Biomedical subjects

Tomas Kirchhausen

Publications and source records attributed to Tomas Kirchhausen.

At least 19 recordsLinked to original sources

PubMed2007

Association of Dishevelled with the clathrin AP-2 adaptor is required for Frizzled endocytosis and planar cell polarity signaling.

Upon activation by Wnt, the Frizzled receptor is internalized in a process that requires the recruitment of Dishevelled. We describe a novel interaction between Dishevelled2 (Dvl2) and micro2-adaptin, a subunit of the clathrin adaptor AP-2; this interaction is required to engage activated Frizzled4 with the endocytic machinery and for its internalization. The interaction of Dvl2 with AP-2 requires simultaneous association of the DEP domain and a peptide YHEL motif within Dvl2 with the C terminus of micro2. Dvl2 mutants in the YHEL motif fail to associate with micro2 and AP-2, and prevent Frizzled4 internalization. Corresponding Xenopus Dishevelled mutants show compromised ability to interfere with gastrulation mediated by the planar cell polarity (PCP) pathway. Conversely, a Dvl2 mutant in its DEP domain impaired in PCP signaling exhibits defective AP-2 interaction and prevents the internalization of Frizzled4. We suggest that the direct interaction of Dvl2 with AP-2 is important for Frizzled internalization and Frizzled/PCP signaling.

Adaptor Protein Complex 2↗

PubMed2006

Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events.

Macropinocytosis, a form of bulk uptake of fluid and solid cargo into cytoplasmic vacuoles, called macropinosomes, has been studied mostly in relation to antigen presentation. Early membrane traffic events occurring in this process are, however, largely unknown. Using human dendritic cells we show that a marked increase in the rate of macropinocytosis occurs a few minutes after application of two markers (small latex beads or dextran), depends on a slow intracellular Ca2+ concentration ([Ca2+](i)) rise that precedes the PI3K-dependent step, and is preceded and accompanied by exocytosis of enlargeosomes compensating in part for the macropinocytic plasma membrane internalization. Unexpectedly, macropinosomes themselves, which share markers with endosomes, undergo Ca2+ -dependent exocytosis so that, after approximately 20 minutes of continuous bead or dextran uptake, an equilibrium is reached preventing cells from overloading themselves with the organelles. Large [Ca2+](i) increases induced by ionomycin trigger rapid (<1 minute) exocytic regurgitation of all macropinosomes, whereas endosomes remain apparently unaffected. We conclude that, in dendritic cells, the rate of macropinocytosis is not constant but increases in a regulated fashion, as previously shown in other cell types. Moreover, macropinosomes are not simple containers that funnel cargo to an endocytic pathway, but unique organelles, distinct from endosomes by their competence for regulated exocytosis and other membrane properties.

Calcium↗

PubMed2006

Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly.

Clathrin-coated vesicles mediate vesicular traffic in cells. Three-dimensional image reconstructions of homogenous populations of in vitro assembled clathrin coats have yielded a molecular model for clathrin and its interactions with some of its partners. The intrinsic averaging required for those calculations has precluded detailed analysis of heterogeneous populations of clathrin-coated vesicles isolated from cells. We have therefore used cryo-electron tomography to study the lattice organization of individual clathrin-coated vesicles and the disposition of the captured vesicle with respect to the surrounding coat. We find a wide range of designs for the clathrin lattice, with different patterns of pentagonal, hexagonal, and occasionally heptagonal facets. Many coats, even smaller ones, enclose membrane vesicles, which are generally offset from the center of the clathrin shell. The electron density distribution between the coat and the underlying vesicle is not uniform, and the number of apparent contacts that anchor the clathrin lattice to the vesicle membrane is significantly less than the number of clathrin heavy chains in the assembly. We suggest that the eccentric position of the vesicle reflects the polarity of assembly, from initiation of coat formation to membrane pinching.

Animals↗

PubMed2006

Role of lipids and actin in the formation of clathrin-coated pits.

Assembly of clathrin-coated pits and their maturation into coated vesicles requires coordinated interactions between specific lipids and several structural and regulatory proteins. In the presence of primary alcohols, phospholipase D generates phosphatidylalcohols instead of PA, reducing stimulation of phosphatidyl inositol 5-kinase (PI5K) and hence decreasing formation of phosphoinositide-4,5-biphosphate (PIP(2)). Using live-cell imaging, we have shown that acute treatment of cells with 1-butanol or other small primary alcohols induces rapid disassembly of coated pits at the plasma membrane and blocks appearance of new ones. Addition of exogenous PIP(2) reverses this effect. Coated pits and vesicles reappear synchronously upon removal of 1-butanol; we have used this synchrony to assess the role of actin in coated vesicle assembly. Prolonged inhibition of actin polymerization by latrunculin A or cytochalasin D reduced by approximately 50% the frequency of coated pit formation without affecting maturation into coated vesicles. As in control cells, removal of 1-butanol in the continued presence of an actin depolymerizer led to synchronous appearance of new pits, which matured normally. Thus, remodeling of the actin cytoskeleton is not essential for clathrin-coated vesicle assembly but may indirectly affect the nucleation of clathrin-coated pits.

1-Butanol↗

PubMed2006

Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy.

Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.

Clathrin↗

PubMed2006

A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating.

Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.

Animals↗

PubMed2006

Synthesis of a 10,000-membered library of molecules resembling carpanone and discovery of vesicular traffic inhibitors.

Split-and-pool synthesis of a 10,000-membered library of molecules resembling the natural product carpanone has been achieved. The synthesis features development of solid-phase multicomponent reactions between nitrogen nucleophiles, enones, and hydroxylamines, and a solid-phase application of the Huisgen cycloaddition affording substituted triazoles. The synthesis was performed in high-capacity (500 microm) polystyrene beads using a one bead-one stock solution strategy that enabled phenotypic screens of the resulting library. Using whole-cell fluorescence imaging, we discovered a series of molecules from the carpanone-based library that inhibit exocytosis from the Golgi apparatus. The most potent member of this series has an IC(50) of 14 microM. We also report structure-activity relationships for the molecules exhibiting this interesting phenotype. These inhibitors of exocytosis may be useful reagents for the study of vesicular traffic.

Biological Products↗

PubMed2006

The Cdc42 inhibitor secramine B prevents cAMP-induced K+ conductance in intestinal epithelial cells.

Cyclic 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.

Benzazepines↗

PubMed2006

Dynasore, a cell-permeable inhibitor of dynamin.

Dynamin is essential for clathrin-dependent coated vesicle formation. It is required for membrane budding at a late stage during the transition from a fully formed pit to a pinched-off vesicle. Dynamin may also fulfill other roles during earlier stages of vesicle formation. We have screened about 16,000 small molecules and have identified 1, named here dynasore, that interferes in vitro with the GTPase activity of dynamin1, dynamin2, and Drp1, the mitochondrial dynamin, but not of other small GTPases. Dynasore acts as a potent inhibitor of endocytic pathways known to depend on dynamin by rapidly blocking coated vesicle formation within seconds of dynasore addition. Two types of coated pit intermediates accumulate during dynasore treatment, U-shaped, half formed pits and O-shaped, fully formed pits, captured while pinching off. Thus, dynamin acts at two steps during clathrin coat formation; GTP hydrolysis is probably needed at both steps.

Cell Membrane Permeability↗

PubMed2005

Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro.

Inspired by the usefulness of small molecules to study membrane traffic, we used high-throughput synthesis and phenotypic screening to discover secramine, a molecule that inhibits membrane traffic out of the Golgi apparatus by an unknown mechanism. We report here that secramine inhibits activation of the Rho GTPase Cdc42, a protein involved in membrane traffic, by a mechanism dependent upon the guanine dissociation inhibitor RhoGDI. RhoGDI binds Cdc42 and antagonizes its membrane association, nucleotide exchange and effector binding. In vitro, secramine inhibits Cdc42 binding to membranes, GTP and effectors in a RhoGDI-dependent manner. In cells, secramine mimics the effects of dominant-negative Cdc42 expression on protein export from the Golgi and on Golgi polarization in migrating cells. RhoGDI-dependent Cdc42 inhibition by secramine illustrates a new way to inhibit Rho GTPases with small molecules and provides a new means to study Cdc42, RhoGDI and the cellular processes they mediate.

Actins↗

PubMed2005

Possible role of deep tubular invaginations of the plasma membrane in MHC-I trafficking.

Tubules and vesicles are membrane carriers involved in traffic along the endocytic and secretory routes. The small GTPase Arf6 regulates a recycling branch of short dynamic tubular intermediates used by major histocompatibility class I (MHC-I) molecules to traffic through vesicles between endosomes and the plasma membrane. We observed that Arf6 also affects a second network of very long and stable tubules containing MHC-I, many of which correspond to deep invaginations of the plasma membrane. Treatment with wortmannin, an inhibitor of phosphatidylinositol-3-phosphate kinase, prevents formation of the short dynamic tubules while increasing the number of the long and very stable ones. Expression of NefAAAA, a mutant form of HIV Nef, increases the number of cells containing the stable tubules, and is used here as a tool to facilitate their study. Photoactivation of NefAAAA-PA-GFP demonstrates that this molecule traffics from endosomes to the tubules. Finally, live-cell imaging also shows internalization of MHC-I molecules into these tubules, suggesting that this is an additional route for MHC-I traffic.

ADP-Ribosylation Factor 6↗

PubMed2005

Endocytosis is not required for the selective lipid uptake mediated by murine SR-BI.

The scavenger receptor class B, type I (SR-BI) mediates the cellular selective uptake of cholesteryl esters and other lipids from high-density lipoproteins (HDL) and low-density lipoproteins (LDL). This process, unlike classical receptor-mediated endocytosis, does not result in lipoprotein degradation. Instead, the lipid depleted particles are released into the medium. Here we show that selective lipid uptake mediated by murine SR-BI can be uncoupled from the endocytosis of HDL or LDL particles. We found that blocking selective lipid uptake by incubating cells with the small chemical inhibitors BLT-1 or BLT-4 did not affect endocytosis of HDL. Similarly, blocking endocytosis by hyperosmotic sucrose or K+ depletion did not prevent selective lipid uptake from HDL or LDL. These findings suggest that mSR-BI-mediated selective uptake occurs at the cell surface upon the association of lipoproteins with mSR-BI and does not require endocytosis of HDL or LDL particles.

Animals↗

PubMed2005

Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis.

Perforin delivers granzymes to induce target-cell apoptosis. At high concentrations, perforin multimerizes in the plasma membrane to form pores. However, whether granzymes enter target cells via membrane pores is uncertain. Here we find that perforin at physiologically relevant concentrations and during cell-mediated lysis creates pores in the target-cell membrane, transiently allowing Ca(2+) and small dyes into the cell. The Ca(2+) flux triggers a wounded membrane-repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. Perforin also triggers the rapid endocytosis of granzymes into large EEA-1-staining vesicles. The restoration of target-cell membrane integrity by triggering the repair response is necessary for target cells subjected to cytotoxic T lymphocyte attack to avoid necrosis and undergo the slower process of programmed cell death. Thus, the target cell actively participates in determining its own fate during cell-mediated death.

Apoptosis↗

PubMed2005

Single-molecule live-cell imaging of clathrin-based endocytosis.

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the first real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have visualized the formation of coats by monitoring the incorporation of fluorescently tagged clathrin or its adaptor AP-2 (adaptor protein 2), and have followed clathrin-mediated uptake of transferrin, single LDL (low-density lipoprotein) and single reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no preferred nucleation sites. A proportion of the nucleation events appear to be abortive. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit, and loading appears to commit that pit to finish assembly. Our data led to a model in which coated pits initiate randomly, but collapse with high likelihood unless stabilized, presumably by cargo capture.

Adaptor Protein Complex 2↗

PubMed2004

The transcription factor NFAT3 mediates neuronal survival.

Neuronal apoptosis is critical for normal development of the mammalian nervous system and also contributes to the pathogenesis of ischemic and degenerative diseases of the brain. Apoptosis of neurons is tightly regulated by extrinsic signals including growth factors and neuronal activity, but the intracellular mechanisms by which these signals promote neuronal survival are incompletely understood. We report that the transcription factor NFAT3 plays a critical role in mediating survival of granule neurons of the developing cerebellum. NFAT3 accumulated in the nucleus of primary granule neurons under survival conditions of serum growth factors and neuronal activity that was elicited by depolarization with high K(+). In contrast, deprivation of serum and K(+), which leads to neuronal apoptosis, triggered NFAT3 nuclear export. Treatment of granule neurons with Li(+), an inhibitor of the NFAT export kinase GSK3, prevented the nuclear export of NFAT3 and increased granule cell survival even under pro-apoptotic conditions. Thus, the nuclear localization of NFAT3 correlated tightly with granule neuron survival. Consistent with a pro-survival function for NFAT3, genetic knockdown of NFAT3 by RNA interference in primary granule neurons led to increased apoptosis even in neurons cultured under survival conditions. Conversely, expression of a constitutively active form of NFAT protected neurons against apoptosis induced by serum withdrawal and low K(+). Taken together, these results reveal an essential function for NFAT3-mediated transcription in neuronal survival that may play important roles in the developing and mature brain.

Active Transport, Cell Nucleus↗

PubMed2004

Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating.

Clathrin-coated pits invaginate from specific membrane compartments and pinch off as coated vesicles. These vesicles then uncoat rapidly once released. The Hsc70 molecular chaperone effects the uncoating reaction, and is guided to appropriate locations on clathrin lattices by the J-domain-containing co-chaperone molecule auxilin. This raises the question of how a local event such as ATP hydrolysis by Hsc70 can catalyse a global disassembly. Here, we have used electron cryomicroscopy to determine 12-A-resolution structures of in-vitro-assembled clathrin coats in association with a carboxy-terminal fragment of auxilin that contains both the clathrin-binding region and the J domain. We have located the auxilin fragment by computing differences between these structures and those lacking auxilin (described in an accompanying paper). Auxilin binds within the clathrin lattice near contacts between an inward-projecting C-terminal helical tripod and the crossing of two 'ankle' segments; it also contacts the terminal domain of yet another clathrin 'leg'. It therefore recruits Hsc70 to the neighbourhood of a set of critical interactions. Auxilin binding produces a local change in heavy-chain contacts, creating a detectable global distortion of the clathrin coat. We propose a mechanism by which local destabilization of the lattice promotes general uncoating.

Animals↗

PubMed2004

Molecular model for a complete clathrin lattice from electron cryomicroscopy.

Clathrin-coated vesicles are important vehicles of membrane traffic in cells. We report the structure of a clathrin lattice at subnanometre resolution, obtained from electron cryomicroscopy of coats assembled in vitro. We trace most of the 1,675-residue clathrin heavy chain by fitting known crystal structures of two segments, and homology models of the rest, into the electron microscopy density map. We also define the position of the central helical segment of the light chain. A helical tripod, the carboxy-terminal parts of three heavy chains, projects inward from the vertex of each three-legged clathrin triskelion, linking that vertex to 'ankles' of triskelions centred two vertices away. Analysis of coats with distinct diameters shows an invariant pattern of contacts in the neighbourhood of each vertex, with more variable interactions along the extended parts of the triskelion 'legs'. These invariant local interactions appear to stabilize the lattice, allowing assembly and uncoating to be controlled by events at a few specific sites.

Animals↗

PubMed2004

Crystal structure of the clathrin adaptor protein 1 core.

The heterotetrameric adaptor proteins (AP complexes) link the outer lattice of clathrin-coated vesicles with membrane-anchored cargo molecules. We report the crystal structure of the core of the AP-1 complex, which functions in the trans-Golgi network (TGN). Packing of complexes in the crystal generates an exceptionally long (1,135-A) unit-cell axis, but the 6-fold noncrystallographic redundancy yields an excellent map at 4-A resolution. The AP-1 core comprises N-terminal fragments of the two large chains, beta1 and gamma, and the intact medium and small chains, micro1 and sigma1. Its molecular architecture closely resembles that of the core of AP-2, the plasma-membrane-specific adaptor, for which a structure has been determined. Both structures represent an "inactive" conformation with respect to binding of cargo with a tyrosine-based sorting signal. TGN localization of AP-1 depends on the small GTPase, Arf1, and the phosphoinositide, PI-4-P. We show that directed mutations of residues at a particular corner of the gamma chain prevent recruitment to the TGN in cells and diminish PI-4-P-dependent, but not Arf1-dependent, liposome binding in vitro.

Adaptor Protein Complex 1↗