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  • 1. Bagnato, Paola
    et al.
    Castagnino, Alessia
    Cortese, Katia
    Bono, Maria
    Grasso, Silvia
    Bellese, Grazia
    Daniele, Tiziana
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Defilippi, Paola
    Castagnola, Patrizio
    Tacchetti, Carlo
    Cooperative but distinct early co-signaling events originate from ERBB2 and ERBB1 receptors upon trastuzumab treatment in breast cancer cells2017Ingår i: Oncotarget, E-ISSN 1949-2553, Vol. 8, nr 36, s. 60109-60122Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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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  • 2. Cortese, Katia
    et al.
    Howes, Mark T.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Tagliatti, Erica
    Bagnato, Paola
    Petrelli, Annalisa
    Bono, Maria
    McMahon, Harvey T.
    Parton, Robert G.
    Tacchetti, Carlo
    The HSP90 inhibitor geldanamycin perturbs endosomal structure and drives recycling ErbB2 and transferrin to modified MVBs/lysosomal compartments2013Ingår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 24, nr 2, s. 129-144Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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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  • 3. Daste, Frederic
    et al.
    Walrant, Astrid
    Holst, Mikkel R.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Gadsby, Jonathan R.
    Mason, Julia
    Lee, Ji-Eun
    Brook, Daniel
    Mettlen, Marcel
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lee, Steven F.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Gallop, Jennifer L.
    Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature2017Ingår i: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 216, nr 11, s. 3745-3765Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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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  • 4. Daumke, Oliver
    et al.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vallis, Yvonne
    Martens, Sascha
    Butler, P Jonathan G
    McMahon, Harvey T
    Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling2007Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 449, nr 7164, s. 923-927Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 5. Doherty, Gary J
    et al.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    GRAF1-dependent endocytosis2009Ingår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 37, nr 5, s. 1061-1065Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 6. Doherty, Gary J.
    et al.
    Åhlund, Monika K.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Howes, Mark T.
    Moren, Bjorn
    Parton, Robert G.
    McMahon, Harvey T.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading2011Ingår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, nr 22, s. 4380-4389Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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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  • 7. Eberth, Alexander
    et al.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gremer, Lothar
    Dvorsky, Radovan
    Koessmeier, Katja T
    McMahon, Harvey T
    Ahmadian, Mohammad Reza
    A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family2009Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 417, nr 1, s. 371-377Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 8.
    Francis, Monika K.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Holst, Mikkel R.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Vidal-Quadras, Maite
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Henriksson, Sara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Santarella-Mellwig, Rachel
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF12015Ingår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, nr 22, s. 4183-4195Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 9.
    Francis, Monika K.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Krupp, Nikolai
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Behrmann, Elmar
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    GRAF1 sculpts membrane through a regulated oligomerisation reactionManuskript (preprint) (Övrigt vetenskapligt)
  • 10.
    Fryklund, Claes
    et al.
    Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Neuhaus, Mathis
    Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Morén, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Borreguero-Muñoz, Andrea
    Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Stenkula, Karin G.
    Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Expansion of the Inguinal Adipose Tissue Depot Correlates With Systemic Insulin Resistance in C57BL/6J Mice2022Ingår i: Frontiers in Cell and Developmental Biology, E-ISSN 2296-634X, Vol. 10, artikel-id 942374Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To accommodate surplus energy, the adipose tissue expands by increasing adipocyte size (hypertrophy) and number (hyperplasia). The presence of hypertrophic adipocytes is a key characteristic of adipose tissue dysfunction. High-fat diet (HFD) fed C57BL/6J mice are a commonly used model to study obesity and obesity-related complications. In the present study, we have characterized adipose plasticity, at both the cellular and tissue level, by examining the temporal development of systemic insulin resistance and adiposity in response to HFD-feeding for 4, 8, and 12 weeks (4w, 8w, and 12w). Within the same time frame, we examined systemic metabolic flexibility and adipose plasticity when switching from HFD- to chow-diet during the last 2 weeks of diet intervention (referred to as the reverse (REV) group: 4wREV (2w HFD+2w chow), 8wREV (6w HFD+2w chow), 12wREV (10w HFD+2w chow)). In response to HFD-feeding over time, the 12w group had impaired systemic insulin sensitivity compared to both the 4w and 8w groups, accompanied by an increase in hypertrophic inguinal adipocytes and liver triglycerides. After reversing from HFD- to chow-feeding, most parameters were completely restored to chow control levels for 4wREV and 8wREV groups. In contrast, the 12wREV group had a significantly increased number of hypertrophic adipocytes, liver triglycerides accumulation, and impaired systemic insulin sensitivity compared to chow-fed mice. Further, image analysis at the single-cell level revealed a cell-size dependent organization of actin filaments for all feeding conditions. Indeed, the impaired adipocyte size plasticity in the 12wREV group was accompanied by increased actin filamentation and reduced insulin-stimulated glucose uptake compared with chow-fed mice. In summary, these results demonstrate that the C57BL/6J HFD-feeding model has a large capacity to restore adipocyte cell size and systemic insulin sensitivity, and that a metabolic tipping point occurs between 8 and 12w of HFD-feeding where this plasticity deteriorates. We believe these findings provide substantial understanding of C57BL/6J mice as an obesity model, and that an increased pool of hypertrophic ING adipocytes could contribute to aggravated insulin resistance.

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  • 11. Hoernke, M.
    et al.
    Mohan, J.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kahra, Dana
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Westenhoff, S.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Schwieger, C.
    Determining membrane bound protein structures by infrared reflection-absorption spectroscopy2017Ingår i: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, s. S161-S161Artikel i tidskrift (Övrigt vetenskapligt)
  • 12. Hoernke, M
    et al.
    Mohan, Jagan
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Blomberg, J
    Kahra, Dana
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Daumke, O
    Westenhof, S
    Schweiger, C
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    TP driven stabilization of a membrane bound open confirmation of the ATPase EHD2 restrains caveolae dynamicsManuskript (preprint) (Övrigt vetenskapligt)
  • 13. Hoernke, Maria
    et al.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mohan, Jagan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Westenhoff, Sebastian
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Schwieger, Christian
    Structural Mechanism in a Membrane Remodelling ATP-ASE2016Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, nr 3, s. 578A-578AArtikel i tidskrift (Övrigt vetenskapligt)
  • 14. Hoernke, Maria
    et al.
    Mohan, Jagan
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kahra, Dana
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Westenhoff, Sebastian
    Schwieger, Christian
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation2017Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 22, s. E4360-E4369Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.

  • 15.
    Holst, Mikkel Roland
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Vidal-Quadras, Maite
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Song, Jie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lundborg, Magnus
    Landström, Maréne
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion2017Ingår i: Cell Reports, E-ISSN 2211-1247, Vol. 20, nr 8, s. 1893-1905Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cellular blebbing, caused by local alterations in cellsurface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrinindependent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension- mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol- interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.

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  • 16. Howes, Mark T
    et al.
    Kirkham, Matthew
    Riches, James
    Cortese, Katia
    Walser, Piers J
    Simpson, Fiona
    Hill, Michelle M
    Jones, Alun
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. MRC Laboratory of Molecular Biology, Cambridge, England.
    Lindsay, Margaret R
    Hernandez-Deviez, Delia J
    Hadzic, Gordana
    McCluskey, Adam
    Bashir, Rumasia
    Liu, Libin
    Pilch, Paul
    McMahon, Harvey
    Robinson, Phillip J
    Hancock, John F
    Mayor, Satyajit
    Parton, Robert G
    Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells2010Ingår i: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 190, nr 4, s. 675-691Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.

  • 17.
    Hubert, Madlen
    et al.
    Department of Pharmacy, Uppsala University, Uppsala, Sweden.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Liu, Kang Cheng
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Caveolae biogenesis and lipid sorting at the plasma membrane2022Ingår i: Plasma membrane shaping / [ed] Shiro Suetsugu, Academic Press, 2022, s. 219-228Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    The plasma membrane of many cell types, in particular, endothelia, smooth muscle cells, and adipocytes, contains numerous small invaginations termed caveolae. In nonmuscle cells, caveolae are formed by lipid-driven assembly of the integral membrane protein caveolin 1 (Cav1) and the peripherally attached protein cavin1. Accessory proteins such as Eps15 homology domain-containing 2 (EHD2) control the cell surface association of caveolae, together providing a unique invaginated membrane structure with distinct dynamics and protein and lipid compositions. These features enable caveolae to survey the plasma membrane integrity and to adjust membrane tension, and sort lipids according to the cellular requirements. Currently, characteristics of the protein and lipid interface of caveola are being unraveled, and this chapter is focused on the present knowledge of caveolae biogenesis and dynamics and describes methods that are being used to study the role of caveolae in lipid flux and lipid composition at the cell surface.

  • 18.
    Hubert, Madlen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Caveolae dynamics is strongly influenced by the lipid composition of the plasma membrane2017Ingår i: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, s. S121-S121Artikel i tidskrift (Övrigt vetenskapligt)
  • 19.
    Hubert, Madlen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Department of Pharmacy, Uppsala University, BMC P.O. Box 580, SE-751 23 Uppsala, Sweden.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Keeping in touch with the membrane; protein- and lipid-mediated confinement of caveolae to the cell surface2020Ingår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 48, s. 155-163Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Caveolae are small Omega-shaped invaginations of the plasma membrane that play important roles in mechanosensing, lipid homeostasis and signaling. Their typical morphology is characterized by a membrane funnel connecting a spherical bulb to the membrane. Membrane funnels (commonly known as necks and pores) are frequently observed as transient states during fusion and fission of membrane vesicles in cells. However, caveolae display atypical dynamics where the membrane funnel can be stabilized over an extended period of time, resulting in cell surface constrained caveolae. In addition, caveolae are also known to undergo flattening as well as short-range cycles of fission and fusion with the membrane, requiring that the membrane funnel closes or opens up, respectively. This mini-review considers the transition between these different states and highlights the role of the protein and lipid components that have been identified to control the balance between surface association and release of caveolae.

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  • 20.
    Hubert, Madlen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Vegesna, Naga Venkata Gayathri
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Ahnlund, Maria
    Johansson, Annika I.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Moodie, Lindon W. K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lipid accumulation controls the balance between surface connection and scission of caveolae2020Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 9, artikel-id e55038Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Caveolae are bulb-shaped invaginations of the plasma membrane (PM) that undergo scission and fusion at the cell surface and are enriched in specific lipids. However, the influence of lipid composition on caveolae surface stability is not well described or understood. Accordingly, we inserted specific lipids into the cell PM via membrane fusion and studied their acute effects on caveolae dynamics. We demonstrate that sphingomyelin stabilizes caveolae to the cell surface, whereas cholesterol and glycosphingolipids drive caveolae scission from the PM. Although all three lipids accumulated specifically in caveolae, cholesterol and sphingomyelin were actively sequestered, whereas glycosphingolipids diffused freely. The ATPase EHD2 restricts lipid diffusion and counteracts lipid-induced scission. We propose that specific lipid accumulation in caveolae generates an intrinsically unstable domain prone to scission if not restrained by EHD2 at the caveolae neck. This work provides a mechanistic link between caveolae and their ability to sense the PM lipid composition.

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  • 21.
    Håberg, Karin
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Lundmark, Richard
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Carlsson, Sven
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    SNX18 is an SNX9 paralog that acts as a membrane tubulator in AP-1-positive endosomal trafficking.2008Ingår i: Journal of Cell Science, ISSN 0021-9533, Vol. 121, nr Pt 9, s. 1495-505Artikel i tidskrift (Refereegranskat)
  • 22.
    Kindstedt, Elin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Koskinen Holm, Cecilia
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Sulniute, Rima
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Martinez-Carrasco, Irene
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lundberg, Pernilla
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    CCL11, a novel mediator of inflammatory bone resorption2017Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 7, nr 1, artikel-id 5334Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Normal bone homeostasis, which is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts is perturbed by inflammation. Inchronic inflammatory disease with disturbed bone remodelling, e.g. rheumatoid arthritis, patients show increased serum levels of the chemokine eotaxin-1 (CCL11). Herein, we demonstrate an inflammatory driven expression of CCL11 in bone tissue and a novel role of CCL11 in osteoclast migration and resorption. Using an inflammatory bone lesion model and primary cell cultures, we discovered that osteoblasts express CCL11 in vivo and in vitro and that expression increased during inflammatory conditions. Osteoclasts did not express CCL11, but the high affinity receptor CCR3 was significantly upregulated during osteoclast differentiation and found to colocalise with CCL11. Exogenous CCL11 was internalised in osteoclast and stimulated the migration of pre-osteoclast and concomitant increase in bone resorption. Our data pinpoints that the CCL11/CCR3 pathway could be a new target for treatment of inflammatory bone resorption.

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  • 23.
    Kong, Ziqing
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Jia, Shaodong
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Anna Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Appelblad, Patrik
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi. Merck Chemicals and Life Science AB, Solna, Sweden.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Moritz, Thomas
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry2018Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, nr 11, artikel-id e66Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Information about the intracellular concentration of dNTPs and NTPs is important for studies of the mechanisms of DNA replication and repair, but the low concentration of dNTPs and their chemical similarity to NTPs present a challenge for their measurement. Here, we describe a new rapid and sensitive method utilizing hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the simultaneous determination of dNTPs and NTPs in biological samples. The developed method showed linearity (R2 > 0.99) in wide concentration ranges and could accurately quantify dNTPs and NTPs at low pmol levels. The intra-day and inter-day precision were below 13%, and the relative recovery was between 92% and 108%. In comparison with other chromatographic methods, the current method has shorter analysis times and simpler sample pre-treatment steps, and it utilizes an ion-pair-free mobile phase that enhances mass-spectrometric detection. Using this method, we determined dNTP and NTP concentrations in actively dividing and quiescent mouse fibroblasts.

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  • 24.
    Larsson, Elin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Analysis of protein and lipid interactions using liposome co-sedimentation assays2020Ingår i: Caveolae: methods and protocols / [ed] Cedric M. Blouin, Humana Press, 2020, , s. 9s. 119-127Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    The dynamic assembly of proteins at the membrane interphase is key to many cell biological processes such as the generation and stabilization of caveolae at the cell surface via coat proteins. The liposome co-sedimentation assay has been widely used for studies of protein and lipid interactions and has provided important information about binding mechanisms, lipid-binding specificity, and curvature preference of proteins. Here, we describe this technique in detail and how it can be used as a tool to address the membrane-binding ability and lipid specificity of caveolae-associated proteins.

  • 25.
    Larsson, Elin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Morén, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    McMahon, Kerrie-Ann
    Institute for Molecular Bioscience, The University of Queensland, QLD, Brisbane, Australia.
    Parton, Robert G.
    Institute for Molecular Bioscience, The University of Queensland, QLD, Brisbane, Australia; Centre for Microscopy and Microanalysis, The University of Queensland, QLD, Brisbane, Australia.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Dynamin2 functions as an accessory protein to reduce the rate of caveola internalization2023Ingår i: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 222, nr 4, artikel-id e202205122Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Caveolae are small membrane invaginations that generally are stably attached to the plasma membrane. Their release is believed to depend on the GTPase dynamin 2 (Dyn2), in analogy with its role in fission of clathrin-coated vesicles. The mechanistic understanding of caveola fission is, however, sparse. Here, we used microscopy-based tracking of individual caveolae in living cells to determine the role of Dyn2 in caveola dynamics. We report that Dyn2 stably associated with the bulb of a subset of caveolae, but was not required for formation or fission of caveolae. Dyn2-positive caveolae displayed longer plasma membrane duration times, whereas depletion of Dyn2 resulted in shorter duration times and increased caveola fission. The stabilizing role of Dyn2 was independent of its GTPase activity and the caveola stabilizing protein EHD2. Thus, we propose that, in contrast to the current view, Dyn2 is not a core component of the caveolae machinery, but rather functions as an accessory protein that restrains caveola internalization.

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  • 26.
    Larsson, Elin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Morén, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Shah, Claudio
    Max-Delbrück-Center for Molecular Medicine.
    Daumke, Oliver
    Max-Delbrück-Center for Molecular Medicine.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    An EH-domain switching mechanism regulates stable membrane association of EHD2Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    EHD2 is a dimeric ATPase known to stabilise the surface connection of the characteristic small invaginations of the cell surface termed caveolae. EHD2 oligomerises into rings around lipid membranes thereby controlling their shape. Here, we have analysed the domain interactions and mechanism that control the stable membrane association of EHD2 at caveolae. We have found that the N-terminus of EHD2, which is buried in the core protein and obstruct assembly, has to be relieved by an EH domain dependent mechanism. The binding between the EH domain and a loop in the GTPase domain of EHD2 was required for stable membrane association, but the loop in itself was not sufficient for specific recruitment to caveolae. A positively charged stretch in the EH domain is proposed to bind to lipids and thereby influence the exchange rate of EHD2. Taken together, we propose a stringent regulatory mechanism for the assembly of EHD2 involving switching of the EH domain position to release the N-terminus and facilitate oligomerisation.

  • 27.
    Liu, Kang-Cheng
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Pace, Hudson
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Medicinsk och klinisk genetik. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hossain, Shakhawath
    Department of Pharmacy, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Kabedev, Aleksei
    Department of Pharmacy, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Shukla, Ankita
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Jerschabek, Vanessa
    Institute of Physical Chemistry, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
    Mohan, Jagan
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Bergström, Christel A.S.
    Department of Pharmacy, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Bally, Marta
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Schwieger, Christian
    Institute of Physical Chemistry, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Department of Pharmacy, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Membrane insertion mechanism of the caveola coat protein Cavin12022Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 119, nr 25, artikel-id 2202295119Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

  • 28.
    Lundmark, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Carlsson, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sorting nexin 9 participates in clathrin-mediated endocytosis through interactions with the core components2003Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, nr 47, s. 46772-46781Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the alpha and beta2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis.

  • 29.
    Lundmark, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Carlsson, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The beta-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-22002Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 362, nr 3, s. 597-607Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the beta-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the beta-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the beta-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel beta-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.

  • 30.
    Lundmark, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Carlsson, Sven R
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis.2010Ingår i: Seminars in Cell and Developmental Biology, ISSN 1084-9521, E-ISSN 1096-3634, Vol. 21, nr 4, s. 363-70Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.

  • 31.
    Lundmark, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Carlsson, Sven R
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Expression and properties of sorting nexin 9 in dynamin-mediated endocytosis2005Ingår i: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 404, s. 545-556Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sorting nexin 9 (SNX9) is identified as an important regulator of dynamin function in clathrin-mediated endocytosis. SNX9 recruits dynamin to the plasma membrane and promotes its GTPase activity, resulting in membrane constriction and ultimate transport vesicle scission. This chapter describes procedures to express recombinant SNX9, to biochemically characterize the cytosolic complex between SNX9 and dynamin, and to identify additional interacting partners of SNX9. Assays are presented to investigate the requirements for SNX9-dependent membrane recruitment of dynamin in vitro and in vivo.

  • 32.
    Lundmark, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Carlsson, Sven R
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9.2004Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 41, s. 42694-42702Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The endocytic proteins sorting nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase.

  • 33.
    Lundmark, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Carlsson, Sven R
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    SNX9 - a prelude to vesicle release2009Ingår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 122, nr 1, s. 5-11Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The sorting nexin SNX9 has, in the past few years, been singled out as an important protein that participates in fundamental cellular activities. SNX9 binds strongly to dynamin and is partly responsible for the recruitment of this GTPase to sites of endocytosis. SNX9 also has a high capacity for modulation of the membrane and might therefore participate in the formation of the narrow neck of endocytic vesicles before scission occurs. Once assembled on the membrane, SNX9 stimulates the GTPase activity of dynamin to facilitate the scission reaction. It has also become clear that SNX9 has the ability to activate the actin regulator N-WASP in a membrane-dependent manner to coordinate actin polymerization with vesicle release. In this Commentary, we summarize several aspects of SNX9 structure and function in the context of membrane remodeling, discuss its interplay with various interaction partners and present a model of how SNX9 might work in endocytosis.

  • 34.
    Lundmark, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Doherty, Gary J
    Howes, Mark T
    Cortese, Katia
    Vallis, Yvonne
    Parton, Robert G
    McMahon, Harvey T
    The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway2008Ingår i: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 18, nr 22, s. 1802-1808Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Clathrin-independent endocytosis is an umbrella term for a variety of endocytic pathways that internalize numerous cargoes independently of the canonical coat protein Clathrin [1, 2]. Electron-microscopy studies have defined the pleiomorphic CLathrin-Independent Carriers (CLICs) and GPI-Enriched Endocytic Compartments (GEECs) as related major players in such uptake [3, 4]. This CLIC/GEEC pathway relies upon cellular signaling and activation through small G proteins, but mechanistic insight into the biogenesis of its tubular and tubulovesicular carriers is lacking. Here we show that the Rho-GAP-domain-containing protein GRAF1 marks, and is indispensable for, a major Clathrin-independent endocytic pathway. This pathway is characterized by its ability to internalize bacterial exotoxins, GPI-linked proteins, and extracellular fluid. We show that GRAF1 localizes to PtdIns(4,5)P2-enriched, tubular, and punctate lipid structures via N-terminal BAR and PH domains. These membrane carriers are relatively devoid of caveolin1 and flotillin1 but are associated with activity of the small G protein Cdc42. This study provides the first specific noncargo marker for CLIC/GEEC endocytic membranes and demonstrates how GRAF1 can coordinate small G protein signaling and membrane remodeling to facilitate internalization of CLIC/GEEC pathway cargoes.

  • 35.
    Lundmark, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Doherty, Gary J
    Vallis, Yvonne
    Peter, Brian J
    McMahon, Harvey T
    Arf family GTP loading is activated by, and generates, positive membrane curvature.2008Ingår i: The Biochemical journal, ISSN 1470-8728, Vol. 414, nr 2, s. 189-94Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Small G-proteins belonging to the Arf (ADP-ribosylation factor) family serve as regulatory proteins for numerous cellular processes through GTP-dependent recruitment of effector molecules. In the present study we demonstrate that proteins in this family regulate, and are regulated by, membrane curvature. Arf1 and Arf6 were shown to load GTP in a membrane-curvature-dependent manner and stabilize, or further facilitate, changes in membrane curvature through the insertion of an amphipathic helix.

  • 36.
    Lundmark, Richard
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk och translationell biologi.
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk och translationell biologi.
    Pulkkinen, I. A. Lauri
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk och translationell biologi.
    The adaptable caveola coat generates a plasma membrane sensory system2024Ingår i: Current Opinion in Cell Biology, ISSN 0955-0674, E-ISSN 1879-0410, Vol. 88, artikel-id 102371Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Caveolae are atypical plasma membrane invaginations that take part in lipid sorting and regulation of oxidative and mechanical plasma membrane stress. Caveola formation requires caveolin, cavin, and specific lipid types. The recent advances in understanding the structure and assembly of caveolin and cavin complexes within the membrane context have clarified the fundamental processes underlying caveola biogenesis. In addition, the curvature of the caveola membrane is controlled by the regulatory proteins EHD2, pacsin2, and dynamin2, which also function to restrain the scission of caveolae from the plasma membrane (PM). Here, this is integrated with novel insights on caveolae as lipid and mechanosensing complexes that can dynamically flatten or disassemble to counteract mechanical, and oxidative stress.

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  • 37. Marchès, O
    et al.
    Batchelor, M
    Shaw, RK
    Patel, A
    Cummings, N
    Nagai, T
    Sasakawa, C
    Carlsson, Sven R
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Cougoule, C
    Caron, E
    Knutton, S
    Connerton, I
    Frankel, G
    EspF of enteropathogenic Escherichia coli binds sorting nexin 92006Ingår i: Journal of Bacteriology, Vol. 188, nr 8, s. 3110-3115Artikel i tidskrift (Refereegranskat)
  • 38. Matthaeus, Claudia
    et al.
    Lahmann, Ines
    Kunz, Severine
    Jonas, Wenke
    Melo, Arthur Alves
    Lehmann, Martin
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Kern, Matthias
    Blueher, Matthias
    Olschowski, Hannah
    Kompa, Julian
    Bruegger, Britta
    Mueller, Dominik N.
    Haucke, Volker
    Schuermann, Annette
    Birchmeier, Carmen
    Daumke, Oliver
    EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake2020Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 117, nr 13, s. 7471-7481Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.

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  • 39. Melo, Arthur Alves
    et al.
    Hegde, Balachandra G.
    Shah, Claudio
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Isas, J. Mario
    Kunz, Severine
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Langen, Ralf
    Daumke, Oliver
    Structural insights into the activation mechanism of dynamin-like EHD ATPases2017Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 22, s. 5629-5634Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Eps15 (epidermal growth factor receptor pathway substrate 15)homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50 degrees rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

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  • 40.
    Mohan, Jagan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Hubert, Madlen
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Schweiger, C
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Structural arrangement of membrane-bound cavinManuskript (preprint) (Övrigt vetenskapligt)
  • 41.
    Mohan, Jagan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Morén, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Larsson, Elin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).