Sarah R Chadwick, Samuel Stack-Couture, Matthew D Berg, Sonja Di Gregorio, Bryan Lung, Julie Genereaux, Robyn D Moir, Christopher J Brandl, Ian M Willis, Erik L Snapp, Patrick Lajoie
{"title":"TUDCA modulates drug bioavailability to regulate resistance to acute ER stress in <i>Saccharomyces cerevisiae</i>.","authors":"Sarah R Chadwick, Samuel Stack-Couture, Matthew D Berg, Sonja Di Gregorio, Bryan Lung, Julie Genereaux, Robyn D Moir, Christopher J Brandl, Ian M Willis, Erik L Snapp, Patrick Lajoie","doi":"10.1091/mbc.E24-04-0147","DOIUrl":"https://doi.org/10.1091/mbc.E24-04-0147","url":null,"abstract":"<p><p>Cells counter accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) through activation of the Unfolded Protein Response (UPR). Small molecules termed chemical chaperones can promote protein folding to alleviate ER stress. The bile acid tauroursodeoxycholic acid (TUDCA), has been described as a chemical chaperone. While promising in models of protein folding diseases, TUDCA's mechanism of action remains unclear. Here, we found TUDCA can rescue growth of yeast treated with the ER stressor tunicamycin (Tm), even in the absence of a functional UPR. In contrast, TUDCA failed to rescue growth on other ER stressors. Nor could TUDCA attenuate chronic UPR associated with specific gene deletions or over-expression of a misfolded mutant secretory protein. Neither pretreatment with or delayed addition of TUDCA conferred protection against Tm. Importantly, attenuation of Tm-induced toxicity required TUDCA's critical micelle forming concentration, suggesting a mechanism where TUDCA directly sequesters drugs. Indeed, in several assays, TUDCA treated cells closely resembled cells treated with lower doses of Tm. In addition, we found TUDCA can inhibit dyes from labeling intracellular compartments. Thus, our study challenges the model of TUDCA as a chemical chaperone and suggests that TUDCA decreases drug bioavailability, allowing cells to adapt to ER stress.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24040147"},"PeriodicalIF":3.1,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142813545","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}
Jason R Schrad, Gang Fu, Whitney E Hable, Alexandra M Tayar, Kenneth Oliveira, Daniela Nicastro
{"title":"Cryo-electron tomography of eel sperm flagella reveals a molecular \"minimum system\" for motile cilia.","authors":"Jason R Schrad, Gang Fu, Whitney E Hable, Alexandra M Tayar, Kenneth Oliveira, Daniela Nicastro","doi":"10.1091/mbc.E24-08-0351","DOIUrl":"https://doi.org/10.1091/mbc.E24-08-0351","url":null,"abstract":"<p><p>Cilia and flagella play a crucial role in the development and function of eukaryotes. The activity of thousands of dyneins is precisely regulated to generate flagellar motility. The complex proteome (600+ proteins) and architecture of the structural core of flagella, the axoneme, have made it challenging to dissect the functions of the different complexes, like the regulatory machinery. Previous reports suggested that the flagellum of American eel sperm lacks many of the canonical axonemal complexes yet is still motile. Here, we use cryo-electron tomography for molecular characterization of this proposed \"minimal\" motile flagellum. We observed different diameters for the eel sperm flagellum: narrow at the base and wider towards the flagellar tip. Subtomogram averaging revealed the 3D-structure of the eel sperm flagellum. As expected, major complexes were missing, e.g., outer dynein arms, radial spokes, and the central-pair-complex, but we found molecular remnants of most complexes. We also identified bend-direction specific patterns in the inter-DMT distance in actively beating eel sperm flagella and we propose a model for the regulation of dynein activity during their motility. Together, our results shed light on the structure and function of the eel sperm flagellum and provide insight into the minimum requirements for ciliary beating. [Media: see text].</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"mbcE24080351"},"PeriodicalIF":3.1,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142813542","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}
Oghosa H Akenuwa, Jinmo Gu, Andreas Nebenführ, Steven M Abel
{"title":"Morphometric analysis of actin networks.","authors":"Oghosa H Akenuwa, Jinmo Gu, Andreas Nebenführ, Steven M Abel","doi":"10.1091/mbc.E24-06-0248","DOIUrl":"10.1091/mbc.E24-06-0248","url":null,"abstract":"<p><p>The organization of cytoskeletal elements is pivotal for coordinating intracellular transport in eukaryotic cells. Several quantitative measures based on image analysis have been proposed to characterize morphometric features of fluorescently labeled actin networks. While helpful in detecting differences in actin organization between treatments or genotypes, the accuracy of these measures could not be rigorously assessed due to a lack of ground-truth data to which they could be compared. To overcome this limitation, we utilized coarse-grained computer simulations of actin filaments and cross-linkers to generate synthetic actin networks with varying levels of bundling. We converted the simulated networks into pseudofluorescence images similar to images obtained using confocal microscopy. Using both published and novel analysis procedures, we extracted a series of morphometric parameters and benchmarked them against analogous measures based on the ground-truth actin configurations. Our analysis revealed a set of parameters that reliably reports on actin network density, orientation, ordering, and bundling. Application of these morphometric parameters to root epidermal cells of <i>Arabidopsis thaliana</i> revealed subtle changes in network organization between wild-type and mutant cells. This work provides robust measures that can be used to quantify features of actin networks and characterize changes in actin organization for different experimental conditions.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar146"},"PeriodicalIF":3.1,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504323","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}
Ramyajit Mitra, Raghabendra Adhikari, Saniya S Davis, Benita L McVicker, Pamela L Tuma
{"title":"Distinct peroxisome populations differentially respond to alcohol-associated hepatic injury.","authors":"Ramyajit Mitra, Raghabendra Adhikari, Saniya S Davis, Benita L McVicker, Pamela L Tuma","doi":"10.1091/mbc.E24-06-0252","DOIUrl":"10.1091/mbc.E24-06-0252","url":null,"abstract":"<p><p>Although peroxisomes are known to oxidize ethanol, metabolize lipids, and regulate oxidative stress, they remain understudied in the context of ethanol-induced liver injury. We examined peroxisome early responses to alcohol-induced oxidative stress and lipid overload. Analysis of peroxisomes labeled with catalase, an ethanol oxidizing enzyme, or ABCD3, a fatty acid transporter, revealed that distinct peroxisome populations differentially respond to ethanol. We determined that ethanol exposure induced a reversible, time-dependent, saturable increase in functional peroxisomes labeled with either marker. This increase was due to ethanol-induced oxidative stress. In cells treated with oleic acid (to mimic fatty liver), only ABCD3-positive peroxisomes proliferated, and preferentially colocalized with lipid droplets in cells treated with oleic acid alone and/or with ethanol. In cells overexpressing the tubulin-specific acetyltransferase, αTAT1, we determined that peroxisome-lipid droplet contacts were mediated by acetylated microtubules. Peroxisome proliferation was also observed in ethanol-fed mouse and rat livers, but was absent in fibrotic mouse models of liver injury and in samples from individuals with alcohol-induced cirrhosis suggesting that alcohol exposure promotes an early hepatoprotective rise in peroxisomes that is lost as the condition progresses to fibrosis. Our studies further suggest that peroxisome proliferator-activated receptor agonists may be an effective intervention for early ethanol-associated liver disease.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar156"},"PeriodicalIF":3.1,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142624066","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}
Smit A Patel, Sungjin Park, Dantong Zhu, Elizabeth E Torr, Ashley-Grace Dureke, Alina McIntyre, Nadiya Muzyka, Jackson Severson, Ahna R Skop
{"title":"Extracellular vesicles, including large translating vesicles called midbody remnants, are released during the cell cycle.","authors":"Smit A Patel, Sungjin Park, Dantong Zhu, Elizabeth E Torr, Ashley-Grace Dureke, Alina McIntyre, Nadiya Muzyka, Jackson Severson, Ahna R Skop","doi":"10.1091/mbc.E23-10-0384","DOIUrl":"10.1091/mbc.E23-10-0384","url":null,"abstract":"<p><p>Extracellular vesicles (EVs) play crucial roles in cell-cell communication, but the biogenesis of large EVs has remained elusive. Here, we show that the biogenesis of large EVs (>800 nm-2 µm) occurs predominantly through the completion of successful cytokinesis, and the majority of large EVs are midbody remnants (MBRs) with translation activity, and the unique marker MKLP1. Blocking the cell cycle or cytokinesis, genetically or chemically, significantly decreases MBRs and large (800 nm-2 µm), medium (500-800 nm), and small (<300 nm) EVs, suggesting that proliferative cells can also generate all sizes of EVs. The canonical EV markers including CD9, CD63, CD81 localize to the spindle midzone, midbody, and MBRs, suggesting that these markers are not specific for detecting EVs exclusively. Importantly, all commonly used EV isolation methods isolate MBRs, confounding previous EV research. Last, isolated MBRs maintain translation activity regardless of the isolation method. We propose a model for the biogenesis of EVs throughout the cell cycle and suggest that some large EVs are primarily generated from mitotic cells. The discovery of MBRs as a unique class of large, translating EVs has implications for using them as cancer diagnostic markers and for engineering them for therapeutic cargo delivery during mitosis.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar155"},"PeriodicalIF":3.1,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142624067","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":"Spatiotemporal analysis of F-actin polymerization with micropillar arrays reveals synchronization between adhesion sites.","authors":"Sarit Hollander, Yuanning Guo, Haguy Wolfenson, Assaf Zaritsky","doi":"10.1091/mbc.E24-06-0276","DOIUrl":"10.1091/mbc.E24-06-0276","url":null,"abstract":"<p><p>We repurposed micropillar arrays to quantify spatiotemporal inter-adhesion communication. Following the observation that integrin adhesions formed around pillar tops we relied on the precise repetitive spatial control of the pillars to reliably monitor F-actin dynamics in mouse embryonic fibroblasts as a model for spatiotemporal adhesion-related intracellular signaling. Using correlation-based analyses, we revealed localized information flows propagating between adjacent pillars that were integrated over space and time to synchronize the adhesion dynamics within the entire cell. Probing the mechanical regulation, we discovered that stiffer pillars or partial actomyosin contractility inhibition enhances inter-adhesion F-actin synchronization, and that inhibition of Arp2/3, but not formin, reduces synchronization. Our results suggest that adhesions can communicate and highlight the potential of using micropillar arrays as a tool to measure spatiotemporal intracellular signaling.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"br23"},"PeriodicalIF":3.1,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504324","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}
Fatemeh Rajabi, Rebecca Smith, Win-Yan Liu-Bordes, Michael Schertzer, Sebastien Huet, Arturo Londoño-Vallejo
{"title":"DNA damage-induced EMT controlled by the PARP-dependent chromatin remodeler ALC1 promotes DNA repair efficiency through RAD51 in tumor cells.","authors":"Fatemeh Rajabi, Rebecca Smith, Win-Yan Liu-Bordes, Michael Schertzer, Sebastien Huet, Arturo Londoño-Vallejo","doi":"10.1091/mbc.E24-08-0370","DOIUrl":"10.1091/mbc.E24-08-0370","url":null,"abstract":"<p><p>Epithelial-to-mesenchymal transition (EMT) allows cancer cells to metastasize while acquiring resistance to apoptosis and chemotherapeutic agents with significant implications for patients' prognosis and survival. Despite its clinical relevance, the mechanisms initiating EMT during cancer progression remain poorly understood. We demonstrate that DNA damage triggers EMT and that activation of poly (ADP-ribose) polymerase (PARP) and the PARP-dependent chromatin remodeler ALC1 (CHD1L) was required for this response. Our results suggest that this activation directly facilitates access to the chromatin of EMT transcriptional factors (TFs) which then initiate cell reprogramming. We also show that EMT-TFs bind to the RAD51 promoter to stimulate its expression and to promote DNA repair by homologous recombination. Importantly, a clinically relevant PARP inhibitor reversed or prevented EMT in response to DNA damage while resensitizing tumor cells to other genotoxic agents. Overall, our observations shed light on the intricate relationship between EMT, DNA damage response, and PARP inhibitors, providing potential insights for in cancer therapeutics.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar151"},"PeriodicalIF":4.3,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142591306","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}
Jared Wollman, Kevin Wanniarachchi, Bijaya Pradhan, Lu Huang, Jason G Kerkvliet, Adam D Hoppe, Natalie W Thiex
{"title":"Mannose receptor (MRC1) mediates uptake of dextran by bone marrow-derived macrophages.","authors":"Jared Wollman, Kevin Wanniarachchi, Bijaya Pradhan, Lu Huang, Jason G Kerkvliet, Adam D Hoppe, Natalie W Thiex","doi":"10.1091/mbc.E24-08-0355","DOIUrl":"10.1091/mbc.E24-08-0355","url":null,"abstract":"<p><p>Macrophages survey their environment using receptor-mediated endocytosis and pinocytosis. Receptor-mediated endocytosis allows internalization of specific ligands, whereas pinocytosis nonselectively internalizes extracellular fluids and solutes. CRISPR/Cas9 whole-genome screens were used to identify genes regulating constitutive and growth factor-stimulated dextran uptake in murine bone marrow-derived macrophages (BMDM). The mannose receptor c-type 1 (MRC1/CD206) was a top hit in the screen. Targeted gene disruptions of <i>Mrc1</i> reduced dextran uptake but had little effect on fluid-phase uptake of Lucifer yellow. Other screen hits also differentially affected the uptake of dextran and Lucifer yellow, indicating internalization by separate mechanisms. Visualization of dextran and Lucifer yellow uptake by microscopy showed enrichment of dextran in small puncta, which was inhibitable by mannan, a ligand of MRC1. In contrast, Lucifer yellow predominantly was internalized in larger macropinosomes<i>.</i> In addition, IL4-treated BMDMs internalized more dextran than untreated BMDM correlating with increased MRC1 expression. Therefore, dextran is not an effective marker for pinocytosis in BMDMs since it is internalized by receptor-mediated process. Numerous genes that regulate dextran internalization in primary murine macrophages were identified in the whole-genome screens, which can inform understanding of the regulation of MRC1 expression and MRC1-mediated uptake in macrophages.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar153"},"PeriodicalIF":3.1,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142591309","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}
Pearl A Leon Guerrero, Jeffrey P Rasmussen, Eric Peterman
{"title":"Calcium dynamics of skin-resident macrophages during homeostasis and tissue injury.","authors":"Pearl A Leon Guerrero, Jeffrey P Rasmussen, Eric Peterman","doi":"10.1091/mbc.E24-09-0420","DOIUrl":"10.1091/mbc.E24-09-0420","url":null,"abstract":"<p><p>Immune cells depend on rapid changes in intracellular calcium activity to modulate cell function. Skin contains diverse immune cell types and is critically dependent on calcium signaling for homeostasis and repair, yet the dynamics and functions of calcium in skin immune cells remain poorly understood. Here, we characterize calcium activity in Langerhans cells, skin-resident macrophages responsible for surveillance and clearance of cellular debris after tissue damage. Langerhans cells reside in the epidermis and extend dynamic dendrites in close proximity to adjacent keratinocytes and somatosensory peripheral axons. We find that homeostatic Langerhans cells exhibit spontaneous and transient changes in calcium activity, with calcium flux occurring primarily in the cell body and rarely in the dendrites. Triggering somatosensory axon degeneration increases the frequency of calcium activity in Langerhans cell dendrites. By contrast, we show that Langerhans cells exhibit a sustained increase in intracellular calcium following engulfment of damaged keratinocytes. Altering intracellular calcium activity leads to a decrease in engulfment efficiency of keratinocyte debris. Our findings demonstrate that Langerhans cells exhibit context-specific changes in calcium activity and highlight the utility of skin as an accessible model for imaging calcium dynamics in tissue-resident macrophages.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"br26"},"PeriodicalIF":3.1,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142624064","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}
Timon Beck, Lize-Mari van der Linden, Wade M Borcherds, Kyoohyun Kim, Raimund Schlüßler, Paul Müller, Titus M Franzmann, Conrad Möckel, Ruchi Goswami, Mark Leaver, Tanja Mittag, Simon Alberti, Jochen Guck
{"title":"Optical characterization of molecular interaction strength in protein condensates.","authors":"Timon Beck, Lize-Mari van der Linden, Wade M Borcherds, Kyoohyun Kim, Raimund Schlüßler, Paul Müller, Titus M Franzmann, Conrad Möckel, Ruchi Goswami, Mark Leaver, Tanja Mittag, Simon Alberti, Jochen Guck","doi":"10.1091/mbc.E24-03-0128","DOIUrl":"10.1091/mbc.E24-03-0128","url":null,"abstract":"<p><p>Biomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques-Brillouin microscopy and quantitative phase imaging (QPI)-to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by fused in sarcoma (FUS) and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical cross-linking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.</p>","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":" ","pages":"ar154"},"PeriodicalIF":3.1,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142624068","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}