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Journal of Cell Biology
Journal Prestige (SJR): 6.479  Citation Impact (citeScore): 7 Number of Followers: 55  Subscription journalISSN (Print) 0021-9525 - ISSN (Online) 1540-8140 Published by Rockefeller University Press  [3 journals]
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- Correction: The UVSSA complex alleviates MYC-driven transcription stress
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Abstract: Vol. 220, No. 2 10.1083/jcb.201807163 January 6, 2021
PubDate: Thu, 23 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.20180716302092023c
Issue No: Vol. 222, No. 3 (2023)
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- Mitochondrial membrane biogenesis: A new pathway for lipid transport
mediated by PERK/E-Syt1 complex-
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Abstract: Despite decades of extensive research, mitochondrial lipid transport is a process far from fully understood. In this issue, Sassano et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202206008) identified a new complex, composed of E-Syt1 and PERK, which mediates lipid transport at ER–mitochondria contact sites and regulates mitochondrial functions in human cells.
PubDate: Thu, 23 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202301132
Issue No: Vol. 222, No. 3 (2023)
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- PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid
transport and respiration-
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Abstract: The integrity of ER–mitochondria appositions ensures transfer of ions and phospholipids (PLs) between these organelles and exerts crucial effects on mitochondrial bioenergetics. Malfunctions within the ER–mitochondria contacts altering lipid trafficking homeostasis manifest in diverse pathologies, but the molecular effectors governing this process remain ill-defined. Here, we report that PERK promotes lipid trafficking at the ER–mitochondria contact sites (EMCS) through a non-conventional, unfolded protein response-independent, mechanism. PERK operates as an adaptor for the recruitment of the ER–plasma membrane tether and lipid transfer protein (LTP) Extended-Synaptotagmin 1 (E-Syt1), within the EMCS. In resting cells, the heterotypic E-Syt1-PERK interaction endorses transfer of PLs between the ER and mitochondria. Weakening the E-Syt1-PERK interaction or removing the lipid transfer SMP-domain of E-Syt1, compromises mitochondrial respiration. Our findings unravel E-Syt1 as a PERK interacting LTP and molecular component of the lipid trafficking machinery of the EMCS, which critically maintains mitochondrial homeostasis and fitness.
PubDate: Thu, 23 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202206008
Issue No: Vol. 222, No. 3 (2023)
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- The septin cytoskeleton: Heteromer composition defines filament function
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Abstract: Septins are an evolutionarily conserved protein family whose members form hetero-oligomeric complexes that assemble into filaments and higher-order structures. In this issue, Martins et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202203016) and Cannon et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202204063) report that heteromer composition impacts the physiological role of septin filaments in yeast and human cells.
PubDate: Thu, 23 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202302010
Issue No: Vol. 222, No. 3 (2023)
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- Judith Agudo: Beware of your inner self-immune attack
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Abstract: Judith Agudo studies the mechanisms that adult and cancer stem cells use to evade the immune response with the goals of engineering autoimmunity- and allograft-resistant stem cells and improving the response of cancer stem cells to immunotherapy.
PubDate: Tue, 21 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202302027
Issue No: Vol. 222, No. 3 (2023)
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- An in vivo phosphoregulation paradox for focal adhesions
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Abstract: Focal adhesions (FAs) dynamics regulate single cell migration. In this issue, Xue et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202206078) show that Y118 phosphorylation on Paxilin, a key FA protein, limits migration of cells in vivo. Unphosphorylated Paxilin is necessary for FA disassembly and cell motility. Their findings directly contradict results from in vitro experiments, emphasizing the need for recreating the in vivo complexity to understand how cells behave in their native environments.
PubDate: Thu, 16 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202301060
Issue No: Vol. 222, No. 3 (2023)
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- Mitochondria on the move: Horizontal mitochondrial transfer in disease and
health-
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Abstract: Mammalian genes were long thought to be constrained within somatic cells in most cell types. This concept was challenged recently when cellular organelles including mitochondria were shown to move between mammalian cells in culture via cytoplasmic bridges. Recent research in animals indicates transfer of mitochondria in cancer and during lung injury in vivo, with considerable functional consequences. Since these pioneering discoveries, many studies have confirmed horizontal mitochondrial transfer (HMT) in vivo, and its functional characteristics and consequences have been described. Additional support for this phenomenon has come from phylogenetic studies. Apparently, mitochondrial trafficking between cells occurs more frequently than previously thought and contributes to diverse processes including bioenergetic crosstalk and homeostasis, disease treatment and recovery, and development of resistance to cancer therapy. Here we highlight current knowledge of HMT between cells, focusing primarily on in vivo systems, and contend that this process is not only (patho)physiologically relevant, but also can be exploited for the design of novel therapeutic approaches.
PubDate: Thu, 16 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202211044
Issue No: Vol. 222, No. 3 (2023)
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- Plastin 3 rescues BDNF-TrkB signaling in spinal muscular atrophy
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Abstract: In this issue, Hennlein and colleagues (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202204113) show that F-actin–bundling protein Plastin 3 is drastically reduced in motor neurons with spinal muscular atrophy, whereas virus-mediated overexpression of Plastin 3 restores actin cytoskeleton and promotes BDNF–TrkB signaling in the growth cones of spinal motor neurons.
PubDate: Tue, 14 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202301036
Issue No: Vol. 222, No. 3 (2023)
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- A gene duplication of a septin reveals a developmentally regulated
filament length control mechanism-
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Abstract: Septins are a family of conserved filament-forming proteins that function in multiple cellular processes. The number of septin genes within an organism varies, and higher eukaryotes express many septin isoforms due to alternative splicing. It is unclear if different combinations of septin proteins in complex alter the polymers’ biophysical properties. We report that a duplication event within the CDC11 locus in Ashbya gossypii gave rise to two similar but distinct Cdc11 proteins: Cdc11a and Cdc1b. CDC11b transcription is developmentally regulated, producing different amounts of Cdc11a- and Cdc11b-complexes in the lifecycle of Ashbya gossypii. Deletion of either gene results in distinct cell polarity defects, suggesting non-overlapping functions. Cdc11a and Cdc11b complexes have differences in filament length and membrane-binding ability. Thus, septin subunit composition has functional consequences on filament properties and cell morphogenesis. Small sequence differences elicit distinct biophysical properties and cell functions of septins, illuminating how gene duplication could be a driving force for septin gene expansions seen throughout the tree of life.
PubDate: Tue, 14 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202204063
Issue No: Vol. 222, No. 3 (2023)
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- Clathrin coats partially preassemble and subsequently bend during
endocytosis-
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Abstract: Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle.Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area before they become rapidly and continuously bent, and this mechanism is confirmed in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane.
PubDate: Fri, 03 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202206038
Issue No: Vol. 222, No. 3 (2023)
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- Allosteric regulation of exocyst: Discrete activation of tethering by two
spatial signals-
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Abstract: The exocyst imparts spatial control during exocytic vesicle tethering through its interactions with proteins and lipids on the vesicle and the plasma membrane. One such interaction is with the vesicle tether Sro7, although the outcome of this interaction is poorly understood. Here, we describe how Sro7 binding to the Exo84 subunit results in activation of the exocyst complex which leads to an increase in avidity for the Rab GTPase Sec4 and an increase in exocyst-mediated vesicle tethering. Gain-of-function (GOF) mutations in Exo84 that mimic Sro7 activation replicate these biochemical changes and result in allosteric changes within the complex. Direct comparison of GOF mutants which mimic Sro7- and Rho/Cdc42-activation of the exocyst reveals distinct mechanisms and outcomes. We propose a model by which these two activation pathways reside within the same tethering complex but remain insulated from one another. Structural modeling suggests a related mechanism for Sro7 activation of the exocyst in yeast and Ral GTPase activation of the exocyst in animal cells.
PubDate: Thu, 02 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202206108
Issue No: Vol. 222, No. 3 (2023)
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- Lack of Paxillin phosphorylation promotes single-cell migration in vivo
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Abstract: Focal adhesions are structures that physically link the cell to the extracellular matrix for cell migration. Although cell culture studies have provided a wealth of information regarding focal adhesion biology, it is critical to understand how focal adhesions are dynamically regulated in their native environment. We developed a zebrafish system to visualize focal adhesion structures during single-cell migration in vivo. We find that a key site of phosphoregulation (Y118) on Paxillin exhibits reduced phosphorylation in migrating cells in vivo compared to in vitro. Furthermore, expression of a non-phosphorylatable version of Y118-Paxillin increases focal adhesion disassembly and promotes cell migration in vivo, despite inhibiting cell migration in vitro. Using a mouse model, we further find that the upstream kinase, focal adhesion kinase, is downregulated in cells in vivo, and cells expressing non-phosphorylatable Y118-Paxillin exhibit increased activation of the CRKII-DOCK180/RacGEF pathway. Our findings provide significant new insight into the intrinsic regulation of focal adhesions in cells migrating in their native environment.
PubDate: Wed, 01 Feb 2023 00:00:00 GMT
DOI: 10.1083/jcb.202206078
Issue No: Vol. 222, No. 3 (2023)
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- Tubulin isotype regulation maintains asymmetric requirement for
α-tubulin over β-tubulin-
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Abstract: How cells regulate α- and β-tubulin to meet the demand for αβ-heterodimers and avoid consequences of monomer imbalance is not understood. We investigate the role of gene copy number and how shifting expression of α- or β-tubulin genes impacts tubulin proteostasis and microtubule function in Saccharomyces cerevisiae. We find that α-tubulin gene copy number is important for maintaining excess α-tubulin protein compared to β-tubulin protein. Excess α-tubulin prevents accumulation of super-stoichiometric β-tubulin, which leads to loss of microtubules, formation of non-microtubule assemblies of tubulin, and disrupts cell proliferation. In contrast, sub-stoichiometric β-tubulin or overexpression of α-tubulin has minor effects. We provide evidence that yeast cells equilibrate α-tubulin protein concentration when α-tubulin isotype expression is increased. We propose an asymmetric relationship between α- and β-tubulins, in which α-tubulins are maintained in excess to supply αβ-heterodimers and limit the accumulation of β-tubulin monomers.
PubDate: Tue, 31 Jan 2023 00:00:00 GMT
DOI: 10.1083/jcb.202202102
Issue No: Vol. 222, No. 3 (2023)
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- Nuclear-enriched protein phosphatase 4 ensures outer kinetochore assembly
prior to nuclear dissolution-
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Abstract: A landmark event in the transition from interphase to mitosis in metazoans is nuclear envelope breakdown (NEBD). Important mitotic events occur prior to NEBD, including condensation of replicated chromosomes and assembly of kinetochores to rapidly engage spindle microtubules. Here, we show that nuclear-enriched protein phosphatase 4 (PP4) ensures robust assembly of the microtubule-coupling outer kinetochore prior to NEBD. In the absence of PP4, chromosomes exhibit extended monopolar orientation after NEBD and subsequently mis-segregate. A secondary consequence of diminished outer kinetochore assembly is defective sister chromatid resolution. After NEBD, a cytoplasmic activity compensates for PP4 loss, leading to outer kinetochore assembly and recovery of chromosomes from monopolar orientation to significant bi-orientation. The Ndc80-Ska microtubule-binding module of the outer kinetochore is required for this recovery. PP4 associates with the inner kinetochore protein CENP-C; however, disrupting the PP4–CENP-C interaction does not perturb chromosome segregation. These results establish that PP4-dependent outer kinetochore assembly prior to NEBD is critical for timely and proper engagement of chromosomes with spindle microtubules.
PubDate: Tue, 31 Jan 2023 00:00:00 GMT
DOI: 10.1083/jcb.202208154
Issue No: Vol. 222, No. 3 (2023)
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- A nucleotide binding–independent role for γ-tubulin in microtubule
capping and cell division-
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Abstract: The γ-tubulin ring complex (γ-TuRC) has essential roles in centrosomal and non-centrosomal microtubule organization during vertebrate mitosis. While there have been important advances in understanding γ-TuRC-dependent microtubule nucleation, γ-TuRC capping of microtubule minus-ends remains poorly characterized. Here, we utilized biochemical reconstitutions and cellular assays to characterize the human γ-TuRC’s capping activity. Single filament assays showed that the γ-TuRC remained associated with a nucleated microtubule for tens of minutes. In contrast, caps at dynamic microtubule minus-ends displayed lifetimes of ∼1 min. Reconstituted γ-TuRCs with nucleotide-binding deficient γ-tubulin (γ-tubulinΔGTP) formed ring-shaped complexes that did not nucleate microtubules but capped microtubule minus-ends with lifetimes similar to those measured for wild-type complexes. In dividing cells, microtubule regrowth assays revealed that while knockdown of γ-tubulin suppressed non-centrosomal microtubule formation, add-back of γ-tubulinΔGTP could substantially restore this process. Our results suggest that γ-TuRC capping is a nucleotide-binding-independent activity that plays a role in non-centrosomal microtubule organization during cell division.
PubDate: Wed, 25 Jan 2023 00:00:00 GMT
DOI: 10.1083/jcb.202204102
Issue No: Vol. 222, No. 3 (2023)
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- Developmental pruning of sensory neurites by mechanical tearing in
Drosophila-
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Abstract: Mechanical forces actively shape cells during development, but little is known about their roles during neuronal morphogenesis. Developmental neurite pruning, a critical circuit specification mechanism, often involves neurite abscission at predetermined sites by unknown mechanisms. Pruning of Drosophila sensory neuron dendrites during metamorphosis is triggered by the hormone ecdysone, which induces local disassembly of the dendritic cytoskeleton. Subsequently, dendrites are severed at positions close to the soma by an unknown mechanism. We found that ecdysone signaling causes the dendrites to become mechanically fragile. Severing occurs during periods of increased pupal morphogenetic tissue movements, which exert mechanical forces on the destabilized dendrites. Tissue movements and dendrite severing peak during pupal ecdysis, a period of strong abdominal contractions, and abolishing ecdysis causes non-cell autonomous dendrite pruning defects. Thus, our data establish mechanical tearing as a novel mechanism during neurite pruning.
PubDate: Tue, 17 Jan 2023 00:00:00 GMT
DOI: 10.1083/jcb.202205004
Issue No: Vol. 222, No. 3 (2023)
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- Myelination generates aberrant ultrastructure that is resolved by
microglia-
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Abstract: To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one “eat me” signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement.
PubDate: Fri, 13 Jan 2023 00:00:00 GMT
DOI: 10.1083/jcb.202204010
Issue No: Vol. 222, No. 3 (2023)
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- Meiotic resetting of the cellular Sod1 pool is driven by protein
aggregation, degradation, and transient LUTI-mediated repression-
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Abstract: Gametogenesis requires packaging of the cellular components needed for the next generation. In budding yeast, this process includes degradation of many mitotically stable proteins, followed by their resynthesis. Here, we show that one such case—Superoxide dismutase 1 (Sod1), a protein that commonly aggregates in human ALS patients—is regulated by an integrated set of events, beginning with the formation of pre-meiotic Sod1 aggregates. This is followed by degradation of a subset of the prior Sod1 pool and clearance of Sod1 aggregates. As degradation progresses, Sod1 protein production is transiently blocked during mid-meiotic stages by transcription of an extended and poorly translated SOD1 mRNA isoform, SOD1LUTI. Expression of SOD1LUTI is induced by the Unfolded Protein Response, and it acts to repress canonical SOD1 mRNA expression. SOD1LUTI is no longer expressed following the meiotic divisions, enabling a resurgence of canonical mRNA and synthesis of new Sod1 protein such that gametes inherit a full complement of Sod1 protein. Failure to aggregate and degrade Sod1 results in reduced gamete fitness in the presence of oxidants, highlighting the importance of this regulation. Investigation of Sod1 during yeast gametogenesis, an unusual cellular context in which Sod1 levels are tightly regulated, could shed light on conserved aspects of its aggregation and degradation, with relevance to understanding Sod1’s role in human disease.
PubDate: Mon, 09 Jan 2023 00:00:00 GMT
DOI: 10.1083/jcb.202206058
Issue No: Vol. 222, No. 3 (2023)
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- Plastin 3 rescues cell surface translocation and activation of TrkB in
spinal muscular atrophy-
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Abstract: Plastin 3 (PLS3) is an F-actin-bundling protein that has gained attention as a modifier of spinal muscular atrophy (SMA) pathology. SMA is a lethal pediatric neuromuscular disease caused by loss of or mutations in the Survival Motor Neuron 1 (SMN1) gene. Pathophysiological hallmarks are cellular maturation defects of motoneurons prior to degeneration. Despite the observed beneficial modifying effect of PLS3, the mechanism of how it supports F-actin-mediated cellular processes in motoneurons is not yet well understood. Our data reveal disturbed F-actin-dependent translocation of the Tropomyosin receptor kinase B (TrkB) to the cell surface of Smn-deficient motor axon terminals, resulting in reduced TrkB activation by its ligand brain-derived neurotrophic factor (BDNF). Improved actin dynamics by overexpression of hPLS3 restores membrane recruitment and activation of TrkB and enhances spontaneous calcium transients by increasing Cav2.1/2 “cluster-like” formations in SMA axon terminals. Thus, our study provides a novel role for PLS3 in supporting correct alignment of transmembrane proteins, a key mechanism for (moto)-neuronal development.
PubDate: Fri, 06 Jan 2023 00:00:00 GMT
DOI: 10.1083/jcb.202204113
Issue No: Vol. 222, No. 3 (2023)
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- A covalently linked probe to monitor local membrane properties surrounding
plasma membrane proteins-
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Abstract: Functional membrane proteins in the plasma membrane are suggested to have specific membrane environments that play important roles to maintain and regulate their function. However, the local membrane environments of membrane proteins remain largely unexplored due to the lack of available techniques. We have developed a method to probe the local membrane environment surrounding membrane proteins in the plasma membrane by covalently tethering a solvatochromic, environment-sensitive dye, Nile Red, to a GPI-anchored protein and the insulin receptor through a flexible linker. The fluidity of the membrane environment of the GPI-anchored protein depended upon the saturation of the acyl chains of the lipid anchor. The local environment of the insulin receptor was distinct from the average plasma membrane fluidity and was quite dynamic and heterogeneous. Upon addition of insulin, the local membrane environment surrounding the receptor specifically increased in fluidity in an insulin receptor-kinase dependent manner and on the distance between the dye and the receptor.
PubDate: Mon, 26 Dec 2022 00:00:00 GMT
DOI: 10.1083/jcb.202206119
Issue No: Vol. 222, No. 3 (2022)
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- CLEM Site , a software for automated phenotypic screens using light
microscopy and FIB-SEM-
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Abstract: In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume ultrastructural imaging. We present a toolset for adherent cells that enables tracking and finding cells, previously identified in light microscopy (LM), in the FIB-SEM, along with the automatic acquisition of high-resolution volume datasets. We detect the underlying grid pattern in both modalities (LM and EM), to identify common reference points. A combination of computer vision techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope’s field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 µm accuracy while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing volume acquisition in multiple locations autonomously is possible in EM.
PubDate: Fri, 23 Dec 2022 00:00:00 GMT
DOI: 10.1083/jcb.202209127
Issue No: Vol. 222, No. 3 (2022)
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- Human septins organize as octamer-based filaments and mediate
actin-membrane anchoring in cells-
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Abstract: Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin–membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.
PubDate: Fri, 23 Dec 2022 00:00:00 GMT
DOI: 10.1083/jcb.202203016
Issue No: Vol. 222, No. 3 (2022)
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- Kinesin-1 autoinhibition facilitates the initiation of dynein cargo
transport-
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Abstract: The functional significance of Kinesin-1 autoinhibition has been unclear. Kinesin-1 transports multiple cargoes including cytoplasmic dynein to microtubule plus ends. From a genetic screen for Aspergillus mutants defective in dynein-mediated early endosome transport, we identified a kinesin-1 mutation kinAK895* at the C-terminal IAK motif involved in autoinhibition. The kinA∆IAK and kinAK895E mutants exhibited a similar defect in dynein-mediated early endosome transport, verifying the importance of kinesin-1 autoinhibition in dynein-mediated transport. Kinesin-1 autoinhibition is not critical for dynein accumulation at microtubule plus ends or for the secretory vesicle cargoes of kinesin-1 to reach the hyphal tip. However, it facilitates dynein to initiate early endosome transport. This is unrelated to a direct competition between dynein and kinesin-1 on early endosomes because kinesin-3 rather than kinesin-1 drives the plus-end-directed early endosome movement. This effect of kinesin-1 autoinhibition on dynein-mediated early endosome transport is related to cargo adapter-mediated dynein activation but at a step beyond the switching of dynein from its autoinhibited conformation.
PubDate: Fri, 16 Dec 2022 00:00:00 GMT
DOI: 10.1083/jcb.202205136
Issue No: Vol. 222, No. 3 (2022)
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- Hyperstabilization of T cell microvilli contacts by chimeric antigen
receptors-
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Abstract: T cells typically recognize their ligands using a defined cell biology—the scanning of their membrane microvilli (MV) to palpate their environment—while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.
PubDate: Thu, 15 Dec 2022 00:00:00 GMT
DOI: 10.1083/jcb.202205118
Issue No: Vol. 222, No. 3 (2022)
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