Abstract
To navigate through diverse tissues, migrating cells must balance persistent self-propelled motion with adaptive behaviors to circumvent obstacles. We identify a curvature-sensing mechanism ...underlying obstacle evasion in immune-like cells. Specifically, we propose that actin polymerization at the advancing edge of migrating cells is inhibited by the curvature-sensitive BAR domain protein Snx33 in regions with inward plasma membrane curvature. The genetic perturbation of this machinery reduces the cells’ capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. Our results show how cells can read out their surface topography and utilize actin and plasma membrane biophysics to interpret their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR domain proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment.
Besides digesting nutrients, the gut protects the host against invasion by pathogens. Enterocytes may be subjected to damage by both microbial and host defensive responses, causing their death. Here, ...we report a rapid epithelial response that alleviates infection stress and protects the enterocytes from the action of microbial virulence factors. Intestinal epithelia exposed to hemolysin, a pore-forming toxin secreted by Serratia marcescens, undergo an evolutionarily conserved process of thinning followed by the recovery of their initial thickness within a few hours. In response to hemolysin attack, Drosophila melanogaster enterocytes extrude most of their apical cytoplasm, including damaged organelles such as mitochondria, yet do not lyse. We identify two secreted peptides, the expression of which requires CyclinJ, that mediate the recovery phase in which enterocytes regain their original shape and volume. Epithelial thinning and recovery constitute a fast and efficient response to intestinal infections, with pore-forming toxins acting as alarm signals.
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•A pore-forming toxin induces the thinning of both insect and mammalian gut epithelium•Thinning of enterocytes occurs by the limited extrusion of their apical cytoplasm•Extrusion protects enterocytes by purging damaged components and invading bacteria•Enterocyte recovery involves CyclinJ-dependent signaling between enterocytes
Bacterial pore-forming toxins damage host cells. Lee, Lestradet et al. find that enterocytes exposed to these toxins extrude much of their apical cytoplasm, purging themselves of damaged components and bacteria. The thinned epithelium recovers its original shape within hours via a process that requires CyclinJ-dependent signaling between enterocytes.
Three-dimensional live imaging has become an indispensable technique in the fields of cell, developmental and neural biology. Precise spatio-temporal manipulation of biological entities is often ...required for a deeper functional understanding of the underlying biological process. Here we present a home-built integrated framework and optical design that combines three-dimensional light-sheet imaging over time with precise spatio-temporal optical manipulations induced by short infrared laser pulses. We demonstrate their potential for sub-cellular ablation of neurons and nuclei, tissue cauterization and optogenetics by using the Drosophila melanogaster and zebrafish model systems.
Understanding transdifferentiation--the conversion of one differentiated cell type into another--is important from both basic science and clinical perspectives. In Caenorhabditis elegans, an ...epithelial cell named Y is initially part of the rectum but later appears to withdraw, migrate, and then become a motor neuron named PDA. Here, we show that this represents a bona fide transdifferentiation event: Y has epithelial hallmarks without detectable neural characteristics, and PDA has no residual epithelial characteristics. Using available mutants and laser microsurgery, we found that transdifferentiation does not depend on fusion with a neighboring cell or require migration of Y away from the rectum, that other rectal epithelial cells are not competent to transdifferentiate, and that transdifferentiation requires the EGL-5 and SEM-4 transcription factors and LIN-12/Notch signaling. Our results establish Y-to-PDA transdifferentiation as a genetically tractable model for deciphering the mechanisms underlying cellular plasticity in vivo.
Here, high‐throughput tomography (HiTT), a fast and versatile phase‐contrast imaging platform for life‐science samples on the EMBL beamline P14 at DESY in Hamburg, Germany, is presented. A ...high‐photon‐flux undulator beamline is used to perform tomographic phase‐contrast acquisition in about two minutes which is linked to an automated data processing pipeline that delivers a 3D reconstructed data set less than a minute and a half after the completion of the X‐ray scan. Combining this workflow with a sophisticated robotic sample changer enables the streamlined collection and reconstruction of X‐ray imaging data from potentially hundreds of samples during a beam‐time shift. HiTT permits optimal data collection for many different samples and makes possible the imaging of large sample cohorts thus allowing population studies to be attempted. The successful application of HiTT on various soft tissue samples in both liquid (hydrated and also dehydrated) and paraffin‐embedded preparations is demonstrated. Furthermore, the feasibility of HiTT to be used as a targeting tool for volume electron microscopy, as well as using HiTT to study plant morphology, is demonstrated. It is also shown how the high‐throughput nature of the work has allowed large numbers of `identical' samples to be imaged to enable statistically relevant sample volumes to be studied.
High‐throughput tomography, a propagation‐based phase‐contrast X‐ray imaging technique that can visualize 1 mm3 biological samples of various types at high resolution, is presented. 3D reconstructions of the imaged volumes are calculated automatically once data collection is complete. The entire process from pressing start on data collection to viewing the final data takes less than three minutes. This speed, in combination with the use of an automated sample changer to exchange the samples, truly enables high‐throughput X‐ray imaging for the first time.
Polarized intracellular trafficking in epithelia is critical in development, immunity, and physiology to deliver morphogens, defensins, or ion pumps to the appropriate membrane domain. The mechanisms ...that control apical trafficking remain poorly defined. Using Caenorhabditis elegans, we characterize a novel apical secretion pathway involving multivesicularbodies and the release of exosomes at the apical plasma membrane. By means of two different genetic approaches, we show that the membrane-bound V0 sector of the vacuolar H⁺-ATPase (V-ATPase) acts in this pathway, independent of its contribution to the V-ATPase proton pump activity. Specifically, we identified mutations in the V0 "a" subunit VHA-5 that affect either the V0-specific function or the V0+V1 function of the V-ATPase. These mutations allowed us to establish that the V0 sector mediates secretion of Hedgehog-related proteins. Our data raise the possibility that the V0 sector mediates exosome and morphogen release in mammals.
Centriole elimination is an essential process that occurs in female meiosis of metazoa to reset centriole number in the zygote at fertilization. How centrioles are eliminated remains poorly ...understood. Here we visualize the entire elimination process live in starfish oocytes. Using specific fluorescent markers, we demonstrate that the two older, mother centrioles are selectively removed from the oocyte by extrusion into polar bodies. We show that this requires specific positioning of the second meiotic spindle, achieved by dynein-driven transport, and anchorage of the mother centriole to the plasma membrane via mother-specific appendages. In contrast, the single daughter centriole remaining in the egg is eliminated before the first embryonic cleavage. We demonstrate that these distinct elimination mechanisms are necessary because if mother centrioles are artificially retained, they cannot be inactivated, resulting in multipolar zygotic spindles. Thus, our findings reveal a dual mechanism to eliminate centrioles: mothers are physically removed, whereas daughters are eliminated in the cytoplasm, preparing the egg for fertilization.
BAR domains can prevent membrane fission through their ability to shield necks of budding vesicles from fission-inducing factors. However, the physiological role of this inhibitory function and its ...regulation is unknown. Here we identify a checkpoint involving the BAR-domain-containing protein Arfaptin-1 that controls biogenesis of secretory granules at the trans-Golgi network (TGN). We demonstrate that protein kinase D (PKD) phosphorylates Arfaptin-1 at serine 132, which disrupts the ability of Arfaptin-1 to inhibit the activity of ADP ribosylation factor, an important component of the vesicle scission machinery. The physiological significance of this regulatory mechanism is evidenced by loss of glucose-stimulated insulin secretion due to granule scission defects in pancreatic β cells expressing nonphosphorylatable Arfaptin-1. Accordingly, depletion of Arfaptin-1 leads to the generation of small nonfunctional secretory granules. Hence, PKD-mediated Arfaptin-1 phosphorylation is necessary to ensure biogenesis of functional transport carriers at the TGN in regulated secretion.
► Arfaptin-1 prevents premature fission of growing insulin granules ► Arfaptin-1 shields ARF from interaction with fission effectors in the Golgi ► Arfaptin-1 phosphorylation by PKD disrupts its interaction with ARF ► Arfaptin-1 phosphorylation is required for glucose-stimulated insulin secretion
Tight regulation of membrane scission at the Golgi is essential for generation of properly loaded secretory vesicles. Gehart et al. report a checkpoint mechanism in pancreatic beta cells mediated by BAR-domain-containing Arfaptin-1. This mechanism allows PKD signaling to activate membrane scission of insulin granules, coordinating glucose-stimulated insulin secretion.
The mesothelium is a dynamic and specialized tissue layer that covers the somatic cavities (pleural, peritoneal, and pericardial) as well as the surface of the visceral organs such as the lung, ...heart, liver, bowel and tunica vaginalis testis. The potential therapeutic manipulation of visceral organs has been complicated by the carbohydrate surface layer-here, called the mesopolysaccharide (MPS)-that coats the outer layer of the mesothelium. The traditional understanding of MPS structure has relied upon fixation techniques known to degrade carbohydrates. The recent development of carbohydrate-preserving fixation for high resolution imaging techniques has provided an opportunity to re-examine the structure of both the MPS and the visceral mesothelium. In this report, we used high pressure freezing (HPF) as well as serial section transmission electron microscopy to redefine the structure of the MPS expressed on the murine lung, heart and liver surface. Tissue preserved by HPF and examined by transmission electron microscopy demonstrated a pleural MPS layer 13.01±1.1 um deep-a 100-fold increase in depth compared to previously reported data obtained with conventional fixation techniques. At the base of the MPS were microvilli 1.1±0.35 um long and 42±5 nm in diameter. Morphological evidence suggested that the MPS was anchored to the mesothelium by microvilli. In addition, membrane pits 97±17 nm in diameter were observed in the apical mesothelial membrane. The spatial proximity and surface density (29±4.5%) of the pits suggested an active process linked to the structural maintenance of the MPS. The striking magnitude and complex structure of the MPS indicates that it is an important consideration in studies of the visceral mesothelium.
Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron ...microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest).
Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron‐microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high‐pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non‐correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron‐microscopic protocols, making them more accessible to a wider range of researchers.
Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time‐demanding procedure of specimen preparation for any kind of TEM.
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