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| 3. |
- Haglund, Kaisa, et al.
(författare)
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Cindr interacts with anillin to control cytokinesis in Drosophila melanogaster
- 2010
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Ingår i: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 20:10, s. 944-950
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Tidskriftsartikel (refereegranskat)abstract
- Cytokinesis, the final step of cell division, conventionally proceeds to cell separation by abscission, or complete cytokinesis [1, 2], but may in certain tissues be incomplete, yielding daughter cells that are interconnected in syncytia by stable intercellular bridges [3]. The mechanisms that determine complete versus incomplete cytokinesis are not known. Here we report a novel in vivo role of the Drosophila CD2AP/CIN85 ortholog Cindr in both complete and incomplete cytokinesis. We also show evidence for the presence of persistent intercellular bridges in the major larval imaginal disc epithelia. During conventional division of both cultured and embryonic cells, Cindr localizes to cleavage furrows, intercellular bridges, and midbodies. Moreover, in cells undergoing incomplete cytokinesis in the female germline and the somatic ovarian follicle cell and larval imaginal disc epithelia, Cindr localizes to arrested cleavage furrows and stable intercellular bridges, respectively. In these structures, Cindr colocalizes with the essential cytokinesis regulator Anillin. We show that Cindr interacts with Anillin and that depletion of either Cindr or Anillin gives rise to binucleate cells and fewer intercellular bridges in vivo. We propose that Cindr and Anillin cooperate to promote intercellular bridge stability during incomplete cytokinesis in Drosophila melanogaster.
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| 4. |
- Haglund, Kaisa, 1978-
(författare)
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Ubiquitination and Receptor Endocytosis
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein ubiquitination is an evolutionary conserved mechanism that controls a wide variety of cellular functions. Polyubiquitinated proteins are generally degraded in the proteasome, whereas monoubiquitination controls various other cellular processes, including endocytosis and endosomal sorting.Termination of signaling by activated receptor tyrosine kinases (RTKs) largely occurs via their endocytosis and subsequent lysosomal degradation, processes accompanied by receptor ubiquitination. Cbl family proteins are major ubiquitin ligases that promote RTK ubiquitination and downregulation. We showed that epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors are monoubiquitinated at multiple sites following their ligand-induced activation and that a single ubiquitin is sufficient for both receptor internalization and degradation. Cbl also controls EGF receptor (EGFR) downregulation by binding to CIN85, which recruits endophilins to EGFR/Cbl complexes. In the complex with activated EGFRs, Cbl directs monoubiquitination of CIN85, and the entire complex is targeted for degradation in the lysosome. We propose that multiple monoubiquitination of activated receptors and associated protein complexes ensures proper receptor sorting towards the lysosome. Importantly, the functions of Cbl are also negatively controlled in order to maintain cellular homestasis. Sprouty2 blocks EGFR downregulation by sequestering Cbl from activated EGFRs. We showed that Sprouty2 also associates with CIN85 and that this binding is required for efficient inhibition of EGFR ubiquitination and endocytosis. Cbl is also implicated in other aspects of RTK signaling, including organization of the actin cytoskeleton. We found that growth factor receptor signals promote lamellipodia formation in neuronal cells via a complex containing Cbl, the adaptor protein ArgBP2 and Pyk2. The lamellipodia formation required intact lipid rafts and the recruitment of Crk and PI(3)K to tyrosine phosphorylated Cbl.In conclusion, our findings contribute to a better understanding of monoubiquitin signals in downregulation of RTKs and point at a role of Cbl in the regulation of cytoskeleton dynamics.
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| 5. |
- Hämälistö, Saara, et al.
(författare)
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A ZO-1/α5β1-integrin complex regulates cytokinesis downstream of PKCε in NCI-H460 cells plated on fibronectin
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:8, s. e70696-
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Tidskriftsartikel (refereegranskat)abstract
- Recently, we demonstrated that integrin adhesion to the extracellular matrix at the cleavage furrow is essential for cytokinesis of adherent cells. Here, we report that tight junction protein ZO-1 (Zonula Occludens-1) is required for successful cytokinesis in NCI-H460 cells plated on fibronectin. This function of ZO-1 involves interaction with the cytoplasmic domain of α5-integrin to facilitate recruitment of active fibronectin-binding integrins to the base of the cleavage furrow. In the absence of ZO-1, or a functional ZO-1/α5β1-integrin complex, proper actin-dependent constriction between daughter cells is impaired and cells fail cytokinesis. Super-resolution microscopy reveals that in ZO-1 depleted cells the furrow becomes delocalized from the matrix. We also show that PKCε-dependent phosphorylation at Serine168 is required for ZO-1 localization to the furrow and successful cell division. Altogether, our results identify a novel regulatory pathway involving the interplay between ZO-1, α5-integrin and PKCε in the late stages of mammalian cell division.
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| 6. |
- Johansson, Lars-Olof
(författare)
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Engaged in digital service innovation
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The research in this thesis has digital services innovation to support Human-Centred Service Systems (HCSSs) from a practice theory perspective as a foundation. Digital service innovation is understood as service system reconfiguration due to digitalization, with the aim to change the service systemsin a way that increases the value for the involved actors. There are several challenges in digital service innovation; one of the challenges is to address value for a Human-Centered Service System (HCSS), especially since value is the outcome that is determined by the beneficiary. Another challenge is the complexity of sharing and translation of knowledge among heterogeneous actors. The interaction among the involved actors is crucial to understand because it is through human interaction that knowledge is shared and generated.The research has been guided by two research questions: (1) What constitutes value in HCSSs? And (2) How can perceptions of value be aligned in digital service innovation? The presented research expands our understanding of digital service innovation in HCSSs supporting everyday life from a practice perspective. The overall research approach has been engaged scholarship, where the attached insider perspective has been the main focus. The empirical data is collected in two innovation projects (FIND and Free2Ride), the data comes from activities within the projects such as workshops and interviews. One finding in the thesis is the interplay between different levels of value during digital service innovation. Another finding is that beneficiaries and developer stake initiatives to share and translate knowledge. The main contribution of the research is a set of digital service innovation principles. Temporal brokering that leads to leaps in the process of reaching a common understanding and the importance of a learning dimension regarding the roles taken by service beneficiaries are also contributions in this thesis. The research also contributes an exemplification of how learning theories have been applied in order to understand digital service innovation. There are also practical contributions directed to those involved in digital service innovation on a tactical or strategic level. Future research could approach digital service innovation of HCSSs inother service systems and with other perspectives from the practice theory research.
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| 7. |
- Kaur, Namrita, et al.
(författare)
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TECPR1 is activated by damage-induced sphingomyelin exposure to mediate noncanonical autophagy
- 2023
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Ingår i: EMBO Journal. - : EMBO Press. - 0261-4189 .- 1460-2075. ; 42:17
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Tidskriftsartikel (refereegranskat)abstract
- Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin-like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1-containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1-containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage-induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1-ATG5-ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1-ATG5-ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure.
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| 8. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 9. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes
- 2008
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Ingår i: Autophagy. - : Landes Bioscience. - 1554-8627 .- 1554-8635. ; 4:2, s. 151-175
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Forskningsöversikt (refereegranskat)abstract
- Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
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| 10. |
- Knævelsrud, Helene, et al.
(författare)
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Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation
- 2013
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 202:2, s. 331-349
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Tidskriftsartikel (refereegranskat)abstract
- The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.
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| 11. |
- Knævelsrud, Helene, et al.
(författare)
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The membrane-remodeling PX-BAR protein SNX18 is required for autophagy
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Autophagy is a catabolic pathway targeting cytoplasmic material for lysosomal degradation,thereby protecting cells from accumulation of toxic components and enabling cells to survivescarce nutrient supplies. Macroautophagy is characterized by the sequestration of cytoplasmicmaterial into double-membrane vesicles, but the membrane remodeling events required forformation of autophagic vesicles are still not completely understood. However, the class IIIphosphatidylinositol 3-kinase (PI3K)/Vps34 complex and phosphatidylinositol-3-phosphate(PI3P) are of core importance to induction of autophagy. Since PX domain containingproteins are known to bind PI3P and other phosphoinositides and mediate membraneremodeling and trafficking events, we performed an imaging-based siRNA screen targetingPX domain proteins using formation of GFP-LC3 positive autophagosomes as a read-out.The PX-BAR protein SNX18 was found to strongly inhibit autophagosome formation. In linewith this, overexpression of SNX18 increased LC3 lipidation and GFP-LC3 spot formationand we demonstrate that membrane binding of SNX18 is required for efficientautophagosome formation. Moreover, SNX18 colocalizes and interacts with the autophagyassociatedproteins LC3 and TBK1. Our study identified the PX-BAR protein SNX18 to beinvolved in membrane events required for autophagosome formation.
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| 12. |
- Migliano, Simona M., et al.
(författare)
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Removal of hypersignaling endosomes by simaphagy
- 2024
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Ingår i: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 20:4, s. 769-791
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Tidskriftsartikel (refereegranskat)abstract
- Activated transmembrane receptors continue to signal following endocytosis and are only silenced upon ESCRT-mediated internalization of the receptors into intralumenal vesicles (ILVs) of the endosomes. Accordingly, endosomes with dysfunctional receptor internalization into ILVs can cause sustained receptor signaling which has been implicated in cancer progression. Here, we describe a surveillance mechanism that allows cells to detect and clear physically intact endosomes with aberrant receptor accumulation and elevated signaling. Proximity biotinylation and proteomics analyses of ESCRT-0 defective endosomes revealed a strong enrichment of the ubiquitin-binding macroautophagy/autophagy receptors SQSTM1 and NBR1, a phenotype that was confirmed in cell culture and fly tissue. Live cell microscopy demonstrated that loss of the ESCRT-0 subunit HGS/HRS or the ESCRT-I subunit VPS37 led to high levels of ubiquitinated and phosphorylated receptors on endosomes. This was accompanied by dynamic recruitment of NBR1 and SQSTM1 as well as proteins involved in autophagy initiation and autophagosome biogenesis. Light microscopy and electron tomography revealed that endosomes with intact limiting membrane, but aberrant receptor downregulation were engulfed by phagophores. Inhibition of autophagy caused increased intra- and intercellular signaling and directed cell migration. We conclude that dysfunctional endosomes are surveyed and cleared by an autophagic process, simaphagy, which serves as a failsafe mechanism in signal termination.
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| 13. |
- Nahse, Viola, et al.
(författare)
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ATPase activity of DFCP1 controls selective autophagy
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- The endoplasmic reticulum protein DFCP1 is found on omegasomes implicated in autophagosome biogenesis, but its function has remained unknown. Here, Nahse et al. show that DFCP1 is an ATPase that mediates selective autophagy by promoting constriction of large omegasomes. Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that is activated by membrane binding and dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 has a minor effect on bulk autophagic flux, DFCP1 is required to maintain the autophagic flux of p62 under both fed and starved conditions, and this is dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localize to forming omegasomes, these omegasomes fail to constrict properly in a size-dependent manner. Consequently, the release of nascent autophagosomes from large omegasomes is markedly delayed. While knockout of DFCP1 does not affect bulk autophagy, it inhibits selective autophagy, including aggrephagy, mitophagy and micronucleophagy. We conclude that DFCP1 mediates ATPase-driven constriction of large omegasomes to release autophagosomes for selective autophagy.
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| 14. |
- Rusten, Tor Erik, et al.
(författare)
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Fab1 phosphatidylinositol 3-phosphate 5-kinase controls trafficking but not silencing of endocytosed receptors.
- 2006
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 17:9, s. 3989-4001
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Tidskriftsartikel (refereegranskat)abstract
- The trafficking of endocytosed receptors through phosphatidylinositol 3-phosphate [PtdIns(3)P]-containing endosomes is thought to attenuate their signaling. Here, we show that the PtdIns(3)P 5-kinase Fab1/PIKfyve controls trafficking but not silencing of endocytosed receptors. Drosophila fab1 mutants contain undetectable phosphatidylinositol 3,5-bisphosphate levels, show profound increases in cell and organ size, and die at the pupal stage. Mutant larvae contain highly enlarged multivesicular bodies and late endosomes that are inefficiently acidified. Clones of fab1 mutant cells accumulate Wingless and Notch, similarly to cells lacking Hrs, Vps25, and Tsg101, components of the endosomal sorting machinery for ubiquitinated membrane proteins. However, whereas hrs, vps25, and tsg101 mutant cell clones accumulate ubiquitinated cargo, this is not the case with fab1 mutants. Even though endocytic receptor trafficking is impaired in fab1 mutants, Notch, Wingless, and Dpp signaling is unaffected. We conclude that Fab1, despite its importance for endosomal functions, is not required for receptor silencing. This is consistent with the possibility that Fab1 functions at a late stage in endocytic receptor trafficking, at a point when signal termination has occurred.
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