TY - JOUR AB - The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its closest homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study largely extend the current notions of GN function. We show that GN, but not GNL1, localizes to the cell periphery at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that the cell periphery is the major site of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations en route to the cell periphery. A study of GN-GNL1 chimeric ARF-GEFs indicates that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps toward the elucidation of the mechanism underlying unique cellular and development functions of GNOM. AU - Adamowski, Maciek AU - Matijevic, Ivana AU - Friml, Jiří ID - 15033 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Developmental patterning function of GNOM ARF-GEF mediated from the cell periphery VL - 13 ER - TY - JOUR AB - How to generate a brain of correct size and with appropriate cell-type diversity during development is a major question in Neuroscience. In the developing neocortex, radial glial progenitor (RGP) cells are the main neural stem cells that produce cortical excitatory projection neurons, glial cells, and establish the prospective postnatal stem cell niche in the lateral ventricles. RGPs follow a tightly orchestrated developmental program that when disrupted can result in severe cortical malformations such as microcephaly and megalencephaly. The precise cellular and molecular mechanisms instructing faithful RGP lineage progression are however not well understood. This review will summarize recent conceptual advances that contribute to our understanding of the general principles of RGP lineage progression. AU - Hippenmeyer, Simon ID - 12679 IS - 4 JF - Current Opinion in Neurobiology KW - General Neuroscience SN - 0959-4388 TI - Principles of neural stem cell lineage progression: Insights from developing cerebral cortex VL - 79 ER - TY - JOUR AB - Presynaptic inputs determine the pattern of activation of postsynaptic neurons in a neural circuit. Molecular and genetic pathways that regulate the selective formation of subsets of presynaptic inputs are largely unknown, despite significant understanding of the general process of synaptogenesis. In this study, we have begun to identify such factors using the spinal monosynaptic stretch reflex circuit as a model system. In this neuronal circuit, Ia proprioceptive afferents establish monosynaptic connections with spinal motor neurons that project to the same muscle (termed homonymous connections) or muscles with related or synergistic function. However, monosynaptic connections are not formed with motor neurons innervating muscles with antagonistic functions. The ETS transcription factor ER81 (also known as ETV1) is expressed by all proprioceptive afferents, but only a small set of motor neuron pools in the lumbar spinal cord of the mouse. Here we use conditional mouse genetic techniques to eliminate Er81 expression selectively from motor neurons. We find that ablation of Er81 in motor neurons reduces synaptic inputs from proprioceptive afferents conveying information from homonymous and synergistic muscles, with no change observed in the connectivity pattern from antagonistic proprioceptive afferents. In summary, these findings suggest a role for ER81 in defined motor neuron pools to control the assembly of specific presynaptic inputs and thereby influence the profile of activation of these motor neurons. AU - Ladle, David R. AU - Hippenmeyer, Simon ID - 12562 IS - 3 JF - Journal of Neurophysiology KW - Physiology KW - General Neuroscience SN - 0022-3077 TI - Loss of ETV1/ER81 in motor neurons leads to reduced monosynaptic inputs from proprioceptive sensory neurons VL - 129 ER - TY - JOUR AB - Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice and high-resolution phenotyping at the individual cell level. Here, we present a protocol for isolating MADM-labeled cells with high yield for downstream molecular analyses using fluorescence-activated cell sorting (FACS). We describe steps for generating MADM-labeled mice, perfusion, single-cell suspension, and debris removal. We then detail procedures for cell sorting by FACS and downstream analysis. This protocol is suitable for embryonic to adult mice. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).1 AU - Amberg, Nicole AU - Cheung, Giselle T AU - Hippenmeyer, Simon ID - 14683 IS - 1 JF - STAR Protocols KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Neuroscience SN - 2666-1667 TI - Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry VL - 5 ER - TY - JOUR AB - Studies of protein fitness landscapes reveal biophysical constraints guiding protein evolution and empower prediction of functional proteins. However, generalisation of these findings is limited due to scarceness of systematic data on fitness landscapes of proteins with a defined evolutionary relationship. We characterized the fitness peaks of four orthologous fluorescent proteins with a broad range of sequence divergence. While two of the four studied fitness peaks were sharp, the other two were considerably flatter, being almost entirely free of epistatic interactions. Mutationally robust proteins, characterized by a flat fitness peak, were not optimal templates for machine-learning-driven protein design – instead, predictions were more accurate for fragile proteins with epistatic landscapes. Our work paves insights for practical application of fitness landscape heterogeneity in protein engineering. AU - Gonzalez Somermeyer, Louisa AU - Fleiss, Aubin AU - Mishin, Alexander S AU - Bozhanova, Nina G AU - Igolkina, Anna A AU - Meiler, Jens AU - Alaball Pujol, Maria-Elisenda AU - Putintseva, Ekaterina V AU - Sarkisyan, Karen S AU - Kondrashov, Fyodor ID - 11448 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Heterogeneity of the GFP fitness landscape and data-driven protein design VL - 11 ER - TY - JOUR AB - Empirical essays of fitness landscapes suggest that they may be rugged, that is having multiple fitness peaks. Such fitness landscapes, those that have multiple peaks, necessarily have special local structures, called reciprocal sign epistasis (Poelwijk et al. in J Theor Biol 272:141–144, 2011). Here, we investigate the quantitative relationship between the number of fitness peaks and the number of reciprocal sign epistatic interactions. Previously, it has been shown (Poelwijk et al. in J Theor Biol 272:141–144, 2011) that pairwise reciprocal sign epistasis is a necessary but not sufficient condition for the existence of multiple peaks. Applying discrete Morse theory, which to our knowledge has never been used in this context, we extend this result by giving the minimal number of reciprocal sign epistatic interactions required to create a given number of peaks. AU - Saona Urmeneta, Raimundo J AU - Kondrashov, Fyodor AU - Khudiakova, Kseniia ID - 11447 IS - 8 JF - Bulletin of Mathematical Biology KW - Computational Theory and Mathematics KW - General Agricultural and Biological Sciences KW - Pharmacology KW - General Environmental Science KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Mathematics KW - Immunology KW - General Neuroscience SN - 0092-8240 TI - Relation between the number of peaks and the number of reciprocal sign epistatic interactions VL - 84 ER - TY - JOUR AB - Polygenic adaptation is thought to be ubiquitous, yet remains poorly understood. Here, we model this process analytically, in the plausible setting of a highly polygenic, quantitative trait that experiences a sudden shift in the fitness optimum. We show how the mean phenotype changes over time, depending on the effect sizes of loci that contribute to variance in the trait, and characterize the allele dynamics at these loci. Notably, we describe the two phases of the allele dynamics: The first is a rapid phase, in which directional selection introduces small frequency differences between alleles whose effects are aligned with or opposed to the shift, ultimately leading to small differences in their probability of fixation during a second, longer phase, governed by stabilizing selection. As we discuss, key results should hold in more general settings and have important implications for efforts to identify the genetic basis of adaptation in humans and other species. AU - Hayward, Laura AU - Sella, Guy ID - 12157 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience TI - Polygenic adaptation after a sudden change in environment VL - 11 ER - TY - JOUR AB - Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aβ, the peptide associated with Alzheimer’s disease, the mechanism and rate of aggregation have been established for a range of variants and conditions in vitro and in bodily fluids. A key outstanding question is how the relative stabilities of monomers, fibrils and intermediates affect each step in the fibril formation process. By monitoring the kinetics of aggregation of Aβ42, in the presence of urea or guanidinium hydrochloride (GuHCl), we here determine the rates of the underlying microscopic steps and establish the importance of changes in relative stability induced by the presence of denaturant for each individual step. Denaturants shift the equilibrium towards the unfolded state of each species. We find that a non-ionic denaturant, urea, reduces the overall aggregation rate, and that the effect on nucleation is stronger than the effect on elongation. Urea reduces the rate of secondary nucleation by decreasing the coverage of fibril surfaces and the rate of nucleus formation. It also reduces the rate of primary nucleation, increasing its reaction order. The ionic denaturant, GuHCl, accelerates the aggregation at low denaturant concentrations and decelerates the aggregation at high denaturant concentrations. Below approximately 0.25 M GuHCl, the screening of repulsive electrostatic interactions between peptides by the charged denaturant dominates, leading to an increased aggregation rate. At higher GuHCl concentrations, the electrostatic repulsion is completely screened, and the denaturing effect dominates. The results illustrate how the differential effects of denaturants on stability of monomer, oligomer and fibril translate to differential effects on microscopic steps, with the rate of nucleation being most strongly reduced. AU - Weiffert, Tanja AU - Meisl, Georg AU - Curk, Samo AU - Cukalevski, Risto AU - Šarić, Anđela AU - Knowles, Tuomas P. J. AU - Linse, Sara ID - 12251 JF - Frontiers in Neuroscience KW - General Neuroscience SN - 1662-453X TI - Influence of denaturants on amyloid β42 aggregation kinetics VL - 16 ER - TY - JOUR AB - To understand the function of neuronal circuits, it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low throughput, low selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotropic RVdGenvA-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of ‘starter’ AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the mouse hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo. AU - Sumser, Anton L AU - Jösch, Maximilian A AU - Jonas, Peter M AU - Ben Simon, Yoav ID - 12288 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience TI - Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling VL - 11 ER - TY - JOUR AB - To understand how potential gene manipulations affect in vitro microglia, we provide a set of short protocols to evaluate microglia identity and function. We detail steps for immunostaining to determine microglia identity. We describe three functional assays for microglia: phagocytosis, calcium response following ATP stimulation, and cytokine expression upon inflammatory stimuli. We apply these protocols to human induced-pluripotent-stem-cell (hiPSC)-derived microglia, but they can be also applied to other in vitro microglial models including primary mouse microglia. For complete details on the use and execution of this protocol, please refer to Bartalska et al. (2022).1 AU - Hübschmann, Verena AU - Korkut, Medina AU - Siegert, Sandra ID - 12117 IS - 4 JF - STAR Protocols KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Neuroscience SN - 2666-1667 TI - Assessing human iPSC-derived microglia identity and function by immunostaining, phagocytosis, calcium activity, and inflammation assay VL - 3 ER - TY - JOUR AB - Environmental cues influence the highly dynamic morphology of microglia. Strategies to characterize these changes usually involve user-selected morphometric features, which preclude the identification of a spectrum of context-dependent morphological phenotypes. Here we develop MorphOMICs, a topological data analysis approach, which enables semiautomatic mapping of microglial morphology into an atlas of cue-dependent phenotypes and overcomes feature-selection biases and biological variability. We extract spatially heterogeneous and sexually dimorphic morphological phenotypes for seven adult mouse brain regions. This sex-specific phenotype declines with maturation but increases over the disease trajectories in two neurodegeneration mouse models, with females showing a faster morphological shift in affected brain regions. Remarkably, microglia morphologies reflect an adaptation upon repeated exposure to ketamine anesthesia and do not recover to control morphologies. Finally, we demonstrate that both long primary processes and short terminal processes provide distinct insights to morphological phenotypes. MorphOMICs opens a new perspective to characterize microglial morphology. AU - Colombo, Gloria AU - Cubero, Ryan J AU - Kanari, Lida AU - Venturino, Alessandro AU - Schulz, Rouven AU - Scolamiero, Martina AU - Agerberg, Jens AU - Mathys, Hansruedi AU - Tsai, Li-Huei AU - Chachólski, Wojciech AU - Hess, Kathryn AU - Siegert, Sandra ID - 12244 IS - 10 JF - Nature Neuroscience KW - General Neuroscience SN - 1097-6256 TI - A tool for mapping microglial morphology, morphOMICs, reveals brain-region and sex-dependent phenotypes VL - 25 ER - TY - JOUR AB - Self-assembly of nanoparticles can be mediated by polymers, but has so far led almost exclusively to nanoparticle aggregates that are amorphous. Here, we employed Coulombic interactions to generate a range of composite materials from mixtures of charged nanoparticles and oppositely charged polymers. The assembly behavior of these nanoparticle/polymer composites depends on their order of addition: polymers added to nanoparticles give rise to stable aggregates, but nanoparticles added to polymers disassemble the initially formed aggregates. The amorphous aggregates were transformed into crystalline ones by transiently increasing the ionic strength of the solution. The morphology of the resulting crystals depended on the length of the polymer: short polymer chains mediated the self-assembly of nanoparticles into strongly faceted crystals, whereas long chains led to pseudospherical nanoparticle/polymer assemblies, within which the crystalline order of nanoparticles was retained. AU - Bian, Tong AU - Klajn, Rafal ID - 13356 IS - 1 JF - Annals of the New York Academy of Sciences KW - History and Philosophy of Science KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Neuroscience SN - 0077-8923 TI - Morphology control in crystalline nanoparticle–polymer aggregates VL - 1505 ER - TY - JOUR AB - De novo protein synthesis is required for synapse modifications underlying stable memory encoding. Yet neurons are highly compartmentalized cells and how protein synthesis can be regulated at the synapse level is unknown. Here, we characterize neuronal signaling complexes formed by the postsynaptic scaffold GIT1, the mechanistic target of rapamycin (mTOR) kinase, and Raptor that couple synaptic stimuli to mTOR-dependent protein synthesis; and identify NMDA receptors containing GluN3A subunits as key negative regulators of GIT1 binding to mTOR. Disruption of GIT1/mTOR complexes by enhancing GluN3A expression or silencing GIT1 inhibits synaptic mTOR activation and restricts the mTOR-dependent translation of specific activity-regulated mRNAs. Conversely, GluN3A removal enables complex formation, potentiates mTOR-dependent protein synthesis, and facilitates the consolidation of associative and spatial memories in mice. The memory enhancement becomes evident with light or spaced training, can be achieved by selectively deleting GluN3A from excitatory neurons during adulthood, and does not compromise other aspects of cognition such as memory flexibility or extinction. Our findings provide mechanistic insight into synaptic translational control and reveal a potentially selective target for cognitive enhancement. AU - Conde-Dusman, María J AU - Dey, Partha N AU - Elía-Zudaire, Óscar AU - Garcia Rabaneda, Luis E AU - García-Lira, Carmen AU - Grand, Teddy AU - Briz, Victor AU - Velasco, Eric R AU - Andero Galí, Raül AU - Niñerola, Sergio AU - Barco, Angel AU - Paoletti, Pierre AU - Wesseling, John F AU - Gardoni, Fabrizio AU - Tavalin, Steven J AU - Perez-Otaño, Isabel ID - 10301 JF - eLife KW - general immunology and microbiology KW - general biochemistry KW - genetics and molecular biology KW - general medicine KW - general neuroscience SN - 2050-084X TI - Control of protein synthesis and memory by GluN3A-NMDA receptors through inhibition of GIT1/mTORC1 assembly VL - 10 ER - TY - JOUR AB - The sensory and cognitive abilities of the mammalian neocortex are underpinned by intricate columnar and laminar circuits formed from an array of diverse neuronal populations. One approach to determining how interactions between these circuit components give rise to complex behavior is to investigate the rules by which cortical circuits are formed and acquire functionality during development. This review summarizes recent research on the development of the neocortex, from genetic determination in neural stem cells through to the dynamic role that specific neuronal populations play in the earliest circuits of neocortex, and how they contribute to emergent function and cognition. While many of these endeavors take advantage of model systems, consideration will also be given to advances in our understanding of activity in nascent human circuits. Such cross-species perspective is imperative when investigating the mechanisms underlying the dysfunction of early neocortical circuits in neurodevelopmental disorders, so that one can identify targets amenable to therapeutic intervention. AU - Hanganu-Opatz, Ileana L. AU - Butt, Simon J. B. AU - Hippenmeyer, Simon AU - De Marco García, Natalia V. AU - Cardin, Jessica A. AU - Voytek, Bradley AU - Muotri, Alysson R. ID - 9073 IS - 5 JF - The Journal of Neuroscience KW - General Neuroscience SN - 0270-6474 TI - The logic of developing neocortical circuits in health and disease VL - 41 ER - TY - JOUR AB - Vascular dysfunctions are a common feature of multiple age-related diseases. However, modeling healthy and pathological aging of the human vasculature represents an unresolved experimental challenge. Here, we generated induced vascular endothelial cells (iVECs) and smooth muscle cells (iSMCs) by direct reprogramming of healthy human fibroblasts from donors of different ages and Hutchinson-Gilford Progeria Syndrome (HGPS) patients. iVECs induced from old donors revealed upregulation of GSTM1 and PALD1, genes linked to oxidative stress, inflammation and endothelial junction stability, as vascular aging markers. A functional assay performed on PALD1 KD VECs demonstrated a recovery in vascular permeability. We found that iSMCs from HGPS donors overexpressed bone morphogenetic protein (BMP)−4, which plays a key role in both vascular calcification and endothelial barrier damage observed in HGPS. Strikingly, BMP4 concentrations are higher in serum from HGPS vs. age-matched mice. Furthermore, targeting BMP4 with blocking antibody recovered the functionality of the vascular barrier in vitro, hence representing a potential future therapeutic strategy to limit cardiovascular dysfunction in HGPS. These results show that iVECs and iSMCs retain disease-related signatures, allowing modeling of vascular aging and HGPS in vitro. AU - Bersini, Simone AU - Schulte, Roberta AU - Huang, Ling AU - Tsai, Hannah AU - HETZER, Martin W ID - 11055 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Direct reprogramming of human smooth muscle and vascular endothelial cells reveals defects associated with aging and Hutchinson-Gilford progeria syndrome VL - 9 ER - TY - JOUR AB - In recent years, the nuclear pore complex (NPC) has emerged as a key player in genome regulation and cellular homeostasis. New discoveries have revealed that the NPC has multiple cellular functions besides mediating the molecular exchange between the nucleus and the cytoplasm. In this review, we discuss non-transport aspects of the NPC focusing on the NPC-genome interaction, the extreme longevity of the NPC proteins, and NPC dysfunction in age-related diseases. The examples summarized herein demonstrate that the NPC, which first evolved to enable the biochemical communication between the nucleus and the cytoplasm, now doubles as the gatekeeper of cellular identity and aging. AU - Cho, Ukrae H. AU - HETZER, Martin W ID - 11054 IS - 6 JF - Neuron KW - General Neuroscience SN - 0896-6273 TI - Nuclear periphery takes center stage: The role of nuclear pore complexes in cell identity and aging VL - 106 ER - TY - JOUR AB - Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1. AU - Fribourgh, Jennifer L AU - Srivastava, Ashutosh AU - Sandate, Colby R AU - Michael, Alicia Kathleen AU - Hsu, Peter L AU - Rakers, Christin AU - Nguyen, Leslee T AU - Torgrimson, Megan R AU - Parico, Gian Carlo G AU - Tripathi, Sarvind AU - Zheng, Ning AU - Lander, Gabriel C AU - Hirota, Tsuyoshi AU - Tama, Florence AU - Partch, Carrie L ID - 15153 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing VL - 9 ER - TY - JOUR AB - Transposable elements (TEs), the movement of which can damage the genome, are epigenetically silenced in eukaryotes. Intriguingly, TEs are activated in the sperm companion cell – vegetative cell (VC) – of the flowering plant Arabidopsis thaliana. However, the extent and mechanism of this activation are unknown. Here we show that about 100 heterochromatic TEs are activated in VCs, mostly by DEMETER-catalyzed DNA demethylation. We further demonstrate that DEMETER access to some of these TEs is permitted by the natural depletion of linker histone H1 in VCs. Ectopically expressed H1 suppresses TEs in VCs by reducing DNA demethylation and via a methylation-independent mechanism. We demonstrate that H1 is required for heterochromatin condensation in plant cells and show that H1 overexpression creates heterochromatic foci in the VC progenitor cell. Taken together, our results demonstrate that the natural depletion of H1 during male gametogenesis facilitates DEMETER-directed DNA demethylation, heterochromatin relaxation, and TE activation. AU - He, Shengbo AU - Vickers, Martin AU - Zhang, Jingyi AU - Feng, Xiaoqi ID - 12192 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Natural depletion of histone H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation VL - 8 ER - TY - JOUR AB - The inner nuclear membrane (INM) is a subdomain of the endoplasmic reticulum (ER) that is gated by the nuclear pore complex. It is unknown whether proteins of the INM and ER are degraded through shared or distinct pathways in mammalian cells. We applied dynamic proteomics to profile protein half-lives and report that INM and ER residents turn over at similar rates, indicating that the INM’s unique topology is not a barrier to turnover. Using a microscopy approach, we observed that the proteasome can degrade INM proteins in situ. However, we also uncovered evidence for selective, vesicular transport-mediated turnover of a single INM protein, emerin, that is potentiated by ER stress. Emerin is rapidly cleared from the INM by a mechanism that requires emerin’s LEM domain to mediate vesicular trafficking to lysosomes. This work demonstrates that the INM can be dynamically remodeled in response to environmental inputs. AU - Buchwalter, Abigail AU - Schulte, Roberta AU - Tsai, Hsiao AU - Capitanio, Juliana AU - HETZER, Martin W ID - 11060 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Selective clearance of the inner nuclear membrane protein emerin by vesicular transport during ER stress VL - 8 ER - TY - JOUR AB - Eukaryotic cells are densely packed with macromolecular complexes and intertwining organelles, continually transported and reshaped. Intriguingly, organelles avoid clashing and entangling with each other in such limited space. Mitochondria form extensive networks constantly remodeled by fission and fusion. Here, we show that mitochondrial fission is triggered by mechanical forces. Mechano-stimulation of mitochondria – via encounter with motile intracellular pathogens, via external pressure applied by an atomic force microscope, or via cell migration across uneven microsurfaces – results in the recruitment of the mitochondrial fission machinery, and subsequent division. We propose that MFF, owing to affinity for narrow mitochondria, acts as a membrane-bound force sensor to recruit the fission machinery to mechanically strained sites. Thus, mitochondria adapt to the environment by sensing and responding to biomechanical cues. Our findings that mechanical triggers can be coupled to biochemical responses in membrane dynamics may explain how organelles orderly cohabit in the crowded cytoplasm. AU - Helle, Sebastian Carsten Johannes AU - Feng, Qian AU - Aebersold, Mathias J AU - Hirt, Luca AU - Grüter, Raphael R AU - Vahid, Afshin AU - Sirianni, Andrea AU - Mostowy, Serge AU - Snedeker, Jess G AU - Šarić, Anđela AU - Idema, Timon AU - Zambelli, Tomaso AU - Kornmann, Benoît ID - 10370 JF - eLife KW - general immunology and microbiology KW - general biochemistry KW - genetics and molecular biology KW - general medicine KW - general neuroscience SN - 2050-084X TI - Mechanical force induces mitochondrial fission VL - 6 ER - TY - JOUR AB - Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity. AU - Fong, Jiunn CN AU - Rogers, Andrew AU - Michael, Alicia Kathleen AU - Parsley, Nicole C AU - Cornell, William-Cole AU - Lin, Yu-Cheng AU - Singh, Praveen K AU - Hartmann, Raimo AU - Drescher, Knut AU - Vinogradov, Evgeny AU - Dietrich, Lars EP AU - Partch, Carrie L AU - Yildiz, Fitnat H ID - 15154 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience SN - 2050-084X TI - Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms VL - 6 ER -