Person: Kirchhausen, Tomas
Loading...
Email Address
AA Acceptance Date
Birth Date
Research Projects
Organizational Units
Job Title
Last Name
Kirchhausen
First Name
Tomas
Name
Kirchhausen, Tomas
11 results
Search Results
Now showing 1 - 10 of 11
Publication Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds(The Rockefeller University Press, 2013) Martinelli, Roberta; Kamei, Masataka; Sage, Peter; Massol, Ramiro; Varghese, Laya; Sciuto, Tracey; Toporsian, Mourad; Dvorak, Ann; Kirchhausen, Tomas; Springer, Timothy; Carman, ChristopherBasic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these “micro-wounds” to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species–dependent self-restorative actin remodeling dynamics.Publication Structural Correlates of Rotavirus Cell Entry(Public Library of Science, 2014) Abdelhakim, Aliaa H.; Salgado, Eric; Fu, Xiaofeng; Pasham, Mithun; Nicastro, Daniela; Kirchhausen, Tomas; Harrison, StephenCell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex—for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼700 Å inner capsid particle (“double-layered particle”, DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.Publication The Small Molecule Dispergo Tubulates the Endoplasmic Reticulum and Inhibits Export(American Society for Cell Biology, 2013) Lu, Lei; Hannoush, Rami N.; Goess, Brian C.; Varadarajan, Shankar; Shair, Matthew; Kirchhausen, TomasThe mammalian endoplasmic reticulum (ER) is an organelle that maintains a complex, compartmentalized organization of interconnected cisternae and tubules while supporting a continuous flow of newly synthesized proteins and lipids to the Golgi apparatus. Using a phenotypic screen, we identify a small molecule, dispergo, that induces reversible loss of the ER cisternae and extensive ER tubulation, including formation of ER patches comprising densely packed tubules. Dispergo also prevents export from the ER to the Golgi apparatus, and this traffic block results in breakdown of the Golgi apparatus, primarily due to maintenance of the constitutive retrograde transport of its components to the ER. The effects of dispergo are reversible, since its removal allows recovery of the ER cisternae at the expense of the densely packed tubular ER patches. This recovery occurs together with reactivation of ER-to-Golgi traffic and regeneration of a functional Golgi with correct morphology. Because dispergo is the first small molecule that reversibly tubulates the ER and inhibits its export function, it will be useful in studying these complex processes.Publication Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding(eLife Sciences Publications, Ltd, 2017) Adell, Manuel Alonso Y; Migliano, Simona M; Upadhyayula, Srigokul; Bykov, Yury S; Sprenger, Simon; Pakdel, Mehrshad; Vogel, Georg F; Jih, Gloria; Skillern, Wesley; Behrouzi, Reza; Babst, Markus; Schmidt, Oliver; Hess, Michael W; Briggs, John AG; Kirchhausen, Tomas; Teis, DavidThe ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3–45 s lifetimes, the ESCRT-III assemblies accumulated 75–200 Snf7 and 15–50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.Publication Superinfection exclusion is absent during acute Junin virus infection of Vero and A549 cells(Nature Publishing Group, 2015) Gaudin, Raphaël; Kirchhausen, TomasMany viruses have evolved strategies of so-called “superinfection exclusion” to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.Publication Seipin is required for converting nascent to mature lipid droplets(eLife Sciences Publications, Ltd, 2016) Wang, Huajin; Becuwe, Michel; Housden, Benjamin E; Chitraju, Chandramohan; Porras, Ashley J; Graham, Morven M; Liu, Xinran N; Thiam, Abdou Rachid; Savage, David B; Agarwal, Anil K; Garg, Abhimanyu; Olarte, Maria-Jesus; Lin, Qingqing; Fröhlich, Florian; Hannibal-Bach, Hans Kristian; Upadhyayula, Srigokul; Perrimon, Norbert; Kirchhausen, Tomas; Ejsing, Christer S; Walther, Tobias; Farese, RobertHow proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation—the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs. DOI: http://dx.doi.org/10.7554/eLife.16582.001Publication The Cdc42 inhibitor secramine B prevents cAMP-induced K+ conductance in intestinal epithelial cells(Elsevier BV, 2006) Pelish, Henry E.; Ciesla, William; Tanaka, Nori; Reddy, Krishna; Shair, Matthew; Kirchhausen, Tomas; Lencer, WayneCyclic AMP- (cAMP) and calcium-dependent agonists stimulate chloride secretion through the coordinated activation of distinct apical and basolateral membrane channels and ion transporters in mucosal epithelial cells. Defects in the regulation of Cl– transport across mucosal surfaces occur with cystic fibrosis and V. cholerae infection and can be life threatening. Here we report that secramine B, a small molecule that inhibits activation of the Rho GTPase Cdc42, reduced cAMP-stimulated chloride secretion in the human intestinal cell line T84. Secramine B interfered with a cAMP-gated and Ba2+-sensitive K+ channel, presumably KCNQ1/KCNE3. This channel is required to maintain the membrane potential that sustains chloride secretion. In contrast, secramine B did not affect the Ca2+-mediated chloride secretion pathway, which requires a separate K+ channel activity from that of cAMP. Pirl1, another small molecule structurally unrelated to secramine B that also inhibits Cdc42 activation in vitro, similarly inhibited cAMP-dependent but not Ca2+-dependent chloride secretion. These results suggest that Rho GTPases may be involved in the regulation of the chloride secretory response and identify secramine B an inhibitor of cAMP-dependent K+ conductance in intestinal epithelial cells.Publication Regulation of ligand-independent notch signal through intracellular trafficking(Landes Bioscience, 2012) Hori, Kazuya; Sen, Anindya Kumar; Kirchhausen, Tomas; Artavanis-Tsakonas, SpyrosNotch signaling is an evolutionarily conserved mechanism that defines a key cell fate control mechanism in metazoans. Notch signaling relies on the surface interaction between the Notch receptor and membrane bound ligands in an apposing cell. In our recent study, we uncover a non-canonical receptor activation path that relies on a ligand-independent, intracellular activation of the receptor as it travels through the endosomal compartments. We found that Notch receptor, targeted for degradation lysosomal degradation through multivesicular bodies (MVBs) is “diverted” toward activation upon mono-ubiquitination through a synergy between the ubiquitin ligase Deltex, the non-visual β-arrestin Kurtz and the ESCRT-III component Shrub. This activation path is not universal but appears to depend on the cellular context.Publication Formation of the Postmitotic Nuclear Envelope from Extended ER Cisternae Precedes Nuclear Pore Assembly(The Rockefeller University Press, 2011) Lu, Lei; Ladinsky, Mark S.; Kirchhausen, TomasDuring mitosis, the nuclear envelope merges with the endoplasmic reticulum (ER), and nuclear pore complexes are disassembled. In a current model for reassembly after mitosis, the nuclear envelope forms by a reshaping of ER tubules. For the assembly of pores, two major models have been proposed. In the insertion model, nuclear pore complexes are embedded in the nuclear envelope after their formation. In the prepore model, nucleoporins assemble on the chromatin as an intermediate nuclear pore complex before nuclear envelope formation. Using live-cell imaging and electron microscope tomography, we find that the mitotic assembly of the nuclear envelope primarily originates from ER cisternae. Moreover, the nuclear pore complexes assemble only on the already formed nuclear envelope. Indeed, all the chromatin-associated Nup107–160 complexes are in single units instead of assembled prepores. We therefore propose that the postmitotic nuclear envelope assembles directly from ER cisternae followed by membrane-dependent insertion of nuclear pore complexes.Publication Synergy Between the ESCRT-III Complex and Deltex Defines a Ligand-Independent Notch Signal(The Rockefeller University Press, 2011) Hori, Kazuya; Sen, Anindya Kumar; Kirchhausen, Tomas; Artavanis-Tsakonas, SpyrosThe Notch signaling pathway defines a conserved mechanism that regulates cell fate decisions in metazoans. Signaling is modulated by a broad and multifaceted genetic circuitry, including members of the endocytic machinery. Several individual steps in the endocytic pathway have been linked to the positive or negative regulation of the Notch receptor. In seeking genetic elements involved in regulating the endosomal/lysosomal degradation of Notch, mediated by the molecular synergy between the ubiquitin ligase Deltex and Kurtz, the nonvisual \(\beta\)-arrestin in Drosophila, we identified Shrub, a core component of the ESCRT-III complex as a key modulator of this synergy. Shrub promotes the lysosomal degradation of the receptor by mediating its delivery into multivesicular bodies (MVBs). However, the interplay between Deltex, Kurtz, and Shrub can bypass this path, leading to the activation of the receptor. Our analysis shows that Shrub plays a pivotal rate-limiting step in late endosomal ligand-independent Notch activation, depending on the Deltex-dependent ubiquitinylation state of the receptor. This activation mode of the receptor emphasizes the complexity of Notch signal modulation in a cell and has significant implications for both development and disease.