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| 2. |
- Yau, Wai-Lok, et al.
(författare)
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Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication
- 2019
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Ingår i: Journal of Virology. - : AMER SOC MICROBIOLOGY. - 0022-538X .- 1098-5514. ; 93:18
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Tidskriftsartikel (refereegranskat)abstract
- Flavivirus is a positive-sense, single-stranded RNA viral genus, with members causing severe diseases in humans such as tick-borne encephalitis, yellow fever, and dengue fever. Flaviviruses are known to cause remodeling of intracellular membranes into small cavities, where replication of the viral RNA takes place. Nonstructural (NS) proteins are not part of the virus coat and are thought to participate in the formation of these viral replication compartments (RCs). Here, we used tick-borne encephalitis virus (TBEV) as a model for the flaviviruses and developed a stable human cell line in which the expression of NS proteins can be induced without viral RNA replication. The model system described provides a novel and benign tool for studies of the viral components under controlled expression levels. We show that the expression of six NS proteins is sufficient to induce infection-like dilation of the endoplasmic reticulum (ER) and the formation of RC-like membrane invaginations. The NS proteins form a membrane-associated complex in the ER, and electron tomography reveals that the dilated areas of the ER are closely associated with lipid droplets and mitochondria. We propose that the NS proteins drive the remodeling of ER membranes and that viral RNA, RNA replication, viral polymerase, and TBEV structural proteins are not required. IMPORTANCE TBEV infection causes a broad spectrum of symptoms, ranging from mild fever to severe encephalitis. Similar to other flaviviruses, TBEV exploits intracellular membranes to build RCs for viral replication. The viral NS proteins have been suggested to be involved in this process; however, the mechanism of RC formation and the roles of individual NS proteins remain unclear. To study how TBEV induces membrane remodeling, we developed an inducible stable cell system expressing the TBEV NS polyprotein in the absence of viral RNA replication. Using this system, we were able to reproduce RC-like vesicles that resembled the RCs formed in flavivirus-infected cells, in terms of morphology and size. This cell system is a robust tool to facilitate studies of flavivirus RC formation and is an ideal model for the screening of antiviral agents at a lower biosafety level.
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| 3. |
- Bagnato, Paola, et al.
(författare)
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Cooperative but distinct early co-signaling events originate from ERBB2 and ERBB1 receptors upon trastuzumab treatment in breast cancer cells
- 2017
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Ingår i: Oncotarget. - : IMPACT JOURNALS LLC. - 1949-2553. ; 8:36, s. 60109-60122
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Tidskriftsartikel (refereegranskat)abstract
- ERBB2 receptor belongs to the ERBB tyrosine kinase receptor family. At variance to the other family members, ERBB2 is a constitutively active orphan receptor. Upon ligand binding and activation, ERBB receptors form homo-or hetero-dimers with the other family members, including ERBB2, promoting an intracellular signaling cascade. ERBB2 is the preferred dimerization partner and ERBB2 heterodimers signaling is stronger and longer acting compared to heterodimers between other ERBB members. The specific contribution of ERBB2 in heterodimer signaling is still undefined. Here we report the formation of circular dorsal ruffles (CDRs) upon treatment of the ERBB2-overexpressing breast cancer cell lines SK-BR-3 and ZR751 with Trastuzumab, a therapeutic humanized monoclonal antibody directed against ERBB2. We found that in SK-BR-3 cells Trastuzumab leads to surface redistribution of ERBB2 and ERBB1 in CDRs, and that the ERBB2-dependent ERK1/2 phosphorylation and ERBB1 expression are both required for CDR formation. In particular, in these cells CDR formation requires activation of both the protein regulator of actin polymerization N-WASP, mediated by ERK1/2, and of the actin depolymerizing protein cofilin, mediated by ERBB1. Furthermore, we suggest that this latter event may be inhibited by the negative cell motility regulator p140Cap, as we found that p140Cap overexpression led to cofilin deactivation and inhibition of CDR formation. In conclusion, here we show for the first time an ERBB2-specific signaling contribution to an ERBB2/ERBB1 heterodimer, in the activation of a complex biological process such as the formation of CDRs.
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| 4. |
- Cortese, Katia, et al.
(författare)
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The HSP90 inhibitor geldanamycin perturbs endosomal structure and drives recycling ErbB2 and transferrin to modified MVBs/lysosomal compartments
- 2013
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Ingår i: Molecular Biology of the Cell. - : The American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 24:2, s. 129-144
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Tidskriftsartikel (refereegranskat)abstract
- The ErbB2 receptor is a clinically validated cancer target whose internalization and trafficking mechanisms remain poorly understood. HSP90 inhibitors, such as geldanamycin (GA), have been developed to target the receptor to degradation or to modulate downstream signaling. Despite intense investigations, the entry route and postendocytic sorting of ErbB2 upon GA stimulation have remained controversial. We report that ErbB2 levels inversely impact cell clathrin-mediated endocytosis (CME) capacity. Indeed, the high levels of the receptor are responsible for its own low internalization rate. GA treatment does not directly modulate ErbB2 CME rate but it affects ErbB2 recycling fate, routing the receptor to modified multivesicular endosomes (MVBs) and lysosomal compartments, by perturbing early/recycling endosome structure and sorting capacity. This activity occurs irrespective of the cargo interaction with HSP90, as both ErbB2 and the constitutively recycled, HSP90-independent, transferrin receptor are found within modified endosomes, and within aberrant, elongated recycling tubules, leading to modified MVBs/lysosomes. We propose that GA, as part of its anticancer activity, perturbs early/recycling endosome sorting, routing recycling cargoes toward mixed endosomal compartments.
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| 5. |
- Daste, Frederic, et al.
(författare)
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Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature
- 2017
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Ingår i: Journal of Cell Biology. - : ROCKEFELLER UNIV PRESS. - 0021-9525 .- 1540-8140. ; 216:11, s. 3745-3765
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Tidskriftsartikel (refereegranskat)abstract
- The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P-2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3) P is produced by the INPP4A hydrolysis of PI(3,4)P-2, and this is necessary for actin-driven endocytosis. Both Cdc42.guanosine triphosphate and SNX9 activate N-WASP-WIP-and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P-2, and PI(3) P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P-2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3) P production, suggesting PI(3) P kinase inhibitors as a therapeutic strategy in Lowe syndrome.
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| 6. |
- Daumke, Oliver, et al.
(författare)
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Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling
- 2007
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 449:7164, s. 923-927
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Tidskriftsartikel (refereegranskat)abstract
- The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain-containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin-independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.
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| 7. |
- Doherty, Gary J, et al.
(författare)
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GRAF1-dependent endocytosis
- 2009
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Ingår i: Biochemical Society Transactions. - London : Portland Press. - 0300-5127 .- 1470-8752. ; 37:5, s. 1061-1065
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Tidskriftsartikel (refereegranskat)abstract
- The role of endocytosis in controlling a multitude of cell biological events is well established. Molecular and mechanistic characterization of endocytosis has predominantly focused on CME (clathrin-mediated endocytosis), although many other endocytic pathways have been described. it was recently shown that the BAR (Bin/amphiphysin/Rvs) and Rho GAP (GTPase-activating protein) domain-containing protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) is found on prevalent, pleiomorphic endocytic membranes, and is essential for the major, clathrin-independent endocytic pathway that these membranes mediate. This pathway is characterized by its ability to internalize GPI (glycosylphosphatidylinositol)anchored proteins, bacterial toxins and large amounts of extracellular fluid. These membrane carriers are highly dynamic and associated with the activity of the small G-protein Cdc42 (cell division cycle 42). in the present paper, we review the role of GRAF1 in this CLIC (clathrin-independent carrier)/GEEC (GPI-anchored protein-enriched early endocytic compartment) endocytic pathway and discuss the current understanding regarding how this multidomain protein functions at the interface between membrane sculpting, small G-protein signalling and endocytosis.
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| 8. |
- Doherty, Gary J., et al.
(författare)
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The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading
- 2011
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Ingår i: Molecular Biology of the Cell. - Bethesda : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 22:22, s. 4380-4389
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Tidskriftsartikel (refereegranskat)abstract
- The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes.
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| 9. |
- Eberth, Alexander, et al.
(författare)
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A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family
- 2009
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 417:1, s. 371-377
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Tidskriftsartikel (refereegranskat)abstract
- The BAR (Bin/amphiphysin/Rvs) domain defines an emerging superfamily of proteins implicated in fundamental biological processes by sensing and inducing membrane curvature. We identified a novel autoregulatory function for the BAR domain of two related GAPs' (GTPase-activating proteins) of the GRAF (GTPase regulator associated with focal adhesion kinase) subfamily. We demonstrate that the N-terminal fragment of these GAPs including the BAR domain interacts directly with the GAP domain and inhibits its activity. Analysis of various BAR and GAP domains revealed that the BAR domain-mediated inhibition of these GAPs' function is highly specific. These GAPs, in their autoinhibited state, are able to bind and tubulate liposomes in vitro, and to generate lipid tubules in cells. Taken together, we identified BAR domains as cis-acting inhibitory elements that very likely mask the active sites of the GAP domains and thus prevent down-regulation of Rho proteins. Most remarkably, these BAR proteins represent a dual-site system with separate membrane-tubulation and GAP-inhibitory functions that operate simultaneously.
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| 10. |
- Francis, Monika K., et al.
(författare)
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Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1
- 2015
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 128:22, s. 4183-4195
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Tidskriftsartikel (refereegranskat)abstract
- Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase activating protein (GAP) GRAF1, facilitate rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, distinct from clathrin coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane resulting in a corresponding decrease in Cdc42 microdomain association. However, Cdc42 captured in its active state was, via a GAP domain mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers affected in their endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.
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