Ellen C O'Shaughnessy, Mable Lam, Samantha E Ryken, Theresa Wiesner, Kimberly Lukasik, J Bradley Zuchero, Christophe Leterrier, David Adalsteinsson, Stephanie L Gupton
{"title":"pHusion - a robust and versatile toolset for automated detection and analysis of exocytosis.","authors":"Ellen C O'Shaughnessy, Mable Lam, Samantha E Ryken, Theresa Wiesner, Kimberly Lukasik, J Bradley Zuchero, Christophe Leterrier, David Adalsteinsson, Stephanie L Gupton","doi":"10.1242/jcs.261828","DOIUrl":"10.1242/jcs.261828","url":null,"abstract":"<p><p>Exocytosis is a fundamental process used by eukaryotes to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate exocytosis in neuronal morphogenesis, previously we developed computational tools with a graphical user interface to enable the automatic detection and analysis of exocytic events from fluorescence timelapse images. Although these tools were useful, we found the code was brittle and not easily adapted to different experimental conditions. Here, we developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis, written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested pHusion using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types and various exocytic markers, to generate a flexible and intuitive package. Using this system, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11190432/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140848842","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}
Jiwon Lee, Kai Matuschewski, Giel van Dooren, Alexander G Maier, Melanie Rug
{"title":"Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.","authors":"Jiwon Lee, Kai Matuschewski, Giel van Dooren, Alexander G Maier, Melanie Rug","doi":"10.1242/jcs.262162","DOIUrl":"10.1242/jcs.262162","url":null,"abstract":"<p><p>Lipid droplets (LDs) are organelles that are central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and three-dimensional (3D) focused ion beam-scanning electron microscopy (FIB-SEM), we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or breakdown revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498149","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}
Junjun Liu, Frederik Johannes Verweij, Guillaume van Niel, Thierry Galli, Lydia Danglot, Philippe Bun
{"title":"ExoJ - a Fiji/ImageJ2 plugin for automated spatiotemporal detection and analysis of exocytosis.","authors":"Junjun Liu, Frederik Johannes Verweij, Guillaume van Niel, Thierry Galli, Lydia Danglot, Philippe Bun","doi":"10.1242/jcs.261938","DOIUrl":"10.1242/jcs.261938","url":null,"abstract":"<p><p>Exocytosis is a dynamic physiological process that enables the release of biomolecules to the surrounding environment via the fusion of membrane compartments to the plasma membrane. Understanding its mechanisms is crucial, as defects can compromise essential biological functions. The development of pH-sensitive optical reporters alongside fluorescence microscopy enables the assessment of individual vesicle exocytosis events at the cellular level. Manual annotation represents, however, a time-consuming task that is prone to selection biases and human operational errors. Here, we introduce ExoJ, an automated plugin based on Fiji/ImageJ2 software. ExoJ identifies user-defined genuine populations of exocytosis events, recording quantitative features including intensity, apparent size and duration. We designed ExoJ to be fully user-configurable, making it suitable for studying distinct forms of vesicle exocytosis regardless of the imaging quality. Our plugin demonstrates its capabilities by showcasing distinct exocytic dynamics among tetraspanins and vesicular SNARE protein reporters. Assessment of performance on synthetic data shows that ExoJ is a robust tool that is capable of correctly identifying exocytosis events independently of signal-to-noise ratio conditions. We propose ExoJ as a standard solution for future comparative and quantitative studies of exocytosis.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142107731","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":"Super-resolution imaging reveals nucleolar encapsulation by single-stranded DNA.","authors":"Koichiro Maki, Jumpei Fukute, Taiji Adachi","doi":"10.1242/jcs.262039","DOIUrl":"10.1242/jcs.262039","url":null,"abstract":"<p><p>In eukaryotic cell nuclei, specific sets of proteins gather in nuclear bodies and facilitate distinct genomic processes. The nucleolus, a nuclear body, functions as a factory for ribosome biogenesis by accumulating constitutive proteins, such as RNA polymerase I and nucleophosmin 1 (NPM1). Although in vitro assays have suggested the importance of liquid-liquid phase separation (LLPS) of constitutive proteins in nucleolar formation, how the nucleolus is structurally maintained with the intranuclear architecture remains unknown. This study revealed that the nucleolus is encapsulated by a single-stranded (ss)DNA-based molecular complex inside the cell nucleus. Super-resolution lattice-structured illumination microscopy (lattice-SIM) showed that there was a high abundance of ssDNA beyond the 'outer shell' of the nucleolus. Nucleolar disruption and the release of NPM1 were caused by in situ digestion of ssDNA, suggesting that ssDNA has a structural role in nucleolar encapsulation. Furthermore, we identified that ssDNA forms a molecular complex with histone H1 for nucleolar encapsulation. Thus, this study illustrates how an ssDNA-based molecular complex upholds the structural integrity of nuclear bodies to coordinate genomic processes such as gene transcription and replication.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11463959/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142107733","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}
Marie Erard, Cyril Favard, Luke D Lavis, Gaëlle Recher, Hervé Rigneault, Daniel Sage
{"title":"Back to the future - 20 years of progress and developments in photonic microscopy and biological imaging.","authors":"Marie Erard, Cyril Favard, Luke D Lavis, Gaëlle Recher, Hervé Rigneault, Daniel Sage","doi":"10.1242/jcs.262344","DOIUrl":"10.1242/jcs.262344","url":null,"abstract":"<p><p>In 2023, the ImaBio consortium (imabio-cnrs.fr), an interdisciplinary life microscopy research group at the Centre National de la Recherche Scientifique, celebrated its 20th anniversary. ImaBio contributes to the biological imaging community through organization of MiFoBio conferences, which are interdisciplinary conferences featuring lectures and hands-on workshops that attract specialists from around the world. MiFoBio conferences provide the community with an opportunity to reflect on the evolution of the field, and the 2023 event offered retrospective talks discussing the past 20 years of topics in microscopy, including imaging of multicellular assemblies, image analysis, quantification of molecular motions and interactions within cells, advancements in fluorescent labels, and laser technology for multiphoton and label-free imaging of thick biological samples. In this Perspective, we compile summaries of these presentations overviewing 20 years of advancements in a specific area of microscopy, each of which concludes with a brief look towards the future. The full presentations are available on the ImaBio YouTube channel (youtube.com/@gdrimabio5724).</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142501124","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}
Beth A Cimini, Peter Bankhead, Rocco D'Antuono, Elnaz Fazeli, Julia Fernandez-Rodriguez, Caterina Fuster-Barceló, Robert Haase, Helena Klara Jambor, Martin L Jones, Florian Jug, Anna H Klemm, Anna Kreshuk, Stefania Marcotti, Gabriel G Martins, Sara McArdle, Kota Miura, Arrate Muñoz-Barrutia, Laura C Murphy, Michael S Nelson, Simon F Nørrelykke, Perrine Paul-Gilloteaux, Thomas Pengo, Joanna W Pylvänäinen, Lior Pytowski, Arianna Ravera, Annika Reinke, Yousr Rekik, Caterina Strambio-De-Castillia, Daniel Thédié, Virginie Uhlmann, Oliver Umney, Laura Wiggins, Kevin W Eliceiri
{"title":"The crucial role of bioimage analysts in scientific research and publication.","authors":"Beth A Cimini, Peter Bankhead, Rocco D'Antuono, Elnaz Fazeli, Julia Fernandez-Rodriguez, Caterina Fuster-Barceló, Robert Haase, Helena Klara Jambor, Martin L Jones, Florian Jug, Anna H Klemm, Anna Kreshuk, Stefania Marcotti, Gabriel G Martins, Sara McArdle, Kota Miura, Arrate Muñoz-Barrutia, Laura C Murphy, Michael S Nelson, Simon F Nørrelykke, Perrine Paul-Gilloteaux, Thomas Pengo, Joanna W Pylvänäinen, Lior Pytowski, Arianna Ravera, Annika Reinke, Yousr Rekik, Caterina Strambio-De-Castillia, Daniel Thédié, Virginie Uhlmann, Oliver Umney, Laura Wiggins, Kevin W Eliceiri","doi":"10.1242/jcs.262322","DOIUrl":"10.1242/jcs.262322","url":null,"abstract":"<p><p>Bioimage analysis (BIA), a crucial discipline in biological research, overcomes the limitations of subjective analysis in microscopy through the creation and application of quantitative and reproducible methods. The establishment of dedicated BIA support within academic institutions is vital to improving research quality and efficiency and can significantly advance scientific discovery. However, a lack of training resources, limited career paths and insufficient recognition of the contributions made by bioimage analysts prevent the full realization of this potential. This Perspective - the result of the recent The Company of Biologists Workshop 'Effectively Communicating Bioimage Analysis', which aimed to summarize the global BIA landscape, categorize obstacles and offer possible solutions - proposes strategies to bring about a cultural shift towards recognizing the value of BIA by standardizing tools, improving training and encouraging formal credit for contributions. We also advocate for increased funding, standardized practices and enhanced collaboration, and we conclude with a call to action for all stakeholders to join efforts in advancing BIA.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142545667","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}
Vikas A Tillu, Gregory M I Redpath, James Rae, Juanfang Ruan, Yin Yao, Maria L Cagigas, Renee Whan, Edna C Hardeman, Peter W Gunning, Vaishnavi Ananthanarayanan, Robert G Parton, Nicholas Ariotti
{"title":"Precision in situ cryogenic correlative light and electron microscopy of optogenetically positioned organelles.","authors":"Vikas A Tillu, Gregory M I Redpath, James Rae, Juanfang Ruan, Yin Yao, Maria L Cagigas, Renee Whan, Edna C Hardeman, Peter W Gunning, Vaishnavi Ananthanarayanan, Robert G Parton, Nicholas Ariotti","doi":"10.1242/jcs.262163","DOIUrl":"10.1242/jcs.262163","url":null,"abstract":"<p><p>Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense and compacted environment of the cytoplasm remains challenging. Here, we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow that utilizes thin cells grown on a mechanically defined substratum for rapid analysis of organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles that would otherwise be positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142288104","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":"Synchrotron X-ray imaging of soft biological tissues - principles, applications and future prospects.","authors":"Jonas Albers, Angelika Svetlove, Elizabeth Duke","doi":"10.1242/jcs.261953","DOIUrl":"10.1242/jcs.261953","url":null,"abstract":"<p><p>Synchrotron-based tomographic phase-contrast X-ray imaging (SRµCT or SRnCT) is a versatile isotropic three-dimensional imaging technique that can be used to study biological samples spanning from single cells to human-sized specimens. SRµCT and SRnCT take advantage of the highly brilliant and coherent X-rays produced by a synchrotron light source. This enables fast data acquisition and enhanced image contrast for soft biological samples owing to the exploitation of phase contrast. In this Review, we provide an overview of the basics behind the technique, discuss its applications for biologists and provide an outlook on the future of this emerging technique for biology. We introduce the latest advances in the field, such as whole human organs imaged with micron resolution, using X-rays as a tool for virtual histology and resolving neuronal connections in the brain.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11529875/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142501129","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}
Sönke Rudnik, Saskia Heybrock, Etienne Coyaud, Zizhen Xu, Dante Neculai, Brian Raught, Viola Oorschot, Cecilia Heus, Judith Klumperman, Paul Saftig
{"title":"The lysosomal lipid transporter LIMP-2/SCARB2 is part of lysosome-endoplasmic reticulum STARD3-VAPB-dependent contact sites.","authors":"Sönke Rudnik, Saskia Heybrock, Etienne Coyaud, Zizhen Xu, Dante Neculai, Brian Raught, Viola Oorschot, Cecilia Heus, Judith Klumperman, Paul Saftig","doi":"10.1242/jcs.261810","DOIUrl":"https://doi.org/10.1242/jcs.261810","url":null,"abstract":"<p><p>SCARB2/LIMP-2 is an abundant lysosomal membrane protein. Previous studies have shown LIMP-2 functions as a virus receptor, a chaperone for lysosomal enzyme targeting, and a lipid transporter. The large luminal domain of LIMP-2 contains a hydrophobic tunnel that enables transport of phospholipids, sphingosine and cholesterol from the lysosomal lumen to the membrane. The question about the fate of the lipids after LIMP-2-mediated transport is largely unexplored. To elucidate whether LIMP-2 is part of contact sites between lysosomes and the endoplasmic reticulum (ER), we performed a proximity-based interaction screen. This revealed that LIMP-2 interacts with the endosomal protein STARD3 and the ER-resident protein VAPB. Using imaging and co-immunoprecipitation, we demonstrated colocalization and physical interaction between LIMP-2 and these proteins. Moreover, we found that interaction of LIMP-2 with VAPB required the presence of STARD3. Our findings suggest that LIMP-2 is part of ER-lysosome contact sites, possibly facilitating cholesterol transport from the lysosomal to the ER membrane. This suggests a novel mechanism for inter-organelle communication and lipid trafficking mediated by LIMP-2.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142380944","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}
Laura Bel Borja, Samuel J P Taylor, Flavie Soubigou, Federico Pelisch
{"title":"CENP-C-targeted PLK-1 regulates kinetochore function in C. elegans embryos.","authors":"Laura Bel Borja, Samuel J P Taylor, Flavie Soubigou, Federico Pelisch","doi":"10.1242/jcs.262327","DOIUrl":"https://doi.org/10.1242/jcs.262327","url":null,"abstract":"<p><p>Polo-like kinase 1 (PLK-1) is present in centrosomes, the nuclear envelope, and kinetochores and plays a significant role in meiosis and mitosis. PLK-1 depletion or inhibition has severe consequences for spindle assembly, spindle assembly checkpoint (SAC) activation, chromosome segregation, and cytokinesis. BUB-1 targets PLK-1 to the outer kinetochore and, in mammals, the inner kinetochore PLK1 targeting is mediated by the constitutive centromere associated network (CCAN). BUB1-targeted PLK-1 plays a key role in SAC activation and a SAC-independent role through targeting CDC-20. In contrast, whether there is a specific, non-redundant role for inner kinetochore targeted PLK-1 is unknown. Here, we used the C. elegans embryo to study the role of inner kinetochore PLK-1. We found that CENP-C, the sole CCAN component in C. elegans and other species, targets PLK-1 to the inner kinetochore during prometaphase and metaphase. Disruption of the CENP-C/PLK-1 interaction leads to an imbalance in kinetochore components and a defect in chromosome congression, without affecting CDC-20 recruitment. These findings indicate that PLK-1 kinetochore recruitment by CENP-C has at least partially distinct functions than outer kinetochore PLK-1, providing a platform for a better understanding of the different roles played by PLK-1 during mitosis.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142361604","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}