| 1. |
- Gad, Annica K B, et al.
(författare)
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RhoD regulates cytoskeletal dynamics via the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules
- 2012
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Ingår i: Molecular Biology of the Cell. - Stockholm : Karolinska Institutet, Dept of Microbiology, Tumor and Cell Biology. - 1939-4586 .- 1059-1524.
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Tidskriftsartikel (refereegranskat)abstract
- The Rho GTPases have mainly been studied in association with their roles in the regulation of actin filament organization. These studies have shown that the Rho GTPases are essential for basic cellular processes, such as cell migration, contraction, and division. In this paper, we report that RhoD has a role in the organization of actin dynamics that is distinct from the roles of the better-studied Rho members Cdc42, RhoA, and Rac1. We found that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM), as well as the related filamin A–binding protein FILIP1. Of these two RhoD-binding proteins, WHAMM was found to bind to the Arp2/3 complex, while FILIP1 bound filamin A. WHAMM was found to act downstream of RhoD in regulating cytoskeletal dynamics. In addition, cells treated with small interfering RNAs for RhoD and WHAMM showed increased cell attachment and decreased cell migration. These major effects on cytoskeletal dynamics indicate that RhoD and its effectors control vital cytoskeleton-driven cellular processes. In agreement with this notion, our data suggest that RhoD coordinates Arp2/3-dependent and FLNa-dependent mechanisms to control the actin filament system, cell adhesion, and cell migration.
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| 2. |
- Bauerschmitt, Heike, et al.
(författare)
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Ribosome-binding proteins Mdm38 and Mba1 display overlapping functions for regulation of mitochondrial translation
- 2010
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 21:12, s. 1937-1944
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Biogenesis of respiratory chain complexes depends on the expression of mitochondrial-encoded subunits. Their synthesis occurs on membrane-associated ribosomes and is probably coupled to their membrane insertion. Defects in expression of mitochondrial translation products are among the major causes of mitochondrial disorders. Mdm38 is related to Letm1, a protein affected in Wolf-Hirschhorn syndrome patients. Like Mba1 and Oxa1, Mdm38 is an inner membrane protein that interacts with ribosomes and is involved in respiratory chain biogenesis. We find that simultaneous loss of Mba1 and Mdm38 causes severe synthetic defects in the biogenesis of cytochrome reductase and cytochrome oxidase. These defects are not due to a compromised membrane binding of ribosomes but the consequence of a mis-regulation in the synthesis of Cox1 and cytochrome b. Cox1 expression is restored by replacing Cox1-specific regulatory regions in the mRNA. We conclude, that Mdm38 and Mba1 exhibit overlapping regulatory functions in translation of selected mitochondrial mRNAs.
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| 3. |
- Buschke, Susanne, et al.
(författare)
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A decisive function of transforming growth factor-β/Smad signaling in tissue morphogenesis and differentiation of human HaCaT keratinocytes
- 2011
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 22:6, s. 782-794
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism by which transforming growth factor-β (TGFβ) regulates differentiation in human epidermal keratinocytes is still poorly understood. To assess the role of Smad signaling, we engineered human HaCaT keratinocytes either expressing small interfering RNA against Smads2, 3, and 4 or overexpressing Smad7 and verified impaired Smad signaling as decreased Smad phosphorylation, aberrant nuclear translocation, and altered target gene expression. Besides abrogation of TGFβ-dependent growth inhibition in conventional cultures, epidermal morphogenesis and differentiation in organotypic cultures were disturbed, resulting in altered tissue homeostasis with suprabasal proliferation and hyperplasia upon TGFβ treatment. Neutralizing antibodies against TGFβ, similar to blocking the actions of EGF-receptor or keratinocyte growth factor, caused significant growth reduction of Smad7-overexpressing cells, thereby demonstrating that epithelial hyperplasia was attributed to TGFβ-induced "dermis"-derived growth promoting factors. Furthermore impaired Smad signaling not only blocked the epidermal differentiation process or caused epidermal-to-mesenchymal transition but induced a switch to a complex alternative differentiation program, best characterized as mucous/intestinal-type epithelial differentiation. As the same alternative phenotype evolved from both modes of Smad-pathway interference, and reduction of Smad7-overexpression caused reversion to epidermal differentiation, our data suggest that functional TGFβ/Smad signaling, besides regulating epidermal tissue homeostasis, is not only essential for terminal epidermal differentiation but crucial in programming different epithelial differentiation routes.
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| 4. |
- Capaldo, Christopher T., et al.
(författare)
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Tight function zonula occludens-3 regulates cyclin D1-dependent cell proliferation
- 2011
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Ingår i: Molecular Biology of the Cell. - Bethesda, United States : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 22:10, s. 1677-1685
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Tidskriftsartikel (refereegranskat)abstract
- Coordinated regulation of cell proliferation is vital for epithelial tissue homeostasis, and uncontrolled proliferation is a hallmark of carcinogenesis. A growing body of evidence indicates that epithelial tight junctions (TJs) play a role in these processes, although the mechanisms involved are poorly understood. In this study, we identify and characterize a novel plasma membrane pool of cyclin D1 with cell-cycle regulatory functions. We have determined that the zonula occludens (ZO) family of TJ plaque proteins sequesters cyclin D1 at TJs during mitosis, through an evolutionarily conserved class II PSD-95, Dlg, and ZO-1 (PDZ)-binding motif within cyclin D1. Disruption of the cyclin D1/ZO complex through mutagenesis or siRNA-mediated suppression of ZO-3 resulted in increased cyclin D1 proteolysis and G(0)/G(1) cell-cycle retention. This study highlights an important new role for ZO family TJ proteins in regulating epithelial cell proliferation through stabilization of cyclin D1 during mitosis.
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| 5. |
- Chen, Yong, et al.
(författare)
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A Vps21 endocytic module regulates autophagy
- 2014
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Ingår i: Molecular Biology of the Cell. - 1939-4586. ; 25:20, s. 3166-3177
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Tidskriftsartikel (refereegranskat)abstract
- In autophagy, the double-membrane autophagosome delivers cellular components for their degradation in the lysosome. The conserved Ypt/Rab GTPases regulate all cellular trafficking pathways, including autophagy. These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors. Rab7 and its yeast homologue, Ypt7, in the context of such a module, regulate the fusion of both late endosomes and autophagosomes with the lysosome. In yeast, the Rab5-related Vps21 is known for its role in early- to late-endosome transport. Here we show an additional role for Vps21 in autophagy. First, vps21Δ mutant cells are defective in selective and nonselective autophagy. Second, fluorescence and electron microscopy analyses show that vps21Δ mutant cells accumulate clusters of autophagosomal structures outside the vacuole. Third, cells with mutations in other members of the endocytic Vps21 module, including the GEF Vps9 and factors that function downstream of Vps21, Vac1, CORVET, Pep12, and Vps45, are also defective in autophagy and accumulate clusters of autophagosomes. Finally, Vps21 localizes to PAS. We propose that the endocytic Vps21 module also regulates autophagy. These findings support the idea that the two pathways leading to the lysosome—endocytosis and autophagy—converge through the Vps21 and Ypt7 GTPase modules.
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| 6. |
- Chotard, Laëtitia, et al.
(författare)
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TBC-2 regulates RAB-5/RAB-7-mediated endosomal trafficking in Caenorhabditis elegans
- 2010
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 21:13, s. 2285-2296
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Tidskriftsartikel (refereegranskat)abstract
- During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7-positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(-) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(-) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.
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| 7. |
- 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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| 8. |
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| 9. |
- 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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| 10. |
- Dou, Dan, et al.
(författare)
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Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion
- 2014
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 25:21, s. 3363-3374
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Tidskriftsartikel (refereegranskat)abstract
- Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity. This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins. On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the N-out-C-in HA and M2 TMDs but not the N-in-C-out TMDs from the type II membrane protein neuraminidase (NA). To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes. Our results show that the marginally hydrophobic NA TMDs (Delta G(app) > 0 kcal/mol) require the cotranslational insertion process for facilitating their inversion during translocation and a positively charged N-terminal flanking residue and that NA inversion enhances its plasma membrane localization. Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once similar to 70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by similar to 100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins. Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.
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| 11. |
- Ekman, Maria, 1979-, et al.
(författare)
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APC and Smad7 link TGF beta type I receptors to the microtubule system to promote cell migration
- 2012
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Ingår i: Molecular Biology of the Cell. - : American Society of Cell Biology, USA. - 1059-1524 .- 1939-4586. ; 23:11, s. 2109-2121
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Tidskriftsartikel (refereegranskat)abstract
- Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3 beta (GSK-3 beta). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-beta (TGF beta) and is known to inhibit various TGF beta-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen-activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGF beta stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3 beta kinases to facilitate local TGF beta/p38-dependent inactivation of GSK-3 beta, accumulation of beta-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7-APC complex links the TGF beta type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGF beta.
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| 12. |
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| 13. |
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| 14. |
- Heublein, Manfred, et al.
(författare)
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The novel component Kgd4 recruits the E3 subunit to the mitochondrial alpha-ketoglutarate dehydrogenase
- 2014
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 25:21, s. 3342-3349
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Tidskriftsartikel (refereegranskat)abstract
- The mitochondrial citric acid cycle is a central hub of cellular metabolism, providing intermediates for biosynthetic pathways and channeling electrons to the respiratory chain complexes. In this study, we elucidated the composition and organization of the multienzyme complex alpha-ketoglutarate dehydrogenase (alpha-KGDH). In addition to the three classical E1-E3 subunits, we identified a novel component, Kgd4 (Ymr31/MRPS36), which was previously assigned to be a subunit of the mitochondrial ribosome. Biochemical analyses demonstrate that this protein plays an evolutionarily conserved role in the organization of mitochondrial alpha-KGDH complexes of fungi and animals. By binding to both the E1-E2 core and the E3 subunit, Kgd4 acts as a molecular adaptor that is necessary to a form a stable alpha-KGDH enzyme complex. Our work thus reveals a novel subunit of a key citric acid-cycle enzyme and shows how this large complex is organized.
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| 15. |
- Jarrin, Miguel, et al.
(författare)
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A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells
- 2012
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 23:16, s. 3266-3274
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Tidskriftsartikel (refereegranskat)abstract
- In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals.
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| 16. |
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| 17. |
- Li, Zhilun, et al.
(författare)
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Integrin-mediated cell attachment induces a PAK4-dependent feedback loop regulating cell adhesion through modified integrin alpha v beta 5 clustering and turnover
- 2010
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 21:19, s. 3317-3329
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Tidskriftsartikel (refereegranskat)abstract
- Cell-to-extracellular matrix adhesion is regulated by a multitude of pathways initiated distally to the core cell-matrix adhesion machinery, such as via growth factor signaling. In contrast to these extrinsically sourced pathways, we now identify a regulatory pathway that is intrinsic to the core adhesion machinery, providing an internal regulatory feedback loop to fine tune adhesion levels. This autoinhibitory negative feedback loop is initiated by cell adhesion to vitronectin, leading to PAK4 activation, which in turn limits total cell-vitronectin adhesion strength. Specifically, we show that PAK4 is activated by cell attachment to vitronectin as mediated by PAK4 binding partner integrin alpha v beta 5, and that active PAK4 induces accelerated integrin alpha v beta 5 turnover within adhesion complexes. Accelerated integrin turnover is associated with additional PAK4-mediated effects, including inhibited integrin alpha v beta 5 clustering, reduced integrin to F-actin connectivity and perturbed adhesion complex maturation. These specific outcomes are ultimately associated with reduced cell adhesion strength and increased cell motility. We thus demonstrate a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion.
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| 18. |
- MacPherson, Matthew Reid, et al.
(författare)
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Phosphorylation of serine 11 and serine 92 as new positive regulators of human Snail1 function : potential involvement of casein kinase-2 and the cAMP-activated kinase protein kinase A
- 2010
-
Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 21:2, s. 244-253
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Tidskriftsartikel (refereegranskat)abstract
- Snail1 is a major factor for epithelial-mesenchymal transition (EMT), an important event in tumor metastasis and in other pathologies. Snail1 is tightly regulated at transcriptional and posttranscriptional levels. Control of Snail1 protein stability and nuclear export by GSK3beta phosphorylation is important for Snail1 functionality. Stabilization mechanisms independent of GSK3beta have also been reported, including interaction with LOXL2 or regulation of the COP9 signalosome by inflammatory signals. To get further insights into the role of Snail1 phosphorylation, we have performed an in-depth analysis of in vivo human Snail1 phosphorylation combined with mutational studies. We identify new phosphorylation sites at serines 11, 82, and 92 and confirmed previously suggested phosphorylations at serine 104 and 107. Serines 11 and 92 participate in the control of Snail1 stability and positively regulate Snail1 repressive function and its interaction with mSin3A corepressor. Furthermore, serines 11 and 92 are required for Snail1-mediated EMT and cell viability, respectively. PKA and CK2 have been characterized as the main kinases responsible for in vitro Snail1 phosphorylation at serine 11 and 92, respectively. These results highlight serines 11 and 92 as new players in Snail1 regulation and suggest the participation of CK2 and PKA in the modulation of Snail1 functionality.
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| 19. |
- Morén, Björn, et al.
(författare)
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EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization
- 2012
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Ingår i: Molecular Biology of the Cell. - : American society for cell biology. - 1059-1524 .- 1939-4586. ; 23:7, s. 1316-1329
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Tidskriftsartikel (refereegranskat)abstract
- Eps15 homology domain-containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.
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| 20. |
- Nava, Porfirio, et al.
(författare)
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IFN gamma-induced suppression of beta-catenin signaling : evidence for roles of Akt and 14.3.3 zeta
- 2014
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Ingår i: Molecular Biology of the Cell. - Bethesda, United States : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 25:19, s. 2894-2904
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Tidskriftsartikel (refereegranskat)abstract
- The proinflammatory cytokine interferon gamma (IFNgamma ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. beta-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNgamma inhibits IEC proliferation despite sustained activation of Akt/beta-catenin signaling. Here we show that inhibition of Akt/beta-catenin-mediated cell proliferation by IFNgamma is associated with the formation of a protein complex containing phosphorylated beta-catenin 552 (pbeta-cat552) and 14.3.3zeta. Akt1 served as a bimodal switch that promotes or inhibits beta-catenin transactivation in response to IFNgamma stimulation. IFNgamma initially promotes beta-catenin transactivation through Akt-dependent C-terminal phosphorylation of beta-catenin to promote its association with 14.3.3zeta. Augmented beta-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3zeta to translocate 14.3.3zeta/beta-catenin from the nucleus, thereby inhibiting beta-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.
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| 21. |
- Nord, Hanna, et al.
(författare)
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Differential regulation of myosin heavy chains defines new muscle domains in zebrafish
- 2014
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 25:8, s. 1384-1395
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Tidskriftsartikel (refereegranskat)abstract
- Numerous muscle lineages are formed during myogenesis within both slow-and fast-specific cell groups. In this study, we show that six fast muscle-specific myosin heavy chain genes have unique expression patterns in the zebrafish embryo. The expression of tail-specific myosin heavy chain (fmyhc2.1) requires wnt signaling and is essential for fast muscle organization within the tail. Retinoic acid treatment results in reduced wnt signaling, which leads to loss of the fmyhc2.1 domain. Retinoic acid treatment also results in a shift of muscle identity within two trunk domains defined by expression of fmyhc1.2 and fmyhc1.3 in favor of the anteriormost myosin isoform, fmyhc1.2. In summary, we identify new muscle domains along the anteroposterior axis in the zebrafish that are defined by individual nonoverlapping, differentially regulated expression of myosin heavy chain isoforms.
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| 22. |
- Omnus, Deike J., 1981-, et al.
(författare)
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A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy5
- 2011
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 22:15, s. 2754-2765
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Tidskriftsartikel (refereegranskat)abstract
- Regulated proteolysis serves as a mechanism to control cellular processes. The SPS (Ssy1-Ptr3-Ssy5) sensor in yeast responds to extracellular amino acids by endoproteolytically activating transcription factors Stp1 and Stp2 (Stp1/2). The processing endoprotease Ssy5 is regulated via proteasomal degradation of its noncovalently associated N-terminal prodomain. We find that degradation of the prodomain requires a conserved phosphodegron comprising phosphoacceptor sites and ubiquitin-accepting lysine residues. Upon amino acid induction, the phosphodegron is modified in a series of linked events by a set of general regulatory factors involved in diverse signaling pathways. First, an amino acid-induced conformational change triggers phosphodegron phosphorylation by the constitutively active plasma membrane-localized casein kinase I (Yck1/2). Next the prodomain becomes a substrate for polyubiquitylation by the Skp1/Cullin/Grr1 E3 ubiquitin ligase complex (SCF(Grr1)). Finally, the modified prodomain is concomitantly degraded by the 26S proteasome. These integrated events are requisite for unfettering the Ssy5 endoprotease, and thus Stp1/2 processing. The Ssy5 phosphoacceptor motif resembles the Yck1/2- and Grr1-dependent degrons of regulators in the Snf3/Rgt2 glucose-sensing pathway. Our work defines a novel proteolytic activation cascade that regulates an intracellular signaling protease and illustrates how general signaling components are recruited to distinct pathways that achieve conditional and specific signaling outputs.
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| 23. |
- Omnus, Deike J., et al.
(författare)
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Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron
- 2014
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 25:23, s. 3823-3833
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Tidskriftsartikel (refereegranskat)abstract
- The Ssy1-Ptr3-Ssy5 (SPS)-sensing pathway enables yeast to respond to extracellular amino acids. Stp1, the effector transcription factor, is synthesized as a latent cytoplasmic precursor with an N-terminal regulatory domain that restricts its nuclear accumulation. The negative regulatory mechanisms impinging on the N-terminal domain are poorly understood. However, Stp1 latency depends on three inner nuclear membrane proteins, Asi1, Asi2, and Asi3. We report that the N-terminal domain of Stp1 contains a small motif, designated RI, that fully accounts for latency. RI is modular, mediates interactions with the plasma membrane, and can retain histone Htb2 in the cytoplasm. A novel class of STP1 mutations affecting RI were isolated that are less efficiently retained in the cytoplasm but remain under tight negative control by the Asi proteins. Intriguingly, these mutant proteins exhibit enhanced stability in strains lacking ASI1. Our results indicate that RI mediates latency by two distinct activities: it functions as a cytoplasmic retention determinant and an Asi-dependent degron. These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the inner nuclear membrane Asi proteins function in a degradation pathway in the nucleus.
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| 24. |
- Omnus, Deike J., 1981-, et al.
(författare)
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Rts1-protein phosphatase 2A antagonizes Ptr3-mediated activation of the signaling protease Ssy5 by casein kinase I
- 2013
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 24:9, s. 1480-1492
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Tidskriftsartikel (refereegranskat)abstract
- Ligand-induced conformational changes of plasma membrane receptors initiate signals that enable cells to respond to discrete extracellular cues. In response to extracellular amino acids, the yeast Ssy1-Ptr3-Ssy5 sensor triggers the endoproteolytic processing of transcription factors Stp1 and Stp2 to induce amino acid uptake. Activation of the processing protease Ssy5 depends on the signal-induced phosphorylation of its prodomain by casein kinase I (Yck1/2). Phosphorylation is required for subsequent Skp1/Cullin/Grr1 E3 ubiquitin ligase-dependent polyubiquitylation and proteasomal degradation of the inhibitory prodomain. Here we show that Rts1, a regulatory subunit of the general protein phosphatase 2A, and Ptr3 have opposing roles in controlling Ssy5 prodomain phosphorylation. Rts1 constitutively directs protein phosphatase 2A activity toward the prodomain, effectively setting a signaling threshold required to mute Ssy5 activation in the absence of amino acid induction. Ptr3 functions as an adaptor that transduces conformational signals initiated by the Ssy1 receptor to dynamically induce prodomain phosphorylation by mediating the proximity of the Ssy5 prodomain and Yck1/2. Our results demonstrate how pathway-specific and general signaling components function synergistically to convert an extracellular stimulus into a highly specific, tuned, and switch-like transcriptional response that is critical for cells to adapt to changes in nutrient availability.
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| 25. |
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| 26. |
- Raju, Chandrasekhar S., et al.
(författare)
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In neurons, activity-dependent association of dendritically transported mRNA transcripts with the transacting factor CBF-A is mediated by A2RE/RTS elements
- 2011
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 22:11, s. 1864-1877
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Tidskriftsartikel (refereegranskat)abstract
- In neurons certain mRNA transcripts are transported to synapses through mechanisms that are not fully understood. Here we report that the heterogeneous nuclear ribonucleoprotein CBF-A (CArG Box binding Factor A) facilitates dendritic transport and localization of activity-regulated cytoskeleton-associated protein (Arc), brain-derived neurotrophic factor (BDNF), and calmodulin-dependent protein kinase II (CaMKII alpha) mRNAs. We discovered that, in the adult mouse brain, CBF-A has a broad distribution. In the nucleus, CBF-A was found at active transcription sites and interchromosomal spaces and close to nuclear pores. In the cytoplasm, CBF-A localized to dendrites as well as pre- and postsynaptic sites. CBF-A was found in synaptosomal fractions, associated with Arc, BDNF, and CaMKII alpha mRNAs. Electrophoretic mobility shift assays demonstrated a direct interaction mediated via their hnRNP A2 response element (A2RE)/RNA trafficking sequence (RTS) elements located in the 3' untranslated regions. In situ hybridization and microscopy on live hippocampal neurons showed that CBF-A is in dynamic granules containing Arc, BDNF, and CaMKII alpha mRNAs. N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) postsynaptic receptor stimulation led to CBF-A accumulation in dendrites; increased Arc, BDNF, and CaMKII alpha mRNA levels; and increased amounts of transcripts coprecipitating with CBF-A. Finally, CBF-A gene knockdown led to decreased mRNA levels. We propose that CBF-A cotranscriptionally binds RTSs in Arc, BDNF, and CaMKII alpha mRNAs and follows the transcripts from genes to dendrites, promoting activity-dependent nuclear sorting of transport-competent mRNAs.
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| 27. |
- Schmees, Christian, et al.
(författare)
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Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V
- 2012
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 23:13, s. 2571-2582
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Tidskriftsartikel (refereegranskat)abstract
- Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. To what extent the endocytic trafficking routes can contribute to such RTK hyper-activation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation synergized in stimulating PI 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of phosphatidylinositol (PI) 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in non-transformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor (EGF) promoted macropinocytosis of both receptors and increased their activation in non-transformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.
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| 28. |
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| 29. |
- Sellin, Mikael E., et al.
(författare)
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Cell type-specific expression of SEPT3-homology subgroup members controls the subunit number of heteromeric septin complexes
- 2014
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 25:10, s. 1594-1607
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Tidskriftsartikel (refereegranskat)abstract
- Septins are filament-forming proteins important for organizing the cortex of animal and fungal cells. In mammals, 13 septin paralogues were recently shown to assemble into core heterohexamer and heterooctamer complexes, which serve as building blocks for apolar filamentous structures that differ among cell types. To determine how tissue-specific septin paralogue expression may shape core heteromer repertoires and thereby modulate properties of septin filaments, we devised protocols to analyze native septin heteromers with distinct numbers of subunits. Our evidence based on genetically manipulated human cells supports and extends recent concepts of homology subgroup-restricted assembly into distinct categories of apolar heterohexamers and heterooctamers. We also identify a category of tetramers that have a subunit composition equivalent to an octameric building block. These atypical tetramers are prevalent in lymphocytes and neural tissues, in which octamers are abundant but hexamers are rare. Our results can be explained by tissue-specific expression of SEPT3 subgroup members: SEPT3, SEPT9, and SEPT12. These serve as cognate subunits in either heterooctamers or atypical tetramers but exhibit different preferences in various tissues. The identified tissue-specific repertoires of septin heteromers provide insights into how higher-order septin structures with differential properties and stabilities may form in diverse animal cell types.
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| 30. |
- Sellin, Mikael E., et al.
(författare)
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Deciphering the rules governing assembly order of mammalian septin complexes
- 2011
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Ingår i: Molecular Biology of the Cell. - Bethesda : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 22:17, s. 3152-3164
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Tidskriftsartikel (refereegranskat)abstract
- Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9-17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals inter-dependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six-to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.
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| 31. |
- Sellin, Mikael E, et al.
(författare)
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Mammalian SEPT9 isoforms direct microtubule-dependent arrangements of septin core heteromers
- 2012
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 23:21, s. 4242-4255
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Tidskriftsartikel (refereegranskat)abstract
- Septin-family proteins assemble into rod-shaped heteromeric complexes that form higher-order arrangements at the cell cortex, where they serve apparently conserved functions as diffusion barriers and molecular scaffolds. There are 13 confirmed septin paralogues in mammals, which may be ubiquitous or tissue specific. Septin hetero-oligomerization appears homology subgroup directed, which in turn determines the subunit arrangement of six- to eight-subunit core heteromers. Here we address functional properties of human SEPT9, which, due to variable mRNA splicing, exists as multiple isoforms that differ between tissues. Myeloid K562 cells express three SEPT9 isoforms, all of which have an equal propensity to hetero-oligomerize with SEPT7-containing hexamers to generate octameric heteromers. However, due to limiting amounts of SEPT9, K562 cells contain both hexameric and octameric heteromers. To generate cell lines with controllable hexamer-to-octamer ratios and that express single SEPT9 isoforms, we developed a gene product replacement strategy. By this means we identified SEPT9 isoform-specific properties that either facilitate septin heteromer polymerization along microtubules or modulate the size range of submembranous septin disks-a prevalent septin structure in nonadhered cells. Our findings show that the SEPT9 expression level directs the hexamer-to-octamer ratio, and that the isoform composition and expression level together determine higher-order arrangements of septins.
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| 32. |
- Sellin, Mikael E, et al.
(författare)
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Microtubules support a disc-like septin arrangement at the plasma membrane of mammalian cells
- 2011
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Ingår i: Molecular Biology of the Cell. - Bethesda : American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 22:23, s. 4588-4601
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Tidskriftsartikel (refereegranskat)abstract
- Septin family proteins oligomerize through GTP-binding domains into core heteromers, which in turn polymerize at the cleavage furrow of dividing fungal and animal cells. Septin assemblies during the interphase of animal cells remain poorly defined and are the topic of this report. Here we developed protocols for visualization of authentic higher-order assemblies using tagged septins to effectively replace the endogenous gene-product within septin core heteromers in human cells. Our analysis revealed that septins assemble into microtubule-supported disc-like structures at the plasma membrane. In the absence of cell substrate-adhesion, this is the predominant higher-order arrangement in interphase cells and each one of the 7 to 8 septin family members expressed by the two analyzed cell types appears equally represented. However, studies of myeloid and lymphoid cell model systems revealed cell type specific alterations of higher-order septin arrangements in response to substrate-adhesion. Live-cell observations suggested that all higher-order septin assemblies are mutually exclusive with plasma membrane regions undergoing remodeling. The combined data point to a mechanism by which densely arranged cortical microtubules, which are typical for non-adhered spherical cells, support plasma membrane-bound disc-like septin assemblies.
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| 33. |
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| 34. |
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| 35. |
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| 36. |
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| 37. |
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| 38. |
- Wiking, Mikaela, et al.
(författare)
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The Subcellular Protein Atlas
- 2014
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 25
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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