Oddmund Bakke - Academia.edu (original) (raw)
Papers by Oddmund Bakke
Journal of Cell Science, 2021
Rab5 and Rab7a are the main determinants of early and late endosomes and are important regulators... more Rab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch, where Rab5 is gradually exchanged by Rab7a on the same endosome. Here, we provide new insight into the mechanism of endosomal maturation, for which we have discovered a stepwise Rab5 detachment, sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrates a diffusion-like first-phase exchange between the cytosol and the endosomal membrane, and a second phase, in which Rab5 converges into specific domains that detach as a Rab5 indigenous endosome. Consequently, we show that early endosomal maturation regulated through the Rab5-to-Rab7a switch induces the formation of new fully functional Rab5-positive early endosomes. Progression through stepwise early endosomal maturation regulates the direction of transport ...
Journal of Cell Science, 2021
Lysosomal signaling facilitates the migration of immune cells by releasing Ca2+ to activate the a... more Lysosomal signaling facilitates the migration of immune cells by releasing Ca2+ to activate the actin-based motor myosin II at the cell rear. However, how the actomyosin cytoskeleton physically associates to lysosomes is unknown. We have previously identified myosin II as a direct interactor of Rab7b, a small GTPase that mediates the transport from late endosomes/lysosomes to the trans-Golgi network (TGN). Here, we show that Rab7b regulates the migration of dendritic cells (DCs) in one- and three-dimensional environments. DCs are immune sentinels that transport antigens from peripheral tissues to lymph nodes to activate T lymphocytes and initiate adaptive immune responses. We found that the lack of Rab7b reduces myosin II light chain phosphorylation and the activation of the transcription factor EB (TFEB), which controls lysosomal signaling and is required for fast DC migration. Furthermore, we demonstrate that Rab7b interacts with the lysosomal Ca2+ channel TRPML1 (also known as MC...
ABSTRACTInvariant chain (Ii) is traditionally known as the dedicated MHCII chaperone. Recent repo... more ABSTRACTInvariant chain (Ii) is traditionally known as the dedicated MHCII chaperone. Recent reports have broadened our understanding about various tasks that Ii plays including its physiological role in MHCI cross-presentation. Ii bound MHCI via the MHCII scaffolding CLIP peptide may facilitate MHCI trafficking to the endosomal pathway. The sorting function of Ii depends on two leucine-based sorting signals present in the cytoplasmic tail that acts as binding sites for the adaptor proteins AP-1/AP-2. Here we increased the Ii cross-presentation potency by replacing these with an AP3 motif resulting an efficient transport of Ii from TGN to late endosomes. We also replaced the CLIP region of li with a therapeutically relevant peptide, MART-1. We found the Ii AP3mutant-MART1 construct was capable of loading MHCI and stimulate specific T-cell response more efficiently than the wild type counterpart. The results show that Ii with an AP3 binding sorting motif carrying peptide epitope(s) c...
Journal of Cell Science, 2018
Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active... more Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active state and a GDP-bound inactive state. This cycle is regulated by guanine-nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Several efforts have been made in connecting the correct GEFs and GAPs to their specific Rab. Here we aimed to identify GAPs for Rab7b, the small GTPase involved in transport from late endosomes to the trans-Golgi. An siRNA screen targeting proteins containing TBC domains critical for Rab GAPs was performed and coupled to a phenotypic read-out that visualized the distribution of Rab7b. Silencing TBC1D5 provided the strongest phenotype. TBC1D5 was subsequently validated in various in vitro and cell based assays. It localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b in vitro, which is further increased by retromer proteins. Inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive...
Journal of Steroid Biochemistry, 1987
Biochemical Society Transactions, 1987
treatment with 10 pwretinoic acid. The cytokeratin filaments in clone 1 seemed thicker and more d... more treatment with 10 pwretinoic acid. The cytokeratin filaments in clone 1 seemed thicker and more distinct. There was a 4-fold increase in the content of cytokeratin measured with an enzyme-linked immunosorbent assay (ELISA) technique (data not shown). Treatment with retinoic acid led to a flat and well-spread morphology concomitant with changes in the cytokeratin filaments. Retinoic-acid treatment of the negative clone resulted in the appearance of cytokeratin filaments. The filament structure of the two clones became more alike. The intermediate filament-organizing centre seen in cytokeratinpositive control cells was no longer so distinct and the staining of desmosomes had disappeared. After retinoicacid exposure for 8 days, more than 95% of the negative cells became positive. The distribution of microfilament actin in the same clones is shown in Fig. 2. The cytokeratin-positive cells (Fig. 2A) had a uniform distribution of actin filaments with many stress fibres. In the cytokeratin-negative cells (Fig. 2B), there were no stress fibres, but many actin-filament-rich aggregates and ruffles (arrow). The effect of retinoic-acid treatment was most apparent for clone 9. Actin-containing ruffles and aggregates had disappeared and the actin filaments displayed stress fibres instead. The two clones became much more alike. It is suggested that formation of actin stress fibres is due to increased adhesiveness to the substratum (Paulin et al., 1978). The flattening of the retinoic-acid-treated cells also indicates a better attachment to the plastic surface in our experiments. Kitajima et al. (1986) suggest that the stability and architecture of keratin-filament organization may be supported by the microfilament rather than the microtubule cytoskeleton. We did not observe any difference in microtubule organization between the two clones of the NHIK 3025 cell line. Nor does there appear to be any reorganization of the microtubule network after retinoic-acid treatment (data not shown). There was, however, a correlation between the appearance of cytokeratin filaments in the cytokeratinnegative clone and reorganization of actin filaments from ruffles and aggregates to stress fibres in the same cells after retinoic-acid treatment. This may indicate a connection between the two cytoskeleton structures in NHIK 3025 cells.
Journal of Experimental Medicine, 1993
Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molec... more Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the chara...
Journal of Experimental Medicine, 2003
Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 mo... more Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 molecules. Here we show that the adaptor protein complex 3 (AP-3) is required for the efficient presentation of glycolipid antigens that require internalization and processing. AP-3 interacts with mouse CD1d, and cells from mice deficient for AP-3 have increased cell surface levels of CD1d and decreased expression in late endosomes. Spleen cells from AP-3–deficient mice have a reduced ability to present glycolipids to natural killer T (NKT) cells. Furthermore, AP-3–deficient mice have a significantly reduced NKT cell population, although this is not caused by self-tolerance that might result from increased CD1d surface levels. These data suggest that the generation of the endogenous ligand that selects NKT cells may also be AP-3 dependent. However, the function of MHC class II–reactive CD4+ T lymphocytes is not altered by AP-3 deficiency. Consistent with this divergence from the class II p...
Journal of Cell Science, 2012
Organelles in the endocytic pathway interact and communicate through the crucial mechanisms of fu... more Organelles in the endocytic pathway interact and communicate through the crucial mechanisms of fusion and fission. However, any specific link between fusion and fission has not yet been determined. To study the endosomal interactions with high spatial and temporal resolution, the endosomes were enlarged through two mechanistically different methods. Either expression of the MHC class II associated chaperone Invariant chain or Rab5 increased the fusion rate of early endosomes and resulted in enlarged endosomes. Fast homotypic fusions were studied, and immediately after the fusion a highly active and specific tubule formation and fission was observed. These explosive tubule formations following fusion seemed to be a direct effect of fusion. The tubule formations were dependent on microtubule interactions, and specifically controlled by Kif16b and dynein. Our results show that fusion of endosomes is a rapid process that destabilize the membrane and instantly induce molecular motor driv...
Journal of Biological Chemistry, 2002
Cytoplasmic tails of LIMPII and the invariant chain contain similar leucine-based sorting signals... more Cytoplasmic tails of LIMPII and the invariant chain contain similar leucine-based sorting signals, but the invariant chain interacts only with AP1 and AP2, whereas LIMPII interacts strongly with AP3. In a series of in vitro experiments, we investigated the effect of residues upstream of the leucine pairs and demonstrated that these residues determine adapter binding, and certain residues favor interactions with AP3. Furthermore, constructs that interacted stronger with AP3 interacted weakly with AP1 and vice versa. Exchanging residues upstream of the leucine-based signal in LIMPII with those of the invariant chain reduced LIMPII binding to AP3 in vitro, and in vivo the corresponding LIM-PII mutant was rerouted via the plasma membrane like the invariant chain. These preferential interactions of different leucine signals with different AP complexes may thus be the determining step sorting proteins from the trans-Golgi network to their final destinations. Proteins that interact with AP3 are sorted directly to endosomes/lysosomes, whereas proteins that interact with AP1 are sorted via a different route. At the same time, constructs that exhibited specificity for either AP1 or AP3 might still interact with AP2, suggesting that AP2 may recognize a wider variety of leucine signals. This is consistent with the suggested role of AP2 in internalization of proteins containing general leucine-based signals, including proteins that have been missorted to the plasma membrane.
Scientific Reports, 2017
Activation of EGF-R and PDGF-R triggers autophosphorylation and the recruitment of Eps15 and Hrs.... more Activation of EGF-R and PDGF-R triggers autophosphorylation and the recruitment of Eps15 and Hrs. These two endosomal proteins are important for specific receptor sorting. Hrs is recruiting ubiquitinated receptors to early endosomes to further facilitate degradation through the ESCRT complex. Upon receptor activation Hrs becomes phosphorylated and is relocated to the cytosol, important for receptor degradation. In this work we have studied the endosomal binding dynamics of Eps15 and Hrs upon EGF-R and PDGF-R stimulation. By analysing the fluorescence intensity on single endosomes after ligand stimulation we measured a time-specific decrease in the endosomal fluorescence level of Eps15-GFP and Hrs-YFP. Through FRAP experiments we could further register a specific change in the endosomal-membrane to cytosol binding properties of Eps15-GFP and Hrs-YFP. This specific change in membrane fractions proved to be a redistribution of the immobile fraction, which was not shown for the phosphorylation deficient mutants. We here describe a mechanism that can explain the previously observed relocation of Hrs from the endosomes to cytosol after EGF stimulation and show that Eps15 follows a similar mechanism. Moreover, this specific redistribution of the endosomal protein binding dynamics proved to be of major importance for receptor degradation. Receptor tyrosine kinases (RTK) play an important role in the control of fundamental cellular processes, including the cell cycle, cell migration, cell metabolism and survival, cell proliferation and differentiation 1,2. Binding of ligand is the activation signal for all the RTKs, which triggers trans-autophosphorylation of the receptor. This step is crucial for RTK dependent activation and recruitment of a variety of signalling proteins. Binding of ligand leads to ubiquitination of the receptor and recruitment of Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and Epidermal growth factor receptor pathway substrate 15 (Eps15). This process targets RTKs to the lumen of multivesicular bodies (MVBs) for lysosomal degradation 3. Sorting of membrane receptors into MVBs is orchestrated by the sequential recruitment of members of the endosomal-sorting complex required for transport (ESCRT complex) (for review see 3). Epidermal growth factor receptor substrate 15 (Eps15) is an adaptor protein important for endocytosis 4. The N-terminal Eps15 homology (EH) domains bind NPF motifs on a variety of other endocytic adaptor proteins. The central coiled-coil domain mediates Eps15 oligomerization and binding to other proteins including Hrs. The DPF domain of Eps15 binds to adaptor protein-2 (AP-2) and is important in the formation of clathrin-coated vesicles (CCV) 4,5. In the C-terminus of Eps15 the two ubiquitin interacting motifs (UIM domains) are located. Eps15 has been reported to bind directly to ubiquitinated EGF-R through these UIM domains 6. Activation of EGF-R triggers both monoubiquitination and phosphorylation of Eps15 7,8. Hrs is a 115-kDa multidomain coat protein that binds to the endosomal membrane either through the FYVE-(Fab-1, YGL023, Vps27, and EEA1) or the coiled-coiled domain 9-11. Hrs recognizes ubiquitinated receptors through the ubiquitin interacting motif (UIM), and together with signal-transduction adaptor-molecule (STAM) it acts as part of the sorting machinery for degradation via the ESCRT machinery 12,13. Upon EGF-R activation, Hrs is tyrosine phosphorylated and monoubiquitinated 14,15. In this study we have described a downstream effect of EGF-R ligand binding on the phosphorylation and membrane binding kinetics of Hrs and Eps15. To facilitate the analysis of the membrane binding kinetics on single endosomes we enlarged the endosomal size by transfecting the cells with the major histocompatibility complex class-II associated invariant chain (Ii) under the control of an inducible metallothionein promotor.
Journal of Cell …, 2010
Rab7b is a recently identified member of the Rab GTPase protein family and has high similarity to... more Rab7b is a recently identified member of the Rab GTPase protein family and has high similarity to Rab7. It has been reported that Rab7b is lysosome associated, that it is involved in monocytic differentiation and that it promotes lysosomal degradation of TLR4 and TLR9. Here we ...
Ii, invariant chain; G␣M, goat anti-mouse; MDCK, Madin-Darby canine kidney; EM, electron microsco... more Ii, invariant chain; G␣M, goat anti-mouse; MDCK, Madin-Darby canine kidney; EM, electron microscopy; AP-EE, apical early endosomes; BL-EE, basolateral early endosomes; LE-1h, late endosomes containing both 1 h apical and basolateral endocytosed marker; mvb, multivesicular bodies; mvb-1h, multivesicular bodies with 1 h uptake of endocytosed marker; LE-ON, late endosomes with endocytosed markers after both 1 h and overnight incubation; MIIC, MHC class II compartments; UV-flu, UV-inactivated influenza virus particles.
Class II histocompatibility molecules associate with peptides derived from antigens that are proc... more Class II histocompatibility molecules associate with peptides derived from antigens that are processed in endocytic compartments. Antigen presentation to class II-restricted T cells generally requires newly synthesized class II molecules, associated invariant chain, and HLA-DM. Exceptions to these rules have been reported, but without description of an underlying mechanism. Here we show that presentation of immunodominant epitopes in the haemagglutinin protein of influenza virus and in myelin basic protein correlates with recycling of surface HLA-DR molecules. Truncation of either one of the alpha or beta cytoplasmic tails virtually eliminated internalization of HLA-DR molecules and presentation of haemagglutinin from inactive virus particles. In contrast, the invariant chain-dependent presentation of matrix antigen from the same virus particles was unaffected by these truncations. Thus HLA-DR cytoplasmic tails are not required for the conventional presentation pathway, but jointly contribute a signal for an alternative pathway involving internalization of HLA-DR molecules.
Journal of Cell Science
The Invariant chain (Ii, CD74) is a multifunctional regulator of adaptive immune responses and re... more The Invariant chain (Ii, CD74) is a multifunctional regulator of adaptive immune responses and responsible for sorting MHC-I, MHC-II and other Ii-associated molecules to a specific endosomal pathway. When Ii is expressed, endosomal maturation and proteolytic degradation of proteins are delayed and in non-antigen presenting cells the endosomal size increase, but he molecular mechanisms are not known. We identified that a SNARE, Vti1b, is essential for regulating these Ii induced effects. Vti1b binds to Ii and Vti1b is localized at the contact sites of fusing Ii positive endosomes. Furthermore, a tailless Ii that is not internalized from the plasma membrane relocates Vti1b to the plasma membrane. KO of Ii in an antigen presenting cell line was found to speed up endosomal maturation and silencing of Vti1b inhibits the Ii induced maturation delay. Our results suggest that Ii, by interacting with the SNARE Vti1b in antigen presenting cells, direct specific Ii associated SNARE mediated fu...
Journal of Cell Science
Despite progress made in confocal microscopy, even fast systems still have insufficient temporal ... more Despite progress made in confocal microscopy, even fast systems still have insufficient temporal resolution for detailed live cell volume imaging, such as tracking rapid movement of membrane vesicles in three-dimensional space. Depending on the shortfall, this may result in undersampling and/or motion artifacts that ultimately limit the quality of the imaging data. By sacrificing detailed information in the Z-direction, we propose a new imaging modality that involves capturing fast “projections” from the field of depth which shortens imaging time by approximately an order of magnitude as compared to standard volumetric confocal imaging. With faster imaging, radiation exposure to the sample is reduced, resulting in less fluorophore photobleaching and potential photodamage. The implementation minimally requires two synchronized control signals that drive a piezo stage and trigger the camera exposure. The device generating the signals has been tested on spinning disk confocals and inst...
Nature Methods
Measuring rapid kinetics of proteins in living cells requires the capability for fast, accurate m... more Measuring rapid kinetics of proteins in living cells requires the capability for fast, accurate measurements. Researchers hoping to obtain precise kinetic data from fluorescence recovery after photobleaching or photoactivation experiments need an easily controllable system for stimulation of a specific region and subsequent imaging, and the Olympus FluoView FV1000 confocal laser scanning microscope (cLSM) with SIM scanner makes this possible. We describe here how to precisely measure off rates using a cytosolic photoactivatable probe that binds endosomal membranes.
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics
OncoImmunology
Eradication of tumors by the immune system relies on the efficient activation of a T-cell respons... more Eradication of tumors by the immune system relies on the efficient activation of a T-cell response. For many years, the main focus of cancer immunotherapy has been on cytotoxic CD8 T-cell. However, stimulation of CD4 helper T cells is critical for the promotion and maintenance of immune memory, thus a good vaccine should evoke a two-dimensional T-cell response. The invariant chain (Ii) is required for the MHC class II heterodimer to be correctly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells (APC). We previously showed that by replacing the Ii CLIP peptide by an MHC-I cancer peptide, we could efficiently load MHC-I. This prompted us to test whether longer cancer peptides could be loaded on both MHC classes and whether such peptides could be accommodated in the CLIP region of Ii. We here present data showing that expanding the CLIP replacement size leads to T-cell activation. We demonstrate by using long peptides that APCs can present peptides from the same Ii molecule on both MHC-I and-II. In addition, we present evidence that antigen presentation after Ii-loading was superior to an ER-targeted minigene construct, suggesting that ER-localization was not sufficient to obtain efficient MHC-II loading. Finally, we verified that Ii-expressing dendritic cells could prime CD4 + and CD8 + T cells from a naïve population. Taken together our study demonstrates that CLIP peptide replaced Ii constructs fulfill some of the major requirements for an efficient vector for cancer vaccination.
PLOS ONE
Due to their antifungal activity, chitosan and its derivatives have potential to be used for trea... more Due to their antifungal activity, chitosan and its derivatives have potential to be used for treating yeast infections in humans. However, to be considered for use in human medicine, it is necessary to control and know the chemical composition of the compound, which is not always the case for polymeric chitosans. Here, we analyze the antifungal activity of a soluble and well-defined chito-oligosaccharide (CHOS) with an average polymerization degree (DP n) of 32 and fraction of acetylation (F A) of 0.15 (C32) on 52 medically relevant yeast strains. Minimal inhibitory concentrations (MIC) varied widely among yeast species, strains and isolates (from > 5000 to < 9.77 μg mL-1) and inhibition patterns showed a time-and dose-dependencies. The antifungal activity was predominantly fungicidal and was inversely proportional to the pH, being maximal at pH 4.5, the lowest tested pH. Furthermore, antifungal effects of CHOS fractions with varying average molecular weight indicated that those fractions with an intermediate degree of polymerization, i.e. DP 31 and 54, had the strongest inhibitory effects. Confocal imaging showed that C32 adsorbs to the cell surface, with subsequent cell disruption and accumulation of C32 in the cytoplasm. Thus, C32 has potential to be used as a therapy for fungal infections.
Journal of Cell Science, 2021
Rab5 and Rab7a are the main determinants of early and late endosomes and are important regulators... more Rab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch, where Rab5 is gradually exchanged by Rab7a on the same endosome. Here, we provide new insight into the mechanism of endosomal maturation, for which we have discovered a stepwise Rab5 detachment, sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrates a diffusion-like first-phase exchange between the cytosol and the endosomal membrane, and a second phase, in which Rab5 converges into specific domains that detach as a Rab5 indigenous endosome. Consequently, we show that early endosomal maturation regulated through the Rab5-to-Rab7a switch induces the formation of new fully functional Rab5-positive early endosomes. Progression through stepwise early endosomal maturation regulates the direction of transport ...
Journal of Cell Science, 2021
Lysosomal signaling facilitates the migration of immune cells by releasing Ca2+ to activate the a... more Lysosomal signaling facilitates the migration of immune cells by releasing Ca2+ to activate the actin-based motor myosin II at the cell rear. However, how the actomyosin cytoskeleton physically associates to lysosomes is unknown. We have previously identified myosin II as a direct interactor of Rab7b, a small GTPase that mediates the transport from late endosomes/lysosomes to the trans-Golgi network (TGN). Here, we show that Rab7b regulates the migration of dendritic cells (DCs) in one- and three-dimensional environments. DCs are immune sentinels that transport antigens from peripheral tissues to lymph nodes to activate T lymphocytes and initiate adaptive immune responses. We found that the lack of Rab7b reduces myosin II light chain phosphorylation and the activation of the transcription factor EB (TFEB), which controls lysosomal signaling and is required for fast DC migration. Furthermore, we demonstrate that Rab7b interacts with the lysosomal Ca2+ channel TRPML1 (also known as MC...
ABSTRACTInvariant chain (Ii) is traditionally known as the dedicated MHCII chaperone. Recent repo... more ABSTRACTInvariant chain (Ii) is traditionally known as the dedicated MHCII chaperone. Recent reports have broadened our understanding about various tasks that Ii plays including its physiological role in MHCI cross-presentation. Ii bound MHCI via the MHCII scaffolding CLIP peptide may facilitate MHCI trafficking to the endosomal pathway. The sorting function of Ii depends on two leucine-based sorting signals present in the cytoplasmic tail that acts as binding sites for the adaptor proteins AP-1/AP-2. Here we increased the Ii cross-presentation potency by replacing these with an AP3 motif resulting an efficient transport of Ii from TGN to late endosomes. We also replaced the CLIP region of li with a therapeutically relevant peptide, MART-1. We found the Ii AP3mutant-MART1 construct was capable of loading MHCI and stimulate specific T-cell response more efficiently than the wild type counterpart. The results show that Ii with an AP3 binding sorting motif carrying peptide epitope(s) c...
Journal of Cell Science, 2018
Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active... more Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active state and a GDP-bound inactive state. This cycle is regulated by guanine-nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Several efforts have been made in connecting the correct GEFs and GAPs to their specific Rab. Here we aimed to identify GAPs for Rab7b, the small GTPase involved in transport from late endosomes to the trans-Golgi. An siRNA screen targeting proteins containing TBC domains critical for Rab GAPs was performed and coupled to a phenotypic read-out that visualized the distribution of Rab7b. Silencing TBC1D5 provided the strongest phenotype. TBC1D5 was subsequently validated in various in vitro and cell based assays. It localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b in vitro, which is further increased by retromer proteins. Inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive...
Journal of Steroid Biochemistry, 1987
Biochemical Society Transactions, 1987
treatment with 10 pwretinoic acid. The cytokeratin filaments in clone 1 seemed thicker and more d... more treatment with 10 pwretinoic acid. The cytokeratin filaments in clone 1 seemed thicker and more distinct. There was a 4-fold increase in the content of cytokeratin measured with an enzyme-linked immunosorbent assay (ELISA) technique (data not shown). Treatment with retinoic acid led to a flat and well-spread morphology concomitant with changes in the cytokeratin filaments. Retinoic-acid treatment of the negative clone resulted in the appearance of cytokeratin filaments. The filament structure of the two clones became more alike. The intermediate filament-organizing centre seen in cytokeratinpositive control cells was no longer so distinct and the staining of desmosomes had disappeared. After retinoicacid exposure for 8 days, more than 95% of the negative cells became positive. The distribution of microfilament actin in the same clones is shown in Fig. 2. The cytokeratin-positive cells (Fig. 2A) had a uniform distribution of actin filaments with many stress fibres. In the cytokeratin-negative cells (Fig. 2B), there were no stress fibres, but many actin-filament-rich aggregates and ruffles (arrow). The effect of retinoic-acid treatment was most apparent for clone 9. Actin-containing ruffles and aggregates had disappeared and the actin filaments displayed stress fibres instead. The two clones became much more alike. It is suggested that formation of actin stress fibres is due to increased adhesiveness to the substratum (Paulin et al., 1978). The flattening of the retinoic-acid-treated cells also indicates a better attachment to the plastic surface in our experiments. Kitajima et al. (1986) suggest that the stability and architecture of keratin-filament organization may be supported by the microfilament rather than the microtubule cytoskeleton. We did not observe any difference in microtubule organization between the two clones of the NHIK 3025 cell line. Nor does there appear to be any reorganization of the microtubule network after retinoic-acid treatment (data not shown). There was, however, a correlation between the appearance of cytokeratin filaments in the cytokeratinnegative clone and reorganization of actin filaments from ruffles and aggregates to stress fibres in the same cells after retinoic-acid treatment. This may indicate a connection between the two cytoskeleton structures in NHIK 3025 cells.
Journal of Experimental Medicine, 1993
Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molec... more Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the chara...
Journal of Experimental Medicine, 2003
Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 mo... more Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 molecules. Here we show that the adaptor protein complex 3 (AP-3) is required for the efficient presentation of glycolipid antigens that require internalization and processing. AP-3 interacts with mouse CD1d, and cells from mice deficient for AP-3 have increased cell surface levels of CD1d and decreased expression in late endosomes. Spleen cells from AP-3–deficient mice have a reduced ability to present glycolipids to natural killer T (NKT) cells. Furthermore, AP-3–deficient mice have a significantly reduced NKT cell population, although this is not caused by self-tolerance that might result from increased CD1d surface levels. These data suggest that the generation of the endogenous ligand that selects NKT cells may also be AP-3 dependent. However, the function of MHC class II–reactive CD4+ T lymphocytes is not altered by AP-3 deficiency. Consistent with this divergence from the class II p...
Journal of Cell Science, 2012
Organelles in the endocytic pathway interact and communicate through the crucial mechanisms of fu... more Organelles in the endocytic pathway interact and communicate through the crucial mechanisms of fusion and fission. However, any specific link between fusion and fission has not yet been determined. To study the endosomal interactions with high spatial and temporal resolution, the endosomes were enlarged through two mechanistically different methods. Either expression of the MHC class II associated chaperone Invariant chain or Rab5 increased the fusion rate of early endosomes and resulted in enlarged endosomes. Fast homotypic fusions were studied, and immediately after the fusion a highly active and specific tubule formation and fission was observed. These explosive tubule formations following fusion seemed to be a direct effect of fusion. The tubule formations were dependent on microtubule interactions, and specifically controlled by Kif16b and dynein. Our results show that fusion of endosomes is a rapid process that destabilize the membrane and instantly induce molecular motor driv...
Journal of Biological Chemistry, 2002
Cytoplasmic tails of LIMPII and the invariant chain contain similar leucine-based sorting signals... more Cytoplasmic tails of LIMPII and the invariant chain contain similar leucine-based sorting signals, but the invariant chain interacts only with AP1 and AP2, whereas LIMPII interacts strongly with AP3. In a series of in vitro experiments, we investigated the effect of residues upstream of the leucine pairs and demonstrated that these residues determine adapter binding, and certain residues favor interactions with AP3. Furthermore, constructs that interacted stronger with AP3 interacted weakly with AP1 and vice versa. Exchanging residues upstream of the leucine-based signal in LIMPII with those of the invariant chain reduced LIMPII binding to AP3 in vitro, and in vivo the corresponding LIM-PII mutant was rerouted via the plasma membrane like the invariant chain. These preferential interactions of different leucine signals with different AP complexes may thus be the determining step sorting proteins from the trans-Golgi network to their final destinations. Proteins that interact with AP3 are sorted directly to endosomes/lysosomes, whereas proteins that interact with AP1 are sorted via a different route. At the same time, constructs that exhibited specificity for either AP1 or AP3 might still interact with AP2, suggesting that AP2 may recognize a wider variety of leucine signals. This is consistent with the suggested role of AP2 in internalization of proteins containing general leucine-based signals, including proteins that have been missorted to the plasma membrane.
Scientific Reports, 2017
Activation of EGF-R and PDGF-R triggers autophosphorylation and the recruitment of Eps15 and Hrs.... more Activation of EGF-R and PDGF-R triggers autophosphorylation and the recruitment of Eps15 and Hrs. These two endosomal proteins are important for specific receptor sorting. Hrs is recruiting ubiquitinated receptors to early endosomes to further facilitate degradation through the ESCRT complex. Upon receptor activation Hrs becomes phosphorylated and is relocated to the cytosol, important for receptor degradation. In this work we have studied the endosomal binding dynamics of Eps15 and Hrs upon EGF-R and PDGF-R stimulation. By analysing the fluorescence intensity on single endosomes after ligand stimulation we measured a time-specific decrease in the endosomal fluorescence level of Eps15-GFP and Hrs-YFP. Through FRAP experiments we could further register a specific change in the endosomal-membrane to cytosol binding properties of Eps15-GFP and Hrs-YFP. This specific change in membrane fractions proved to be a redistribution of the immobile fraction, which was not shown for the phosphorylation deficient mutants. We here describe a mechanism that can explain the previously observed relocation of Hrs from the endosomes to cytosol after EGF stimulation and show that Eps15 follows a similar mechanism. Moreover, this specific redistribution of the endosomal protein binding dynamics proved to be of major importance for receptor degradation. Receptor tyrosine kinases (RTK) play an important role in the control of fundamental cellular processes, including the cell cycle, cell migration, cell metabolism and survival, cell proliferation and differentiation 1,2. Binding of ligand is the activation signal for all the RTKs, which triggers trans-autophosphorylation of the receptor. This step is crucial for RTK dependent activation and recruitment of a variety of signalling proteins. Binding of ligand leads to ubiquitination of the receptor and recruitment of Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and Epidermal growth factor receptor pathway substrate 15 (Eps15). This process targets RTKs to the lumen of multivesicular bodies (MVBs) for lysosomal degradation 3. Sorting of membrane receptors into MVBs is orchestrated by the sequential recruitment of members of the endosomal-sorting complex required for transport (ESCRT complex) (for review see 3). Epidermal growth factor receptor substrate 15 (Eps15) is an adaptor protein important for endocytosis 4. The N-terminal Eps15 homology (EH) domains bind NPF motifs on a variety of other endocytic adaptor proteins. The central coiled-coil domain mediates Eps15 oligomerization and binding to other proteins including Hrs. The DPF domain of Eps15 binds to adaptor protein-2 (AP-2) and is important in the formation of clathrin-coated vesicles (CCV) 4,5. In the C-terminus of Eps15 the two ubiquitin interacting motifs (UIM domains) are located. Eps15 has been reported to bind directly to ubiquitinated EGF-R through these UIM domains 6. Activation of EGF-R triggers both monoubiquitination and phosphorylation of Eps15 7,8. Hrs is a 115-kDa multidomain coat protein that binds to the endosomal membrane either through the FYVE-(Fab-1, YGL023, Vps27, and EEA1) or the coiled-coiled domain 9-11. Hrs recognizes ubiquitinated receptors through the ubiquitin interacting motif (UIM), and together with signal-transduction adaptor-molecule (STAM) it acts as part of the sorting machinery for degradation via the ESCRT machinery 12,13. Upon EGF-R activation, Hrs is tyrosine phosphorylated and monoubiquitinated 14,15. In this study we have described a downstream effect of EGF-R ligand binding on the phosphorylation and membrane binding kinetics of Hrs and Eps15. To facilitate the analysis of the membrane binding kinetics on single endosomes we enlarged the endosomal size by transfecting the cells with the major histocompatibility complex class-II associated invariant chain (Ii) under the control of an inducible metallothionein promotor.
Journal of Cell …, 2010
Rab7b is a recently identified member of the Rab GTPase protein family and has high similarity to... more Rab7b is a recently identified member of the Rab GTPase protein family and has high similarity to Rab7. It has been reported that Rab7b is lysosome associated, that it is involved in monocytic differentiation and that it promotes lysosomal degradation of TLR4 and TLR9. Here we ...
Ii, invariant chain; G␣M, goat anti-mouse; MDCK, Madin-Darby canine kidney; EM, electron microsco... more Ii, invariant chain; G␣M, goat anti-mouse; MDCK, Madin-Darby canine kidney; EM, electron microscopy; AP-EE, apical early endosomes; BL-EE, basolateral early endosomes; LE-1h, late endosomes containing both 1 h apical and basolateral endocytosed marker; mvb, multivesicular bodies; mvb-1h, multivesicular bodies with 1 h uptake of endocytosed marker; LE-ON, late endosomes with endocytosed markers after both 1 h and overnight incubation; MIIC, MHC class II compartments; UV-flu, UV-inactivated influenza virus particles.
Class II histocompatibility molecules associate with peptides derived from antigens that are proc... more Class II histocompatibility molecules associate with peptides derived from antigens that are processed in endocytic compartments. Antigen presentation to class II-restricted T cells generally requires newly synthesized class II molecules, associated invariant chain, and HLA-DM. Exceptions to these rules have been reported, but without description of an underlying mechanism. Here we show that presentation of immunodominant epitopes in the haemagglutinin protein of influenza virus and in myelin basic protein correlates with recycling of surface HLA-DR molecules. Truncation of either one of the alpha or beta cytoplasmic tails virtually eliminated internalization of HLA-DR molecules and presentation of haemagglutinin from inactive virus particles. In contrast, the invariant chain-dependent presentation of matrix antigen from the same virus particles was unaffected by these truncations. Thus HLA-DR cytoplasmic tails are not required for the conventional presentation pathway, but jointly contribute a signal for an alternative pathway involving internalization of HLA-DR molecules.
Journal of Cell Science
The Invariant chain (Ii, CD74) is a multifunctional regulator of adaptive immune responses and re... more The Invariant chain (Ii, CD74) is a multifunctional regulator of adaptive immune responses and responsible for sorting MHC-I, MHC-II and other Ii-associated molecules to a specific endosomal pathway. When Ii is expressed, endosomal maturation and proteolytic degradation of proteins are delayed and in non-antigen presenting cells the endosomal size increase, but he molecular mechanisms are not known. We identified that a SNARE, Vti1b, is essential for regulating these Ii induced effects. Vti1b binds to Ii and Vti1b is localized at the contact sites of fusing Ii positive endosomes. Furthermore, a tailless Ii that is not internalized from the plasma membrane relocates Vti1b to the plasma membrane. KO of Ii in an antigen presenting cell line was found to speed up endosomal maturation and silencing of Vti1b inhibits the Ii induced maturation delay. Our results suggest that Ii, by interacting with the SNARE Vti1b in antigen presenting cells, direct specific Ii associated SNARE mediated fu...
Journal of Cell Science
Despite progress made in confocal microscopy, even fast systems still have insufficient temporal ... more Despite progress made in confocal microscopy, even fast systems still have insufficient temporal resolution for detailed live cell volume imaging, such as tracking rapid movement of membrane vesicles in three-dimensional space. Depending on the shortfall, this may result in undersampling and/or motion artifacts that ultimately limit the quality of the imaging data. By sacrificing detailed information in the Z-direction, we propose a new imaging modality that involves capturing fast “projections” from the field of depth which shortens imaging time by approximately an order of magnitude as compared to standard volumetric confocal imaging. With faster imaging, radiation exposure to the sample is reduced, resulting in less fluorophore photobleaching and potential photodamage. The implementation minimally requires two synchronized control signals that drive a piezo stage and trigger the camera exposure. The device generating the signals has been tested on spinning disk confocals and inst...
Nature Methods
Measuring rapid kinetics of proteins in living cells requires the capability for fast, accurate m... more Measuring rapid kinetics of proteins in living cells requires the capability for fast, accurate measurements. Researchers hoping to obtain precise kinetic data from fluorescence recovery after photobleaching or photoactivation experiments need an easily controllable system for stimulation of a specific region and subsequent imaging, and the Olympus FluoView FV1000 confocal laser scanning microscope (cLSM) with SIM scanner makes this possible. We describe here how to precisely measure off rates using a cytosolic photoactivatable probe that binds endosomal membranes.
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics
OncoImmunology
Eradication of tumors by the immune system relies on the efficient activation of a T-cell respons... more Eradication of tumors by the immune system relies on the efficient activation of a T-cell response. For many years, the main focus of cancer immunotherapy has been on cytotoxic CD8 T-cell. However, stimulation of CD4 helper T cells is critical for the promotion and maintenance of immune memory, thus a good vaccine should evoke a two-dimensional T-cell response. The invariant chain (Ii) is required for the MHC class II heterodimer to be correctly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells (APC). We previously showed that by replacing the Ii CLIP peptide by an MHC-I cancer peptide, we could efficiently load MHC-I. This prompted us to test whether longer cancer peptides could be loaded on both MHC classes and whether such peptides could be accommodated in the CLIP region of Ii. We here present data showing that expanding the CLIP replacement size leads to T-cell activation. We demonstrate by using long peptides that APCs can present peptides from the same Ii molecule on both MHC-I and-II. In addition, we present evidence that antigen presentation after Ii-loading was superior to an ER-targeted minigene construct, suggesting that ER-localization was not sufficient to obtain efficient MHC-II loading. Finally, we verified that Ii-expressing dendritic cells could prime CD4 + and CD8 + T cells from a naïve population. Taken together our study demonstrates that CLIP peptide replaced Ii constructs fulfill some of the major requirements for an efficient vector for cancer vaccination.
PLOS ONE
Due to their antifungal activity, chitosan and its derivatives have potential to be used for trea... more Due to their antifungal activity, chitosan and its derivatives have potential to be used for treating yeast infections in humans. However, to be considered for use in human medicine, it is necessary to control and know the chemical composition of the compound, which is not always the case for polymeric chitosans. Here, we analyze the antifungal activity of a soluble and well-defined chito-oligosaccharide (CHOS) with an average polymerization degree (DP n) of 32 and fraction of acetylation (F A) of 0.15 (C32) on 52 medically relevant yeast strains. Minimal inhibitory concentrations (MIC) varied widely among yeast species, strains and isolates (from > 5000 to < 9.77 μg mL-1) and inhibition patterns showed a time-and dose-dependencies. The antifungal activity was predominantly fungicidal and was inversely proportional to the pH, being maximal at pH 4.5, the lowest tested pH. Furthermore, antifungal effects of CHOS fractions with varying average molecular weight indicated that those fractions with an intermediate degree of polymerization, i.e. DP 31 and 54, had the strongest inhibitory effects. Confocal imaging showed that C32 adsorbs to the cell surface, with subsequent cell disruption and accumulation of C32 in the cytoplasm. Thus, C32 has potential to be used as a therapy for fungal infections.