loss of F-actin blocks chloroplast division but not furrow ingression, suggesting that pharmacological perturbations can disorganize these events. In this study, we developed an imaging platform to screen natural compounds that perturb cell division while monitoring FtsZ and microtubules and identified 70 unique compounds. One compound, curcumin, has been proposed to bind to both FtsZ and tubulin proteins in bacteria and eukaryotes, respectively. In <i>C. reinhardtii,</i> where both targets coexist and are involved in cell division, curcumin at a specific dose range caused a severe disruption of the FtsZ ring in chloroplast while leaving the furrow-associated microtubule structures largely intact. Time-lapse imaging showed that loss of FtsZ and chloroplast division failure delayed the completion of furrowing but not the initiation, suggesting that the chloroplast division checkpoint proposed in other algae requires FtsZ or is absent altogether in <i>C. reinhardtii.</i></p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"br14"},"PeriodicalIF":3.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143527872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MYC regulation of the miR-92-Robo1 axis in Slit-mediated commissural axon guidance.","authors":"Tanushree Majumder, Bhakti Khot, Harindi Suriyaarachchi, Anagaa Nathan, Guofa Liu","doi":"10.1091/mbc.E24-12-0534","DOIUrl":"10.1091/mbc.E24-12-0534","url":null,"abstract":"<p><p>In the developing spinal cord, translational repression of Robo1 expression by microRNA-92 (miR-92) in precrossing commissural axons (CAs) inhibits Slit/Robo1-mediated repulsion facilitating commissural axon projection and midline crossing; however, the regulatory mechanisms governing miR-92 expression in the developing commissural neurons are currently lacking. Here, we propose that the transcription factor MYC regulates miR-92 expression in the developing spinal cord (of either sex) to control Robo1 levels in precrossing CAs, modulating Slit/Robo1-mediated repulsion and midline crossing. MYC, miR-92, and Robo1 are differentially expressed in the developing chicken spinal cord. MYC binds to the promoter region upstream of the gga-miR-92 gene <i>in vitro</i>. MYC knockdown dramatically decreases miR-92 expression and increases chicken Robo1 (cRobo1) levels. In contrast, overexpression of MYC significantly induces miR-92 expression and reduces cRobo1 levels. MYC knockdown or overexpression results in significant inhibition or induction of miR-92 activity in the developing chicken spinal cord, respectively. Disruption of the MYC-dependent regulation of the miR-92-cRobo1 axis affects Slit2-mediated CA growth cone collapse <i>in vitro</i> and impairs CA projection and midline crossing <i>in vivo</i>. These results elucidate the role of the MYC-miR-92-cRobo1 axis in Slit2/Robo1-mediated CA repulsion and midline crossing.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar50"},"PeriodicalIF":3.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143527874","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Independence of centromeric and pericentromeric chromatin stability on CCAN components.","authors":"Ronald J Biggs, Mingxuan Sun, Kousik Sundararajan, Eline Hendrix, Aaron F Straight, John F Marko","doi":"10.1091/mbc.E24-02-0066","DOIUrl":"10.1091/mbc.E24-02-0066","url":null,"abstract":"<p><p>The chromatin of the centromere provides the assembly site for the mitotic kinetochore that couples microtubule attachment and force production to chromosome movement in mitosis. The chromatin of the centromere is specified by nucleosomes containing the histone H3 variant, CENP-A. The constitutive centromeric-associated network (CCAN) and kinetochore are assembled on CENP-A chromatin to enable chromosome separation. CENP-A chromatin is surrounded by pericentromeric heterochromatin, which itself is bound by the sequence specific binding protein, CENP-B. We performed mechanical experiments on mitotic chromosomes while tracking CENP-A and CENP-B to observe the centromere's stiffness and the role of the CCAN. We degraded CENP-C and CENP-N containing auxin-inducible degrons, which we verified compromises the CCAN via observation of CENP-T loss. Chromosome stretching revealed that the CENP-A domain does not visibly stretch, even in the absence of CENP-C and/or CENP-N. Pericentromeric chromatin deforms upon force application, stretching ∼3-fold less than the entire chromosome. CENP-C and/or CENP-N loss has no impact on pericentromere stretching. Chromosome-disconnecting nuclease treatments showed no structural effects on CENP-A. Our experiments show that the core-centromeric chromatin is more resilient and likely mechanically disconnected from the underlying pericentromeric chromatin, while the pericentric chromatin is deformable yet stiffer than the chromosome arms.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar41"},"PeriodicalIF":3.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143409197","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Polysome profiling is an extensible tool for the analysis of bulk protein synthesis, ribosome biogenesis, and the specific steps in translation.","authors":"Ambar Rodriguez-Martinez, Sara K Young-Baird","doi":"10.1091/mbc.E24-08-0341","DOIUrl":"10.1091/mbc.E24-08-0341","url":null,"abstract":"<p><p>Protein synthesis is an essential and highly regulated cellular process. Here, we demonstrate the versatility of polysome profiling-a methodology traditionally used to assess levels of protein synthesis-to monitor ribosomal integrity and modulation of specific steps in mRNA translation. Using expanded polysome profiling methodologies, we systematically illustrate defects in ribosome biogenesis, translation initiation, and translational elongation in different cellular conditions. We additionally provide instruction for how a modified polysome profiling protocol can be leveraged to identify and characterize the function of factors that regulate protein synthesis. These methodologies are broadly applicable to a range of physiological conditions and human diseases in which ribosome biogenesis or the phases of protein synthesis are distinctly regulated or dysregulated.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mr2"},"PeriodicalIF":3.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Intracellular diffusion in the cytoplasm increases with cell size in fission yeast.","authors":"Catherine Tan, Michael C Lanz, Matthew Swaffer, Jan Skotheim, Fred Chang","doi":"10.1091/mbc.E24-11-0488","DOIUrl":"10.1091/mbc.E24-11-0488","url":null,"abstract":"<p><p>Diffusion in the cytoplasm can greatly impact cellular processes, yet regulation of macromolecular diffusion remains poorly understood. There is increasing evidence that cell size affects the density and macromolecular composition of the cytoplasm. Here, we studied whether cell size affects diffusion at the scale of macromolecules tens of microns in diameter. We analyzed the diffusive motions of intracellular genetically-encoded multimeric 40 nm nanoparticles (cytGEMs) in the cytoplasm of the fission yeast <i>Schizosaccharomyces pombe</i>. Using cell size mutants, we showed that cytGEMs diffusion coefficients decreased in smaller cells and increased in larger cells. This increase in diffusion in large cells may be due to a decrease in the DNA-to-cytoplasm ratio, as diffusion was not affected in large multinucleate cytokinesis mutant cells. In investigating the underlying causes of altered cytGEMs diffusion, we found that the proteomes of large and small cells exhibited size-specific changes, including the subscaling of ribosomal proteins in large cells. Comparison with a similar dataset from human cells revealed that features of size-dependent proteome remodeling were conserved. These studies demonstrate that cell size is an important parameter in determining the biophysical properties and the composition of the cytoplasm.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar51"},"PeriodicalIF":3.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A negative feedback loop between small GTPase Rap1 and mammalian tumor suppressor homologue KrsB regulates cell-substrate adhesion in <i>Dictyostelium</i>.","authors":"Yulia Artemenko, Gengle Niu, Megan E Arnold, Kelsey E Roberts, Bianca N Fernandez, Tiffany Flores, Harper D McClave, Michael Paestella, Jane Borleis, Peter N Devreotes","doi":"10.1091/mbc.E24-11-0507","DOIUrl":"10.1091/mbc.E24-11-0507","url":null,"abstract":"<p><p>Cell adhesion to the substrate influences a variety of cell behaviors and its proper regulation is essential for migration, although details of the molecular pathways regulating cell adhesion during migration are lacking. Rap1 is a small GTPase that regulates adhesion in mammalian cells, as well as in <i>Dictyostelium discoideum</i> social amoeba, which is an established model for studying directed cell migration. In <i>Dictyostelium</i>, Rap1 controls adhesion via its effects on adhesion mediator talin and Ser/Thr kinase Phg2, which inhibits myosin II function. Kinase responsive to stress B (KrsB), a homologue of mammalian tumor suppressor MST1/2 and <i>Drosophila</i> Hippo, also regulates cell adhesion and migration, although the molecular mechanism of KrsB action is not understood. Because KrsB has been shown to interact with active Rap1 by mass spectroscopy, we investigated the genetic interaction between Rap1 and KrsB. Cells lacking KrsB have increased adhesion to the substrate, which leads to reduced movement. Expression of constitutively active Rap1 G12V increased cell spreading and adhesion even in the absence of KrsB, suggesting that Rap1 does not require KrsB to mediate cell adhesion. In contrast, KrsB activation requires Rap1 since dominant-negative Rap1 S17N impaired KrsB phosphorylation, which has been previously shown to be necessary for KrsB activity and its function in adhesion. Even though Rap1 did not require KrsB for its function in adhesion, KrsB negatively regulates Rap1 function as seen by increased cortical localization of active Rap1 in KrsB-null cells. Consistently, Rap1 S17N completely reversed the overadhesive phenotype of KrsB-null cells. Furthermore, chemoattractant-induced activation of downstream effectors of Rap1, TalB and Phg2, was increased in the absence of KrsB. Taken together, these findings suggest that Rap1 leads to activation of KrsB, which inhibits Rap1 and its downstream targets, shutting off adhesion. The existence of a negative feedback loop between Rap1 and KrsB may contribute to the dynamic regulation of cell adhesion that is necessary for rapid amoeboid-type migration.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar43"},"PeriodicalIF":3.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143409182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Calponin-homology domain of GAS2L1 promotes formation of stress fibers and focal adhesions.","authors":"Franco K C Au, Khoi T D Le, Zhitao Liao, Zhijie Lin, Yuehong Shen, Penger Tong, Mingjie Zhang, Robert Z Qi","doi":"10.1091/mbc.E24-10-0444","DOIUrl":"10.1091/mbc.E24-10-0444","url":null,"abstract":"<p><p>Growth arrest-specific 2-like 1 protein (GAS2L1) binds both actin and microtubules through its unique structural domains: a calponin-homology (CH) domain for actin binding and a GAS2-related (GAR) domain for microtubule interaction. In this study, we demonstrate that GAS2L1 promotes stress fiber assembly, enhances focal adhesion formation, and stabilizes cytoskeletal networks against mechanical perturbation through its CH domain. Remarkably, we show that the CH domain dimerizes and induces actin filament bundling and stabilization both in cells and in vitro. The CH and GAR domains interact to form an autoinhibitory module, wherein the GAR domain suppresses CH domain dimerization and actin-bundling activity. Our findings provide novel insights into the regulatory mechanisms of GAS2L1's autoinhibition and identify the CH domain as a critical actin-bundling factor that contributes to the organization of stress fibers and focal adhesions.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar47"},"PeriodicalIF":3.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449699","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The cell biology and genome of <i>Stentor pyriformis</i>, a giant cell that embeds symbiotic algae in a microtubule meshwork.","authors":"Vincent Boudreau, Ashley R Albright, Ben T Larson, Therese M Gerbich, Tanner Fadero, Victoria Yan, Aviva Lucas-DeMott, Jay Yung, Solène L Y Moulin, Carlos Patiño Descovich, Mark M Slabodnick, Adrien Burlacot, Jeremy R Wang, Krishna K Niyogi, Wallace F Marshall","doi":"10.1091/mbc.E24-12-0571","DOIUrl":"10.1091/mbc.E24-12-0571","url":null,"abstract":"<p><p>Endosymbiotic events in which an endosymbiont is retained within a cell that remains capable of phagocytosis, a situation known as mixotrophy, provide potentially important clues about the eukaryotic evolution. Here we describe the cell biology and genome of the giant mixotrophic ciliate <i>Stentor pyriformis</i>. We show that <i>S. pyriformis</i> contains <i>Chlorella variabilis</i> as an endosymbiont that retains the ability to live outside the host. Within the host, the <i>Chlorella</i> cells surrounded by microtubule \"baskets\" near the cell surface. Photosynthetic efficiency of the <i>Chlorella</i> is reduced inside the <i>Stentor</i> cell compared with outside the host, due to increased nonphotochemical quenching. <i>S. pyriformis</i> displays positive phototaxis via directed swimming that requires the presence of the <i>Chlorella</i>, implying a potential flow of information from the symbiont to direct the orientation and swimming of the host cell. We sequenced the <i>S. pyriformis</i> genome and found that it employs a standard genetic code, similar to other <i>Stentor</i> species but different from most other ciliates. We propose that <i>S. pyriformis</i> will serve as a useful model system for studying endosymbiosis, with unique advantages in terms of size and regenerative ability as well as distinct cellular and genomic features compared with other mixotrophic ciliate models.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar44"},"PeriodicalIF":3.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143409200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PP2A-B56 regulates Mid1 protein levels for proper cytokinesis in fission yeast.","authors":"Madeline L Chrupcala, James B Moseley","doi":"10.1091/mbc.E24-08-0382","DOIUrl":"10.1091/mbc.E24-08-0382","url":null,"abstract":"<p><p>Protein phosphorylation regulates many steps in the cell division process including cytokinesis. In fission yeast cells, the anillin-like protein Mid1 sets the cell division plane and is regulated by phosphorylation. Multiple protein kinases act on Mid1, but no protein phosphatases have been shown to regulate Mid1. Here, we discovered that the conserved protein phosphatase PP2A-B56 is required for proper cytokinesis by promoting Mid1 protein levels. We find that <i>par1∆</i> cells lacking the primary B56 subunit divide asymmetrically due to the assembly of misplaced cytokinetic rings that slide toward cell tips. These <i>par1∆</i> mutants have reduced whole-cell levels of Mid1 protein, leading to reduced Mid1 at the cytokinetic ring. Restoring proper Mid1 expression suppresses <i>par1∆</i> cytokinesis defects. This work identifies a new PP2A-B56 pathway regulating cytokinesis through Mid1, with implications for control of cytokinesis in other organisms.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar52"},"PeriodicalIF":3.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567618","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interface integrity in septin protofilaments is maintained by an arginine residue conserved from yeast to man.","authors":"Benjamin Grupp, Jano Benito Graser, Julia Seifermann, Stefan Gerhardt, Justin A Lemkul, Jan Felix Gehrke, Nils Johnsson, Thomas Gronemeyer","doi":"10.1091/mbc.E25-01-0041","DOIUrl":"https://doi.org/10.1091/mbc.E25-01-0041","url":null,"abstract":"<p><p>The septins are conserved, filament-forming, guanine nucleotide binding cytoskeletal proteins. They assemble into palindromic protofilaments which polymerize further into higher-ordered structures that participate in essential intracellular processes such as cytokinesis or polarity establishment. Septins belong structurally to the P-Loop NTPases but, unlike their relatives Ras or Rho, do not mediate signals to effectors through GTP binding and hydrolysis. Biochemical approaches addressing how and why septins utilize nucleotides are hampered by the lack of nucleotide free complexes. Using molecular dynamics simulations, we determined structural alterations and inter-subunit binding free energies in human and yeast septin dimer structures and in their <i>in silico</i> generated apo forms. An interchain salt bridge network around the septin unique β-meander, conserved across all kingdoms of septin containing species, is destabilized upon nucleotide removal, concomitant with disruption of the entire G-interface. Within this network, we confirmed a conserved arginine residue, which coordinates the guanine base of the nucleotide, as the central interaction hub. The essential role of this arginine for interface integrity was experimentally confirmed to be conserved in septins from yeast to human.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE25010041"},"PeriodicalIF":3.1,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730896","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}