Molecular Biology of the Cell最新文献

{"title":"Deep learning-based image classification reveals heterogeneous execution of cell death fates during viral infection.","authors":"Edoardo Centofanti, Alon Oyler-Yaniv, Jennifer Oyler-Yaniv","doi":"10.1091/mbc.E24-10-0438","DOIUrl":"https://doi.org/10.1091/mbc.E24-10-0438","url":null,"abstract":"<p><p>Cell fate decisions, such as proliferation, differentiation, and death, are driven by complex molecular interactions and signaling cascades. While significant progress has been made in understanding the molecular determinants of these processes, historically, cell fate transitions were identified through light microscopy that focused on changes in cell morphology and function. Modern techniques have shifted towards probing molecular effectors to quantify these transitions, offering more precise quantification and mechanistic understanding. However, challenges remain in cases where the molecular signals are ambiguous, complicating the assignment of cell fate. During viral infection, programmed cell death (PCD) pathways, including apoptosis, necroptosis, and pyroptosis, exhibit complex signaling and molecular crosstalk. This can lead to simultaneous activation of multiple PCD pathways, which confounds assignment of cell fate based on molecular information alone. To address this challenge, we employed deep learning-based image classification of dying cells to analyze PCD in single Herpes Simplex Virus-1 (HSV-1)-infected cells. Our approach reveals that despite heterogeneous activation of signaling, individual cells adopt predominantly prototypical death morphologies. Nevertheless, PCD is executed heterogeneously within a uniform population of virus-infected cells and varies over time. These findings demonstrate that image-based phenotyping can provide valuable insights into cell fate decisions, complementing molecular assays. [Media: see text] [Media: see text] [Media: see text] [Media: see text].</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24100438"},"PeriodicalIF":3.1,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143023965","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":"GRASP55 Regulates Sorting and Maturation of the Lysosomal Enzyme β-Hexosaminidase A.","authors":"Sarah Reem Akaaboune, Aadil Javed, Sarah Bui, Alissa Wierenga, Yanzhuang Wang","doi":"10.1091/mbc.E24-10-0452","DOIUrl":"https://doi.org/10.1091/mbc.E24-10-0452","url":null,"abstract":"<p><p>The Golgi apparatus plays a crucial role in the delivery of lysosomal enzymes. Golgi Reassembly Stacking Proteins, GRASP55 and GRASP65, are vital for maintaining Golgi structure and function. GRASP55 depletion results in the missorting and secretion of the lysosomal enzyme cathepsin D (Xiang <i>et al.</i>, 2013), though the mechanisms remain unclear. In this study, we conducted secretomic analyses of GRASP55 knockout (KO) cells and found a significant increase in lysosome-associated proteins in the extracellular medium. Using the lysosomal beta-hexosaminidase subunit alpha (HEXA) as a model, we found that GRASP55 depletion disrupted normal trafficking and processing of HEXA, resulting in increased secretion of the immature (pro-form) HEXA into the extracellular milieu, along with decreased levels of the mature form and enzymatic activity within the cell. GRASP55 depletion significantly reduced the complex formation between HEXA and mannose 6-phosphate (M6P) receptors (MPR), despite no overall change in MPR expression. And finally, we found there was a notable reduction in the expression of GNPTAB, leading to a reduction in M6P modification of HEXA, hindering its efficient targeting to lysosomes. These findings reveal the role of GRASP55 in regulating lysosomal enzyme dynamics, emphasizing its role in the sorting and trafficking of lysosomal proteins.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24100452"},"PeriodicalIF":3.1,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143023971","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":"Anillin tunes contractility and regulates barrier function during Rho flare-mediated tight junction remodeling.","authors":"Zie Craig, Torey R Arnold, Kelsey Walworth, Alexander Walkon, Ann L Miller","doi":"10.1091/mbc.E24-11-0513","DOIUrl":"https://doi.org/10.1091/mbc.E24-11-0513","url":null,"abstract":"<p><p>To preserve barrier function, cell-cell junctions must dynamically remodel during cell shape changes. We have previously described a rapid tight junction repair pathway characterized by local, transient activation of RhoA, termed \"Rho flares\", which repair leaks in tight junctions via promoting local actomyosin-mediated junction remodeling. In this pathway, junction elongation is a mechanical trigger that initiates RhoA activation through an influx of intracellular calcium and recruitment of p115RhoGEF. However, mechanisms that tune the level of RhoA activation and Myosin II contractility during the process remain uncharacterized. Here, we show that the scaffolding protein Anillin localizes to Rho flares and regulates RhoA activity and actomyosin contraction at flares. Knocking down Anillin results in Rho flares with increased intensity but shorter duration. These changes in active RhoA dynamics weaken downstream F-actin and Myosin II accumulation at the site of Rho flares, resulting in decreased junction contraction. Consequently, tight junction breaks are not reinforced following Rho flares. We show that Anillin-driven RhoA regulation is necessary for successfully repairing tight junction leaks and protecting junctions from repeated barrier damage. Together, these results uncover a novel regulatory role for Anillin during tight junction repair and barrier function maintenance. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24110513"},"PeriodicalIF":3.1,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143024036","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":"Roles for the canonical polarity machinery in the <i>de novo</i> establishment of polarity in budding yeast spores.","authors":"Benjamin Cooperman, Michael McMurray","doi":"10.1091/mbc.E24-07-0303","DOIUrl":"https://doi.org/10.1091/mbc.E24-07-0303","url":null,"abstract":"<p><p>The yeast <i>Saccharomyces cerevisiae</i> buds at sites pre-determined by cortical landmarks deposited during prior budding. During mating between haploid cells in the lab, external pheromone cues override the cortical landmarks to drive polarization and cell fusion. By contrast, in haploid gametes (called spores) produced by meiosis, a pre-determined polarity site drives initial polarized morphogenesis independent of mating partner location. Spore membranes are made <i>de novo</i> so existing cortical landmarks were unknown, as were the mechanisms by which the spore polarity site is made and how it works. We find that the landmark canonically required for distal budding, Bud8, stably marks the spore polarity site along with Bud5, a GEF for the GTPase Rsr1 that canonically links cortical landmarks to the conserved Cdc42 polarity machinery. Cdc42 and other GTPase regulators arrive at the site during its biogenesis, after spore membrane closure but apparently at the site where membrane synthesis began, and then these factors leave, pointing to the presence of discrete phases of maturation. Filamentous actin may be required for initial establishment of the site, but thereafter Bud8 accumulates independent of actin filaments. These results suggest a distinct polarization mechanism that may provide insights into gamete polarization in other organisms. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24070303"},"PeriodicalIF":3.1,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143024112","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 Kinesin Kar3 is Required for Endoplasmic Reticulum-Associated Degradation.","authors":"Emmanuel Akoto, Ellen M Doss, Kieran P Claypool, Sophia L Owutey, Kyle A Richards, Katie M Lehman, Mahmoud M Daraghmi, Samantha M Turk, Christopher J Indovina, James A Avaala, Melissa D Evans, Abigail R Scott, Hayden O Schneider, Evan M Rogers, Jason D True, Philip J Smaldino, Eric M Rubenstein","doi":"10.1091/mbc.E24-10-0437","DOIUrl":"https://doi.org/10.1091/mbc.E24-10-0437","url":null,"abstract":"<p><p>Degradation of aberrant, excess, and regulatory proteins at the endoplasmic reticulum (ER) is a conserved feature of eukaryotic cells, disruption of which contributes to disease. While remarkable progress has been made in recent years, mechanisms and genetic requirements for ER-Associated Degradation (ERAD) remain incompletely understood. We recently conducted a screen for genes required for turnover of a model ER translocon-associated substrate of the Hrd1 ubiquitin ligase in <i>Saccharomyces cerevisiae</i>. This screen revealed loss of Kar3 impedes degradation of <i>Deg1</i>*-Sec62, which persistently and aberrantly engages the translocon. Kar3 is a microtubule-associated kinesin 14 family member that impacts multiple aspects of microtubule dynamics during cell division and karyogamy. We investigated involvement of Kar3 and its cofactors in ERAD. Loss of Kar3 hindered ERAD mediated by three ubiquitin ligases but did not impair turnover of a soluble nuclear protein. Further, <i>KAR3</i> deletion caused hypersensitivity to conditions associated with proteotoxic stress. Kar3's cytoplasmic cofactor Vik1 was also required for efficient degradation of <i>Deg1</i>*-Sec62. Our results reveal a profound and underappreciated role for microtubule-associated proteins in ERAD.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24100437"},"PeriodicalIF":3.1,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143024113","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":"Insights into the role of phosphorylation on microtubule crosslinking by PRC1.","authors":"Ellinor Tai, Austin Henglein, Angus Alfieri, Gauri Saxena, Scott Forth","doi":"10.1091/mbc.E24-12-0565","DOIUrl":"https://doi.org/10.1091/mbc.E24-12-0565","url":null,"abstract":"<p><p>The mitotic spindle is composed of distinct networks of microtubules, including interpolar bundles that can bridge sister kinetochore fibers and bundles that organize the spindle midzone in anaphase. The crosslinking protein PRC1 can mediate such bundling interactions between antiparallel microtubules. PRC1 is a substrate of mitotic kinases including CDK/cyclin-B, suggesting that it can be phosphorylated in metaphase and dephosphorylated in anaphase. How these biochemical changes to specific residues regulate its function and ability to organize bundles has been unclear. Here, we perform biophysical analyses on microtubule networks crosslinked by two PRC1 constructs, one a wild-type reflecting a dephosphorylated state, and one phosphomimetic construct with two threonine to glutamic acid substitutions near PRC1's microtubule binding domain. We find that the wild-type construct builds longer and larger bundles that form more rapidly and are much more resistant to mechanical disruption than the phosphomimetic PRC1. Interestingly, microtubule pairs organized by both constructs behave similarly within the same assays. Our results suggest that phosphorylation of PRC1 in metaphase could tune the protein to stabilize smaller and more flexible bundles, while removal of these PTMs in anaphase would promote the assembly of larger, more mechanically robust bundles to resist chromosome and pole separation forces at the spindle midzone.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24120565"},"PeriodicalIF":3.1,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143024028","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":"MAL2 and rab17 selectively redistribute invadopodia proteins to laterally-induced protrusions in hepatocellular carcinoma cells.","authors":"Saniya S Davis, Lauren R Bassaro, Pamela L Tuma","doi":"10.1091/mbc.E24-09-0400","DOIUrl":"https://doi.org/10.1091/mbc.E24-09-0400","url":null,"abstract":"<p><p>MAL2 (myelin and lymphocyte protein 2) and rab17 have been identified as hepatocellular carcinoma tumor suppressors. However, little is known how their functions in hepatic polarized protein sorting/trafficking translates into how they function in the epithelial to mesenchymal transition and/or the mesenchymal to epithelial transition in metastases. To investigate this, we expressed MAL2 and rab17 alone or together in hepatoma-derived Clone 9 cells (that lack endogenous MAL2 and rab17). Like MAL2, we found that rab17 expression led to the formation of actin- and cholesterol-dependent protrusions that correlated to its anti-oncogenic properties. MAL2 or rab17 selectively promoted the redistribution of invadopodia proteins to the protrusion tips that correlated with decreased matrix degradation. MAL2-mediated redistribution required a putative EVH1 recognition motif whereas rab17-mediated redistribution was GTP-dependent. We also determined that MAL2 and rab17 interaction was GTP dependent, but not dependent on the MAL2 EVH1 recognition motifs, and that protrusions formed by their combined expression shared features of those induced by either alone. Finally, we report that MAL2 or rab17 can redirect trafficking of newly synthesized membrane proteins from the Golgi to the induced protrusions and that the EVH1 recognition motif was required in MAL2 and that rab17-mediated trafficking was GTP-dependent.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24090400"},"PeriodicalIF":3.1,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984140","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":"β3 accelerates microtubule plus end maturation through a divergent lateral interface.","authors":"Lisa M Wood, Jeffrey K Moore","doi":"10.1091/mbc.E24-08-0354","DOIUrl":"10.1091/mbc.E24-08-0354","url":null,"abstract":"<p><p>β-tubulin isotypes exhibit similar sequences but different activities, suggesting that limited sequence divergence is functionally important. We investigated this hypothesis for TUBB3/β3, a β-tubulin linked to aggressive cancers and chemoresistance in humans. We created mutant yeast strains with β-tubulin alleles that mimic variant residues in β3 and find that residues at the lateral interface are sufficient to alter microtubule dynamics and response to microtubule targeting agents. In HeLa cells, β3 overexpression decreases the lifetime of microtubule growth, and this requires residues at the lateral interface. These microtubules exhibit a shorter region of EB binding at the plus end, suggesting faster lattice maturation, and resist stabilization by paclitaxel. Resistance requires the H1-S2 and H2-S3 regions at the lateral interface of β3. Our results identify the mechanistic origins of the unique activity of β3 tubulin and suggest that tubulin isotype expression may tune the rate of lattice maturation at growing microtubule plus ends in cells. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24080354"},"PeriodicalIF":3.1,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984157","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":"SYS-1/beta-catenin inheritance and regulation by Wnt-signaling during asymmetric cell division.","authors":"Maria F Valdes Michel, Bryan T Phillips","doi":"10.1091/mbc.E24-10-0441","DOIUrl":"10.1091/mbc.E24-10-0441","url":null,"abstract":"<p><p>Asymmetric cell division (ACD) allows daughter cells of a polarized mother to acquire different developmental fates. In <i>C. elegans</i>, the Wnt/β-catenin Asymmetry (WβA) pathway regulates many embryonic and larval ACDs; here, a Wnt gradient induces an asymmetric distribution of Wnt signaling components within the dividing mother cell. One terminal nuclear effector of the WβA pathway is the transcriptional activator SYS-1/β-catenin. SYS-1 is sequentially negatively regulated during ACD; first by centrosomal regulation and subsequent proteasomal degradation and second by asymmetric activity of the β-catenin \"destruction complex\" in one of the two daughter cells, which decreases SYS-1 levels in the absence of WβA signaling. However, the extent to which mother cell SYS-1 influences cell fate decisions of the daughters is unknown. Here, we quantify inherited SYS-1 in the differentiating daughter cells and the role of SYS-1 inheritance in Wnt-directed ACD. Photobleaching experiments demonstrate the GFP::SYS-1 present in daughter cell nuclei is comprised of inherited and <i>de novo</i> translated SYS-1 pools. We used a photoconvertible DENDRA2::SYS-1, to directly observe the dynamics of inherited SYS-1. Photoconversion during mitosis reveals that SYS-1 clearance at the centrosome preferentially degrades older SYS-1 and that newly localized centrosomal SYS-1 depends on dynein trafficking. Photoconversion of DENDRA2::SYS-1 in the EMS cell during Wnt-driven ACD shows daughter cell inheritance of mother cell SYS-1. Additionally, disrupting centrosomal SYS-1 localization in mother cells increased inherited SYS-1 and, surprisingly, loss of centrosomal SYS-1 also resulted in increased levels of <i>de novo</i> SYS-1 in both EMS daughter cells. Lastly, we show that negative regulation of SYS-1 in daughter cells via the destruction complex member APR-1/APC is key to limit both the <i>de novo</i> and the inherited SYS-1 pools in both the E and the MS cells. We conclude that regulation of both inherited and newly translated SYS-1 via centrosomal processing in the mother cell and daughter cell regulation via Wnt signaling are critical to maintain sister SYS-1 asymmetry during ACD.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24100441"},"PeriodicalIF":3.1,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984152","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":"Fission yeast GPI inositol deacylase Bst1 regulates ER-Golgi transport and functions in late stages of cytokinesis.","authors":"Yanfang Ye, Aysha H Osmani, Zhen-Ru Liu, Addie Kern, Jian-Qiu Wu","doi":"10.1091/mbc.E24-08-0375","DOIUrl":"https://doi.org/10.1091/mbc.E24-08-0375","url":null,"abstract":"<p><p>The Munc13/UNC-13 family protein Ync13 is essential for septum integrity and cytokinesis in fission yeast. To further explore the mechanism of Ync13 functions, spontaneous suppressors of <i>ync13</i> mutants, which can suppress the colony-formation defects and lysis phenotype of <i>ync13</i> mutant cells, are isolated and characterized. One of the suppressor mutants, <i>bst1</i>-<i>s27</i>, shows defects in the cytokinetic contractile ring constriction, septation, and daughter-cell separation, similar to <i>bst1Δ</i> mutant. Bst1, a predicted GPI inositol deacylase, was an uncharacterized protein in fission yeast. It localizes to the nuclear ER and puncta structures in the cytoplasm. The Bst1 puncta overlaps frequently with Anp1, which is a marker of ER-Golgi transport, but rarely with trans-Golgi marker Sec72. The nuclear ER signal of Anp1 increases in <i>bst1Δ</i> mutant, whereas Sec72 localization shows no obvious changes. In addition, more cytoplasmic puncta structures of COPII subunits, Sec13 and Sec24, are observed in <i>bst1Δ</i> mutant, and acid phosphatase secretion is compromised without Bst1. Consistently, the division site targeting of the β-glucanase Eng1 and α-glucanase Agn1 is reduced in <i>bst1Δ</i> and <i>bst1Δ ync13Δ</i> mutant. Taken together, our results suggest that Bst1 regulates ER-Golgi transport and is involved in cytokinesis through regulating the secretion of glucanases.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24080375"},"PeriodicalIF":3.1,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984122","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}