TY - JOUR AB - Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole—a protuberance of the zygote’s vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces. AU - Caballero Mancebo, Silvia AU - Shinde, Rushikesh AU - Bolger-Munro, Madison AU - Peruzzo, Matilda AU - Szep, Gregory AU - Steccari, Irene AU - Labrousse Arias, David AU - Zheden, Vanessa AU - Merrin, Jack AU - Callan-Jones, Andrew AU - Voituriez, Raphaël AU - Heisenberg, Carl-Philipp J ID - 14846 JF - Nature Physics SN - 1745-2473 TI - Friction forces determine cytoplasmic reorganization and shape changes of ascidian oocytes upon fertilization ER - TY - JOUR AB - The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly. AU - Zens, Bettina AU - Fäßler, Florian AU - Hansen, Jesse AU - Hauschild, Robert AU - Datler, Julia AU - Hodirnau, Victor-Valentin AU - Zheden, Vanessa AU - Alanko, Jonna H AU - Sixt, Michael K AU - Schur, Florian KM ID - 15146 IS - 6 JF - Journal of Cell Biology SN - 0021-9525 TI - Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular matrix VL - 223 ER - TY - JOUR AB - When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes. AU - Gaertner, Florian AU - Reis-Rodrigues, Patricia AU - De Vries, Ingrid AU - Hons, Miroslav AU - Aguilera, Juan AU - Riedl, Michael AU - Leithner, Alexander F AU - Tasciyan, Saren AU - Kopf, Aglaja AU - Merrin, Jack AU - Zheden, Vanessa AU - Kaufmann, Walter AU - Hauschild, Robert AU - Sixt, Michael K ID - 10703 IS - 1 JF - Developmental Cell SN - 1534-5807 TI - WASp triggers mechanosensitive actin patches to facilitate immune cell migration in dense tissues VL - 57 ER - TY - JOUR AB - Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair. AU - Inglés Prieto, Álvaro AU - Furthmann, Nikolas AU - Crossman, Samuel H. AU - Tichy, Alexandra Madelaine AU - Hoyer, Nina AU - Petersen, Meike AU - Zheden, Vanessa AU - Bicher, Julia AU - Gschaider-Reichhart, Eva AU - György, Attila AU - Siekhaus, Daria E AU - Soba, Peter AU - Winklhofer, Konstanze F. AU - Janovjak, Harald L ID - 9363 IS - 4 JF - PLoS genetics TI - Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease VL - 17 ER - TY - JOUR AB - Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin–mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells. AU - Johnson, Alexander J AU - Dahhan, Dana A AU - Gnyliukh, Nataliia AU - Kaufmann, Walter AU - Zheden, Vanessa AU - Costanzo, Tommaso AU - Mahou, Pierre AU - Hrtyan, Mónika AU - Wang, Jie AU - Aguilera Servin, Juan L AU - van Damme, Daniël AU - Beaurepaire, Emmanuel AU - Loose, Martin AU - Bednarek, Sebastian Y AU - Friml, Jiří ID - 9887 IS - 51 JF - Proceedings of the National Academy of Sciences TI - The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis VL - 118 ER - TY - JOUR AB - Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets. AU - Nicolai, Leo AU - Schiefelbein, Karin AU - Lipsky, Silvia AU - Leunig, Alexander AU - Hoffknecht, Marie AU - Pekayvaz, Kami AU - Raude, Ben AU - Marx, Charlotte AU - Ehrlich, Andreas AU - Pircher, Joachim AU - Zhang, Zhe AU - Saleh, Inas AU - Marel, Anna-Kristina AU - Löf, Achim AU - Petzold, Tobias AU - Lorenz, Michael AU - Stark, Konstantin AU - Pick, Robert AU - Rosenberger, Gerhild AU - Weckbach, Ludwig AU - Uhl, Bernd AU - Xia, Sheng AU - Reichel, Christoph Andreas AU - Walzog, Barbara AU - Schulz, Christian AU - Zheden, Vanessa AU - Bender, Markus AU - Li, Rong AU - Massberg, Steffen AU - Gärtner, Florian R ID - 8787 JF - Nature Communications TI - Vascular surveillance by haptotactic blood platelets in inflammation and infection VL - 11 ER - TY - JOUR AB - Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz−/− follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity. AU - Xia, Peng AU - Gütl, Daniel J AU - Zheden, Vanessa AU - Heisenberg, Carl-Philipp J ID - 6087 IS - 6 JF - Cell TI - Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity VL - 176 ER - TY - JOUR AB - Background Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. New method Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. Results We show that ∼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. Comparison with existing methods Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. Conclusions These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts. AU - Mckenzie, Catherine AU - Spanova, Miroslava AU - Johnson, Alexander J AU - Kainrath, Stephanie AU - Zheden, Vanessa AU - Sitte, Harald H. AU - Janovjak, Harald L ID - 7406 JF - Journal of Neuroscience Methods SN - 0165-0270 TI - Isolation of synaptic vesicles from genetically engineered cultured neurons VL - 312 ER -