{"title":"Roles for the canonical polarity machinery in the de novo establishment of polarity in budding yeast spores.","authors":"Benjamin Cooperman, Michael McMurray","doi":"10.1091/mbc.E24-07-0303","DOIUrl":"10.1091/mbc.E24-07-0303","url":null,"abstract":"<p><p>The yeast <i>Saccharomyces cerevisiae</i> buds at sites predetermined 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 predetermined 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.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar28"},"PeriodicalIF":3.1,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11974964/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"The dark side of fluorescent protein tagging-the impact of protein tags on biomolecular condensation.","authors":"Edoardo Fatti, Sarah Khawaja, Karsten Weis","doi":"10.1091/mbc.E24-11-0521","DOIUrl":"10.1091/mbc.E24-11-0521","url":null,"abstract":"<p><p>Biomolecular condensation has emerged as an important mechanism to control various cellular processes through the formation of membraneless organelles. Fluorescent protein tags have been extensively used to study the formation and the properties of condensates in vitro and in vivo, but there is evidence that tags may perturb the condensation properties of proteins. In this study, we carefully assess the effects of protein tags on the yeast DEAD-box ATPase Dhh1, a central regulator of processing bodies (P-bodies), which are biomolecular condensates involved in mRNA metabolism. We show that fluorescent tags as well as a polyhistidine tag greatly affect Dhh1 condensation in vitro and lead to condensates with different dynamic properties. Tagging of Dhh1 with various fluorescent proteins in vivo alters the number of P-bodies upon glucose starvation and some tags even show constitutive P-bodies in nonstressed cells. These data raise concerns about the accuracy of tagged protein condensation experiments, highlighting the need for caution when interpreting the results.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"br10"},"PeriodicalIF":3.1,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11974960/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143059457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ellinor Tai, Austin Henglein, Angus Alfieri, Gauri Saxena, Scott Forth
{"title":"Insights into the role of phosphorylation on microtubule cross-linking by PRC1.","authors":"Ellinor Tai, Austin Henglein, Angus Alfieri, Gauri Saxena, Scott Forth","doi":"10.1091/mbc.E24-12-0565","DOIUrl":"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 cross-linking 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 cross-linked 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 post-translational modifications 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":"ar34"},"PeriodicalIF":3.1,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11974947/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Koit Aasumets, Anu Hangas, Georgios Fragkoulis, Cyrielle P J Bader, Direnis Erdinc, Sjoerd Wanrooij, Paulina H Wanrooij, Steffi Goffart, Jaakko L O Pohjoismäki
{"title":"MRE11-independent effects of Mirin on mitochondrial DNA integrity and cellular immune responses.","authors":"Koit Aasumets, Anu Hangas, Georgios Fragkoulis, Cyrielle P J Bader, Direnis Erdinc, Sjoerd Wanrooij, Paulina H Wanrooij, Steffi Goffart, Jaakko L O Pohjoismäki","doi":"10.1091/mbc.E24-01-0002","DOIUrl":"10.1091/mbc.E24-01-0002","url":null,"abstract":"<p><p>Mirin, a chemical inhibitor of MRE11, has been recently reported to suppress immune response triggered by mitochondrial DNA (mtDNA) breakage and release during replication stalling. We show that while Mirin reduces mitochondrial replication fork breakage in mitochondrial 3´-exonuclease MGME1 deficient cells, this effect occurs independently of MRE11. We also discovered that Mirin directly inhibits cellular immune responses, as shown by its suppression of STAT1 phosphorylation in Poly (I:C)-treated cells. Furthermore, Mirin also altered mtDNA supercoiling and accumulation of hemicatenated replication termination intermediates-hallmarks of topoisomerase dysfunction-while mitigating topological changes induced by the overexpression of mitochondrial TOP3A, including TOP3A-dependent strand breakage at the noncoding region of mtDNA. Although Mirin does not seem to inhibit TOP3A activity in vitro, our findings demonstrate its MRE11-independent effects in cells and give insight into the mechanisms of the maintenance of mtDNA integrity.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar11"},"PeriodicalIF":3.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11809308/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142869654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ty J Brown, Jennifer Pichurin, Carlos Ramirez Parrado, Lilian Kabeche, Susan J Baserga
{"title":"A role for the kinetochore protein, NUF2, in ribosome biogenesis.","authors":"Ty J Brown, Jennifer Pichurin, Carlos Ramirez Parrado, Lilian Kabeche, Susan J Baserga","doi":"10.1091/mbc.E24-08-0337","DOIUrl":"10.1091/mbc.E24-08-0337","url":null,"abstract":"<p><p>Ribosome biogenesis (RB) is an intricate and evolutionarily conserved process that takes place mainly in the nucleolus and is required for eukaryotic cells to maintain homeostasis, grow in size, and divide. Our laboratory has identified the NUF2 protein, part of the mitotic kinetochore, in a genome-wide siRNA screen for proteins required for making ribosomes in MCF10A human breast epithelial cells. After rigorous validation and using several biochemical and cell-based assays, we find a role for NUF2 in pre-rRNA transcription, the primary and rate-limiting step of RB. siRNA depletion of other components of the NUF2 kinetochore sub-complex, NDC80, SPC24, and SPC25, also reduce pre-rRNA transcription. Interestingly, essential protein components for pre-rRNA transcription, including the largest subunit of RNA polymerase I, POLR1A, are reduced upon siRNA depletion of NUF2 and its protein partners. Their reduced levels are a likely mechanism for the decrease in pre-rRNA transcription. siRNA depletion of NUF2 and NDC80 also cause increased TP53 and <i>CDKN1A</i> (p21) mRNA levels, which can be restored by codepletion of RPL5, indicating activation of the nucleolar stress pathway (NSP). These results reveal a new connection between proteins with a known role in mitosis to the function of the nucleolus in RB during interphase.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar16"},"PeriodicalIF":3.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11809303/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142869645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Karis A Clark, Andrew J White, Wojciech Paslawski, Kellianne D Alexander, Shaoning Peng, Tracy L Young-Pearse, Per Svenningsson, Dennis J Selkoe, Gary P H Ho
{"title":"Parkinson disease-associated toxic exposures selectively up-regulate vesicular glutamate transporter vGlut2 in a model of human cortical neurons.","authors":"Karis A Clark, Andrew J White, Wojciech Paslawski, Kellianne D Alexander, Shaoning Peng, Tracy L Young-Pearse, Per Svenningsson, Dennis J Selkoe, Gary P H Ho","doi":"10.1091/mbc.E24-08-0376","DOIUrl":"10.1091/mbc.E24-08-0376","url":null,"abstract":"<p><p>Parkinson disease (PD) is the second most common neurodegenerative disease, characterized by both motor and cognitive features. Motor symptoms primarily involve midbrain dopaminergic neurons, while cognitive dysfunction involves cortical neurons. Environmental factors are important contributors to PD risk. In rodents, rare midbrain dopaminergic neurons that coexpress the vesicular glutamate transporter 2 (vGlut2) are resistant to various toxins that induce dopaminergic neurodegeneration. However, it is unclear how, and with what degree of specificity, cortical glutamatergic neurons respond to PD-associated exposures with respect to vGlut2. Here, we found that vGlut2 in stem cell-derived human cortical-like glutamatergic neurons was up-regulated in a highly specific manner to certain PD-related chemicals, such as rotenone, but not others, such as paraquat. Further, exposure to recombinant preformed fibrils of alpha-synuclein (αS), a protein accumulating in PD, also increased vGlut2, while fibrils from non-PD-related proteins did not. This effect did not involve templated aggregation of endogenous αS. Finally, the knockdown of vGlut2 sensitized cortical neurons to rotenone, supporting a functional role in resilience. Thus, up-regulation of vGlut2 occurs in a highly selective manner in response to specific PD-associated exposures in a model of cortical glutamatergic neurons, a key cell type for understanding PD dementia.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"br4"},"PeriodicalIF":3.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11809304/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142922089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Stephen R Tymanskyj, Althea Escorce, Siddharth Karthikeyan, Le Ma
{"title":"Optogenetic control of receptor-mediated growth cone dynamics in neurons.","authors":"Stephen R Tymanskyj, Althea Escorce, Siddharth Karthikeyan, Le Ma","doi":"10.1091/mbc.E23-07-0268","DOIUrl":"10.1091/mbc.E23-07-0268","url":null,"abstract":"<p><p>Development of neuronal connections is spatially and temporally controlled by extracellular cues which often activate their cognate cell surface receptors and elicit localized cellular responses. Here, we demonstrate the use of an optogenetic tool to activate receptor signaling locally to induce actin-mediated growth cone remodeling in neurons. Based on the light-induced interaction between Cryptochrome 2 (CRY2) and CIB1, we generated a bicistronic vector to co-expresses CRY2 fused to the intracellular domain of a guidance receptor and a membrane-anchored CIB1. When expressed in primary neurons, activation of the growth inhibitory PlexA4 receptor induced growth cone collapse, while activation of the growth stimulating TrkA receptor increased growth cone size. Moreover, local activation of either receptor not only elicited the predicted response in light-activated growth cones but also an opposite response in neighboring no-light-exposed growth cones of the same neuron. Finally, this tool was used to reorient growth cones toward or away from the site of light activation and to stimulate local actin polymerization for branch initiation along axonal shafts. These studies demonstrate the use of an optogenetic tool for precise spatial and temporal control of receptor signaling in neurons and support its future application in investigating cellular mechanisms of neuronal development and plasticity.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"br5"},"PeriodicalIF":3.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11809317/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142869661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Maik C Bischoff, Jenevieve E Norton, Erika A Munguia, Sarah E Clark, Noah J Gurley, Rebecca Korankye, Emmanuel Addai Gyabaah, Taino Encarnacion, Christopher J Serody, Corbin D Jones, Mark Peifer
{"title":"A large reverse-genetic screen identifies numerous regulators of testis nascent myotube collective cell migration and collective organ sculpting.","authors":"Maik C Bischoff, Jenevieve E Norton, Erika A Munguia, Sarah E Clark, Noah J Gurley, Rebecca Korankye, Emmanuel Addai Gyabaah, Taino Encarnacion, Christopher J Serody, Corbin D Jones, Mark Peifer","doi":"10.1091/mbc.E24-10-0456","DOIUrl":"10.1091/mbc.E24-10-0456","url":null,"abstract":"<p><p>Collective cell migration is critical for morphogenesis, homeostasis, and wound healing. Migrating mesenchymal cells form tissues that shape the body's organs. We developed a powerful model, exploring how <i>Drosophila</i> nascent myotubes migrate onto the testis during pupal development, forming the muscles ensheathing it and creating its characteristic spiral shape. To define genes regulating this, we used RNA sequencing (RNA-seq) to identify genes expressed in myotubes during migration. Using this dataset, we curated a list of 131 ligands, receptors, and cytoskeletal regulators, including all Rho/Ras/Rap1 regulators, as candidates. We then expressed 279 short hairpin RNAs (shRNAs) targeting these genes and examined adult testes. We identified 29 genes with diverse roles in morphogenesis. Some have phenotypes consistent with defective migration, while others alter testis shape in different ways, revealing the underlying logic of testis morphogenesis. We followed up on the Rho-family GEF dPix in detail. dPix knockdown drastically reduced migration and thus muscle coverage. Our data suggest different isoforms of dPix play distinct roles in this process and reveal a role for its partner Git. We also explored whether dPix regulates Cdc42 activity or cell adhesion. Our RNA-seq dataset and genetic analysis provide an important resource for the community to explore cell migration and organ morphogenesis.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar21"},"PeriodicalIF":3.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11809313/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142922087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Traveling-wave chemotaxis of neutrophil-like HL-60 cells.","authors":"Motohiko Ishida, Masahito Uwamichi, Akihiko Nakajima, Satoshi Sawai","doi":"10.1091/mbc.E24-06-0245","DOIUrl":"10.1091/mbc.E24-06-0245","url":null,"abstract":"<p><p>The question of how changes in chemoattractant concentration translate into the chemotactic response of immune cells serves as a paradigm for the quantitative understanding of how cells perceive and process temporal and spatial information. Here, using a microfluidic approach, we analyzed the migration of neutrophil-like HL-60 cells to a traveling wave of the chemoattractants N-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4). We found that under a pulsatile wave that travels at a speed of 95 and 170 µm/min, cells move forward in the front of the wave but slow down and randomly orient at the back due to temporal decrease in the attractant concentration. Under a slower wave, cells reorient and migrate at the back of the wave; thus, cell displacement is canceled out or even becomes negative as cells chase the receding wave. Fluorescence resonance energy transfer (FRET)-based analysis indicated that these patterns of movement correlated well with spatiotemporal changes in Cdc42 activity. Furthermore, pharmacological perturbations showed that (re)orientation in front and back of the wave had different susceptibility to Cdc42 and ROCK inhibition. These results suggest that pulsatile attractant waves may recruit or disperse neutrophils, depending on their speed and degree of cell polarization.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar17"},"PeriodicalIF":3.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11809305/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142881974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Bioelectricity is a universal multifaced signaling cue in living organisms.","authors":"GuangJun Zhang, Michael Levin","doi":"10.1091/mbc.E23-08-0312","DOIUrl":"10.1091/mbc.E23-08-0312","url":null,"abstract":"<p><p>The cellular electrical signals of living organisms were discovered more than a century ago and have been extensively investigated in the neuromuscular system. Neuronal depolarization and hyperpolarization are essential for our neuromuscular physiological and pathological functions. Bioelectricity is being recognized as an ancient, intrinsic, fundamental property of all living cells, and it is not limited to the neuromuscular system. Instead, emerging evidence supports a view of bioelectricity as an instructional signaling cue for fundamental cellular physiology, embryonic development, regeneration, and human diseases, including cancers. Here, we highlight the current understanding of bioelectricity and share our views on the challenges and perspectives.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":"36 2","pages":"pe2"},"PeriodicalIF":3.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11809311/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053025","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}