| 1. |
- Nordenfelt, Pontus, et al.
(författare)
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Different Requirements for Early and Late Phases of Azurophilic Granule-Phagosome Fusion
- 2009
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Ingår i: TRAFFIC. - : Wiley. - 1398-9219 .- 1600-0854. ; 10:12, s. 1881-1893
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Tidskriftsartikel (refereegranskat)abstract
- Phagocytosis and killing of microorganisms are complex processes that involve tightly regulated membrane traffic events. Because many signaling molecules associate with membrane rafts and because these structures can be found on azurophilic granules, we decided to investigate raft recruitment and the signaling requirements for azurophilic granule secretion during phagosome maturation. At the site of phagocytosis of immunoglobulin G-opsonized prey in human neutrophils, we found that early secretion of azurophilic granules was both raft- and calcium-dependent. Subsequently, rafts at the phagocytic site were internalized with the prey. At the fully formed phagosome, the fusion of azurophilic granules was no longer dependent on rafts or calcium. These findings were found to be true also when using Streptococcus pyogenes bacteria as prey, and depletion of calcium affected the kinetics of bacterial intracellular survival. These findings suggest that the mechanisms for delivery of azurophilic content to nascent and sealed phagosomes, respectively, differ in their dependence on calcium and membrane rafts.
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| 2. |
- Forsmark, Annabelle, 1973, et al.
(författare)
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Quantitative Proteomics of Yeast Post-Golgi Vesicles Reveals a Discriminating Role for Sro7p in Protein Secretion
- 2011
-
Ingår i: Traffic. - : John Wiley & Sons. - 1398-9219 .- 1600-0854. ; 12:6, s. 740-753
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Tidskriftsartikel (refereegranskat)abstract
- We here report the first comparative proteomics of purified yeast post-Golgi vesicles (PGVs). Vesicle samples isolated from PGV-accumulating sec6-4 mutants were treated with isobaric tags (iTRAQ) for subsequent quantitative tandem mass spectrometric analysis of protein content. After background subtraction, a total of 66 vesicle-associated proteins were identified, including known or assumed vesicle residents as well as a fraction not previously known to be PGV associated. Vesicles isolated from cells lacking the polarity protein Sro7p contained essentially the same catalogue of proteins but showed a reduced content of a subset of cargo proteins, in agreement with a previously shown selective role for Sro7p in cargo sorting.
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| 3. |
- Nilsson, Lars, et al.
(författare)
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Caenorhabditis elegans Numb Inhibits Endocytic Recycling by Binding TAT-1 Aminophospholipid Translocase
- 2011
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Ingår i: Traffic. - Malden : Wiley-Blackwell. - 1398-9219 .- 1600-0854. ; 12:12, s. 1839-1849
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Tidskriftsartikel (refereegranskat)abstract
- Numb regulates endocytosis in many metazoans, but the mechanism by which it functions is not completely understood. Here we report that the Caenorhabditis ele-gans Numb ortholog, NUM-1A, a regulator of endocytic recycling, binds the C isoform of transbilayer amphipath transporter-1 (TAT-1), a P4 family adenosine triphosphatase and putative aminophospholipid translocase that is required for proper endocytic trafficking. We demonstrate that TAT-1 is differentially spliced during development and that TAT-1C-specific splicing occurs in the intestine where NUM-1A is known to function. NUM-1A and TAT-1C colocalize in vivo. We have mapped the binding site to an NXXF motif in TAT-1C. This motif is not required for TAT-1C function but is required for NUM-1A's ability to inhibit recycling. We demonstrate that num-1A and tat-1 defects are both suppressed by the loss of the activity of PSSY-1, a phosphatidylserine (PS) synthase. PS is mislocalized in intestinal cells with defects in tat-1 or num-1A function. We propose that NUM-1A inhibits recycling by inhibiting TAT-1C's ability to translocate PS across the membranes of recycling endosomes.
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| 4. |
- Onischenko, Evgeny A, et al.
(författare)
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Annulate Lamellae Play Only a Minor Role in the Storage of Excess Nucleoporins in Drosophila Embryos
- 2004
-
Ingår i: Traffic. - : Blackwell Munksgaard. - 1398-9219 .- 1600-0854. ; 5:3, s. 152-164
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleo-cytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.
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| 5. |
- Pattu, Varsha, et al.
(författare)
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Syntaxin7 is required for lytic granule release from cytotoxic T lymphocytes.
- 2011
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Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 12:7
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Tidskriftsartikel (refereegranskat)abstract
- SNARE proteins are essential fusion mediators for many intracellular trafficking events. Here, we investigate the role of Syntaxin7 (Stx7) in the release of lytic granules from cytotoxic T lymphocytes (CTLs). We show that Stx7 is expressed in CTLs and is preferentially localized to the region of lytic granule release, the immunological synapse (IS). Interference of Stx7 function by expression of a dominant-negative Stx7 construct or by small interfering RNA leads to a dramatic reduction of CTL-mediated killing of target cells. Real-time visualization of individual lytic granules at the IS by evanescent wave microscopy reveals that lytic granules in Stx7-deprived CTLs not only fail to fuse with the plasma membrane but even fail to accumulate at the IS. Surprisingly, the accumulation defect is not caused by an overall reduction in lytic granule number, but by a defect in the trafficking of T cell receptors (TCRs) through endosomes. Subsequent high-resolution nanoscopy shows that Stx7 colocalizes with Rab7 on late endosomes. We conclude from these data that the accumulation of recycling TCRs at the IS is a SNARE-dependent process and that Stx7-mediated processing of recycling TCRs through endosomes is a prerequisite for the cytolytic function of CTLs.
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| 6. |
- Taner, S. B., et al.
(författare)
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Control of immune responses by trafficking cell surface proteins, vesicles and lipid rafts to and from the immunological synapse
- 2004
-
Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 5:9, s. 651-661
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Forskningsöversikt (refereegranskat)abstract
- Supramolecular clusters at the immunological synapse provide a mechanism for structuring complex communication networks between cells of the immune system. Regulating intra- and intercellular trafficking of proteins and lipids to and from the immunological synapse provides an additional level of complexity in determining the functional outcome of immune cell interactions. An emergent principle is that molecules requiring tightly regulated cell surface expression, e.g. negative regulators of cell activation or molecules promoting cytotoxicity, are trafficked to the immunological synapse from intracellular secretory as required lysosomes. Many molecules required for the early stages of the intercellular communication are already present at the cell surface, sometimes in lipid rafts, and are rapidly translocated laterally to the intercellular contact. Our understanding of these events critically depends on utilizing appropriate technologies for probing supramolecular recognition in live cells. Thus, we also present here a critical discussion of the technologies used to study lipid rafts and, more broadly, a map of the spatial and temporal dimensions covered by current live cell physical techniques, highlighting where advances are needed to exceed current spatial and temporal boundaries.
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| 7. |
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| 8. |
- Mizuno, Kouichi, et al.
(författare)
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Rab27b regulates mast cell granule dynamics and secretion
- 2007
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Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 8:7, s. 883-892
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Tidskriftsartikel (refereegranskat)abstract
- The Rab GTPase family regulates membrane domain organization and vesicular transport pathways. Recent studies indicate that one member of the family, Rab27a, regulates transport of lysosome-related organelles in specialized cells, such as melanosomes and lytic granules. Very little is known about the related isoform, Rab27b. Here we used genetically modified mice to study the involvement of the Rab27 proteins in mast cells, which play key roles in allergic responses. Both Rab27a and Rab27b isoforms are expressed in bone marrow-derived mast cells (BMMC) and localize to secretory granules. Nevertheless, secretory defects as measured by β-hexosaminidase release in vitro and passive cutaneous anaphylaxis in vivo were found only in Rab27b and double Rab27 knockout (KO) mice. Immunofluorescence studies suggest that a subset of Rab27b and double Rab27-deficient BMMCs exhibit mild clustering of granules. Quantitative analysis of live-cell time-lapse imaging revealed that BMMCs derived from double Rab27 KO mice showed almost 10-fold increase in granules exhibiting fast movement (>1.5 μm/s), which could be disrupted by nocodazole. These results suggest that Rab27 proteins, particularly Rab27b, play a crucial role in mast cell degranulation and that their action regulates the transition from microtubule to actin-based motility.
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| 9. |
- Abu-Siniyeh, Ahmed, et al.
(författare)
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The aPKC/Par3/Par6 Polarity Complex and Membrane Order Are Functionally Interdependent in Epithelia During Vertebrate Organogenesis
- 2016
-
Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 17:1, s. 66-79
-
Tidskriftsartikel (refereegranskat)abstract
- The differential distribution of lipids between apical and basolateral membranes is necessary for many epithelial cell functions, but how this characteristic membrane organization is integrated within the polarity network during ductal organ development is poorly understood. Here we quantified membrane order in the gut, kidney and liver ductal epithelia in zebrafish larvae at 3-11 days post fertilization (dpf) with Laurdan 2-photon microscopy. We then applied a combination of Laurdan imaging, antisense knock-down and analysis of polarity markers to understand the relationship between membrane order and apical-basal polarity. We found a reciprocal relationship between membrane order and the cell polarity network. Reducing membrane condensation by exogenously added oxysterol or depletion of cholesterol reduced apical targeting of the polarity protein, aPKC. Conversely, using morpholino knock down in zebrafish, we found that membrane order was dependent upon the Crb3 and Par3 polarity protein expression in ductal epithelia. Hence our data suggest that the biophysical property of membrane lipid packing is a regulatory element in apical basal polarity.
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| 10. |
- Dudenhöffer-Pfeifer, Monika, et al.
(författare)
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Different Munc13 isoforms function as priming factors in lytic granule release from murine cytotoxic T lymphocytes.
- 2013
-
Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 14:7
-
Tidskriftsartikel (refereegranskat)abstract
- In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.
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| 11. |
- Echbarthi, Meriem, et al.
(författare)
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Distinct Trafficking of Cell Surface and Endosomal TIM-1 to the Immune Synapse
- 2015
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Ingår i: Traffic : the International Journal of Intracellular Transport. - : Wiley. - 1398-9219 .- 1600-0854. ; 16:11, s. 1193-1207
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Tidskriftsartikel (refereegranskat)abstract
- The T cell costimulatory molecule TIM-1 (T cell/transmembrane, mucin and immunoglobulin domain protein 1) sorts mainly to endosomes in lymphoid cells. At difference from the cell surface protein, endosomal TIM-1 translocates to the immune synapse (IS), where it can contribute to antigen-dependent T cell costimulation. TIM-1 ligands increase the amount of cell surface protein, preventing its traffic to the IS. The bipolar sorting of TIM-1 observed during IS formation is determined by differences in its subcellular location, and probably modulates antigen-driven immune responses. The T-cell/transmembrane, mucin and immunoglobulin domain protein 1 (TIM-1) is a phosphatidlyserine (PtdSer) receptor and a T-cell costimulatory molecule linked to the development of atopic diseases. TIM-1 locates preferentially in intracellular compartments. Here we show that in human and mouse lymphoid cells, TIM-1 localizes in different types of endosomes and that its domain structure is important for protein sorting to intracellular vesicles. The BALB/c mouse TIM-1 protein, which has a longer mucin domain, is sorted more efficiently to endosomes than the shorter C57BL/6 variant. High affinity ligands such as PtdSer increase the amount of cell surface TIM-1; the protein also polarizes toward cell contacts with apoptotic cells. The large pool of intracellular TIM-1 translocates to the immune synapse (IS) with the CD3-TCR (T-cell receptor) complex and colocalizes to the central supramolecular activation cluster (cSMAC). In contrast, cell surface TIM-1 does not traffic to the IS, but is located away from it. The bipolar TIM-1 sorting observed during IS formation is determined by differences in its subcellular location, and might modulate antigen-driven immune responses. © 2015 John Wiley & Sons A/S.
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| 12. |
- Frénal, Karine, et al.
(författare)
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Global analysis of apicomplexan protein S-acyl transferases reveals an enzyme essential for invasion
- 2013
-
Ingår i: Traffic. - : John Wiley & Sons. - 1398-9219 .- 1600-0854. ; 14:8, s. 895-911
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Tidskriftsartikel (refereegranskat)abstract
- The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S-acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp-His-His-Cys cysteine-rich domain (DHHC-CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan-specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite's ability to egress.
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| 13. |
- Karim, Mahmoud Abdul, et al.
(författare)
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Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay
- 2018
-
Ingår i: Traffic. - : City Net Scientific Research Center Ltd. Belgrade, Serbia. - 1398-9219 .- 1600-0854. ; 19:2, s. 138-149
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Tidskriftsartikel (refereegranskat)abstract
- When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS (homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion.
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| 14. |
- Lemaigre, Camille, et al.
(författare)
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N-BAR and F-BAR proteins - Endophilin-A3 and PSTPIP1 - control clathrin-independent endocytosis of L1CAM
- 2023
-
Ingår i: Traffic: the International Journal of Intracellular Transport. - : Wiley. - 1398-9219. ; 24:4, s. 190-212
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Tidskriftsartikel (refereegranskat)abstract
- Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.
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| 15. |
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| 16. |
- Nehru, Vishal, et al.
(författare)
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RhoD Binds the Rab5 Effector Rabankyrin-5 and has a Role in Trafficking of the Platelet-derived Growth Factor Receptor
- 2013
-
Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 14:12, s. 1242-1254
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Tidskriftsartikel (refereegranskat)abstract
- RhoD is a member of the classical Rho GTPases and it has essential roles in the regulation of actin dynamics. RhoD localizes to early endosomes and recycling endosomes, which indicates its important role in the regulation of endosome trafficking. Here, we show that RhoD binds to the Rab5 effector Rabankyrin-5, and RhoD and Rabankyrin-5 colocalize to Rab5-positive endosomes, which suggests a role for Rabankyrin-5 in the coordination of RhoD and Rab5 in endosomal trafficking. Interestingly, depletion of RhoD using siRNA techniques interfered with the internalization of the PDGF receptor and the subsequent activation of the downstream signaling cascades. Our data suggest that RhoD and Rabankyrin-5 have important roles in coordinating RhoD and Rab activities during internalization and trafficking of activated tyrosine kinase receptors.
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| 17. |
- Omar-Hmeadi, Muhmmad, et al.
(författare)
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PtdIns(4,5)P2 is not required for secretory granule docking
- 2018
-
Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 19:6, s. 436-445
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Tidskriftsartikel (refereegranskat)abstract
- Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P-2) by recruitment of a 5-phosphatase strongly inhibited Ca2+-dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by -toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P-2 accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site.
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| 18. |
- Parton, Robert G., et al.
(författare)
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Caveolae : The FAQs
- 2020
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Ingår i: Traffic. - : John Wiley & Sons. - 1398-9219 .- 1600-0854. ; 21:1, s. 181-185
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Tidskriftsartikel (refereegranskat)abstract
- Caveolae are an abundant, but enigmatic, plasma membrane feature of vertebrate cells. In this brief commentary, the authors attempt to answer some key questions related to the formation and function of caveolae based on round‐table discussions at the first EMBO Workshop on Caveolae held in France in May 2019.
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| 19. |
- Ring, Andreas, et al.
(författare)
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Ssy1 functions at the plasma membrane as a receptor of extracellular amino acids independent of plasma membrane‐endoplasmic reticulum junctions
- 2019
-
Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 20:10, s. 775-784
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Tidskriftsartikel (refereegranskat)abstract
- Evidence from multiple laboratories have implicated Ssy1, a non‐transporting amino acid permease, as the receptor component of the yeast plasma membrane (PM)‐localized SPS (Ssy1‐Ptr3‐Ssy5)‐sensor. Upon binding external amino acids, Ssy1 is thought to initiate signaling events leading to the induction of amino acid permease gene expression. In striking contrast, Kralt et al. 2015 (Traffic 16:135‐147) have questioned the role of Ssy1 in amino acid sensing and reported that Ssy1 is a component of the endoplasmic reticulum (ER), where it reportedly participates in the formation of ER‐PM junctions. Here, we have re‐examined the intracellular location of Ssy1 and tested the role of ER‐PM junctions in SPS sensor signaling. We show that the C‐terminal of Ssy1 carries a functional ER‐exit motif required for proper localization of Ssy1 to the PM. Furthermore, ER‐PM junctions are dispensable for PM‐localization and function of Ssy1; Ssy1 localizes to the PM in a Δtether strain lacking ER‐PM junctions (ist2Δ scs2Δ scs22Δ tcb1Δ tcb2Δ tcb3Δ), and this strain retains the ability to initiate signals induced by extracellular amino acids. The data demonstrate that Ssy1 functions as the primary amino acid receptor and that it carries out this function at the PM.
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| 20. |
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| 21. |
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| 22. |
- Sadowski, Lukasz, et al.
(författare)
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Dynamin Inhibitors Impair Endocytosis and Mitogenic Signaling of PDGF
- 2013
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Ingår i: Traffic. - : John Wiley & Sons. - 1398-9219 .- 1600-0854. ; 14:6, s. 725-736
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Tidskriftsartikel (refereegranskat)abstract
- Platelet-derived growth factor (PDGF) isoforms regulate cell proliferation, migration and differentiation both in embryonic development and adult tissue remodeling. At the cellular level, growth-factor signaling is often modulated by endocytosis. Despite important functions of PDGF, its endocytosis remains poorly studied, mainly for lack of tools to track internalized ligand by microscopy. Here, we developed such a tool and quantitatively analyzed internalization and endosomal trafficking of PDGF-BB in human fibroblasts. We further show that PDGF can be internalized in the presence of dynamin inhibitors, arguing that both dynamin-dependent and dynamin-independent pathways can mediate PDGF uptake. Although these routes operate with somewhat different kinetics, they both ultimately lead to lysosomal degradation of PDGF. Although acute inhibition of dynamin activity only moderately affects PDGF endocytosis, it specifically decreases downstream signaling of PDGF via signal transducer and activator of transcription 3 (STAT3). This correlates with reduced expression of MYC and impaired cell entry into S-phase, indicating that dynamin activity is required for PDGF-induced mitogenesis. Our data support a general view that the components governing endocytic trafficking may selectively regulate certain signaling effectors activated by a growth factor.
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| 23. |
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| 24. |
- Zamponi, Nahuel, et al.
(författare)
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Endoplasmic reticulum is the sorting core facility in the Golgi-lacking protozoan Giardia lamblia
- 2017
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Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 18:9, s. 604-621
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Tidskriftsartikel (refereegranskat)abstract
- Our understanding of protein and lipid trafficking in eukaryotic cells has been challenged by the finding of different forms of compartmentalization and cargo processing in protozoan parasites. Here, we show that, in the absence of a Golgi compartment in Giardia, proteins destined for secretion are directly sorted and packaged at specialized ER regions enriched in COPII coatomer complexes and ceramide. We also demonstrated that ER-resident proteins are retained at the ER by the action of a KDEL receptor, which, in contrast to other eukaryotic KDEL receptors, showed no interorganellar dynamic but instead acts specifically at the limit of the ER membrane. Our study suggests that the ER-exit sites and the perinuclear ER-membranes are capable of performing protein-sorting functions. In our view, the description presented here suggests that Giardia adaptation represents an extreme example of reductive evolution without loss of function.
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| 25. |
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| 26. |
- Delacour, Delphine, et al.
(författare)
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Apical sorting by galectin-3-dependent glycoprotein clustering
- 2007
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Ingår i: Traffic: the International Journal of Intracellular Transport. - : Wiley. - 1398-9219. ; 8:4, s. 379-388
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Tidskriftsartikel (refereegranskat)abstract
- Epithelial cells are characterized by their polarized organization based on an apical membrane that is separated from the basolateral membrane domain by tight junctions. Maintenance of this morphology is guaranteed by highly specific sorting machinery that separates lipids and proteins into different carrier populations for the apical or basolateral cell surface. Lipid-raft-independent apical carrier vesicles harbour the beta-galactoside-binding lectin galectin-3, which interacts directly with apical cargo in a glycan-dependent manner. These glycoproteins are mistargeted to the basolateral membrane in galectin-3-depleted cells, dedicating a central role to this lectin in raft-independent sorting as apical receptor. Here, we demonstrate that high-molecular-weight clusters are exclusively formed in the presence of galectin-3. Their stability is sensitive to increased carbohydrate concentrations, and cluster formation as well as apical sorting are perturbed in glycosylation-deficient Madin-Darby canine kidney (MDCK) II cells. Together, our data suggest that glycoprotein cross-linking by galectin-3 is required for apical sorting of non-raft-associated cargo.
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| 27. |
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| 28. |
- Ivarsson, Rosita, et al.
(författare)
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Myosin 5a controls insulin granule recruitment during late-phase secretion.
- 2005
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Ingår i: Traffic: the International Journal of Intracellular Transport. - : Wiley. - 1398-9219. ; 6:11, s. 1027-1035
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Tidskriftsartikel (refereegranskat)abstract
- We have examined the importance of the actin-based molecular motor myosin 5a for insulin granule transport and insulin secretion. Expression of myosin 5a was downregulated in clonal INS-1E cells using RNAinterference. Stimulated hormone secretion was reduced by 46% and single-cell exocytosis, measured by capacitance recordings, was inhibited by 42% after silencing. Silencing of Slac-2c/MYRIP, which links insulin granules to myosin 5a, resulted in similar inhibition of single-cell exocytosis. Antibody inhibition of the myosin 5a-Slac-2c/MYRIP interaction significantly reduced the recruitment of insulin granules for release. The pool of releasable granules independent of myosin 5a activity was estimated to approximately 550 granules. Total internal reflection microscopy was then applied to directly investigate granule recruitment to the plasma membrane. Silencing of myosin 5a inhibited granule recruitment during late phase of insulin secretion. In conclusion, we propose a model where insulin granules are transported through the actin network via both myosin 5a-mediated transport and via passive diffusion, with the former playing the major role during stimulatory conditions.
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| 29. |
- Ivarsson, Rosita, et al.
(författare)
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Temperature-Sensitive Random Insulin Granule Diffusion is a Prerequisite for Recruiting Granules for Release.
- 2004
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Ingår i: Traffic: the International Journal of Intracellular Transport. - : Wiley. - 1398-9219. ; 5:10, s. 750-762
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Tidskriftsartikel (refereegranskat)abstract
- Glucose-evoked insulin secretion exhibits a biphasic time course and is associated with accelerated intracellular granule movement. We combined live confocal imaging of EGFP-labelled insulin granules with capacitance measurements of exocytosis in clonal INS-1 cells to explore the relation between distinct random and directed modes of insulin granule movement, as well as exocytotic capacity. Reducing the temperature from 34 °C to 24 °C caused a dramatic 81% drop in the frequency of directed events, but reduced directed velocities by a mere 25%. The much stronger temperature sensitivity of the frequency of directed events (estimated energy of activation ~ 135 kJ/mol) than that of the granule velocities (~ 22 kJ/mol) suggests that cooling-induced suppression of insulin granule movement is attributable to factors other than reduced motor protein adenosine 5'-triphosphatase activity. Indeed, cooling suppresses random granule diffusion by ~ 50%. In the single cell, the number of directed events depends on the extent of granule diffusion. Finally, single-cell exocytosis exhibits a biphasic pattern corresponding to that observed in vivo, and only the component reflecting 2nd phase insulin secretion is affected by cooling. We conclude that random diffusive movement is a prerequisite for directed insulin granule transport and for the recruitment of insulin granules released during 2nd phase insulin secretion.
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| 30. |
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| 31. |
- Lindquist, Emelie, et al.
(författare)
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Vesicles Are Persistent Features of Different Plastids
- 2016
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Ingår i: Traffic. - : Wiley. - 1398-9219. ; 17:10, s. 1125-1138
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Tidskriftsartikel (refereegranskat)abstract
- Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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| 32. |
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| 33. |
- Potokar, Maja, et al.
(författare)
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Cytoskeleton and vesicle mobility in astrocytes.
- 2007
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Ingår i: Traffic (Copenhagen, Denmark). - : Wiley. - 1398-9219. ; 8:1, s. 12-20
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Tidskriftsartikel (refereegranskat)abstract
- Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.
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| 34. |
- Sciambi, C. J., et al.
(författare)
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A bidirectional kinesin motor in live Drosophila embryos
- 2005
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Ingår i: Traffic. - 1398-9219. ; 6:11, s. 1036-1046
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Tidskriftsartikel (refereegranskat)abstract
- Spindle assembly and elongation involve poleward and away-from-the-pole forces produced by microtubule dynamics and spindle-associated motors. Here, we show that a bidirectional Drosophila Kinesin-14 motor that moves either to the microtubule plus or minus end in vitro unexpectedly causes only minor spindle defects in vivo. However, spindles of mutant embryos are longer than wild type, consistent with increased plus-end motor activity. Strikingly, suppressing spindle dynamics by depriving embryos of oxygen causes the bidirectional motor to show increased accumulation at distal or plus ends of astral microtubules relative to wild type, an effect not observed for a mutant motor defective in motility. Increased motor accumulation at microtubule plus ends may be due to increased slow plus-end movement of the bidirectional motor under hypoxia, caused by perturbation of microtubule dynamics or inactivation of the only other known Drosophila minus-end spindle motor, cytoplasmic dynein. Negative-stain electron microscopy images are consistent with highly cooperative motor binding to microtubules, and gliding assays show dependence on motor density for motility. Mutant effects of the bidirectional motor on spindle function may be suppressed under normal conditions by motor: motor interactions and minus-end movement induced by spindle dynamics. These forces may also bias wild-type motor movement toward microtubule minus ends in live cells. Our findings link motor : motor interactions to function in vivo by showing that motor density, together with cellular dynamics, may influence motor function in live cells.
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| 35. |
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