Expression data from murine wildtype and Sh3gl1-/- B cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE172295 | GEO | 2021/07/19
REPOSITORIES: GEO
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Project description:PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked Single Nucleotide Polymorphisms (SNPs) of PICALM to Alzheimer’s disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. GC-MS studies indicated that loss of PICALM increases total cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters cholesterol homeostasis via increased scavenging of cholesterol. Measurement of LDL receptor endocytosis further showed that PICALM deficient cells express higher LDLR levels resulting in higher lipoprotein internalization. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease. Total RNA was isolated (Qiagen RNeasy® Micro kit, Valencia, CA) from four replicates of PICALM knockout cells transfected with control vector, and from four replicates of PICALM knockout cells transfected with PICALM.
2015-06-17 | E-GEOD-64855 | biostudies-arrayexpress
Project description:PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked Single Nucleotide Polymorphisms (SNPs) of PICALM to Alzheimer’s disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. GC-MS studies indicated that loss of PICALM increases total cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters cholesterol homeostasis via increased scavenging of cholesterol. Measurement of LDL receptor endocytosis further showed that PICALM deficient cells express higher LDLR levels resulting in higher lipoprotein internalization. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease.
2015-06-17 | GSE64855 | GEO
Project description:Clathrin-mediated endocytosis is essential for a wide range of cellular functions. We used a multi- step siRNA-based screening strategy to identify novel regulators of the first step of clathrin- mediated endocytosis, formation of clathrin-coated vesicles (CCVs) at the plasma membrane. A primary genome-wide screen identified 334 hits that caused accumulation of CCV cargo on the cell surface. A secondary screen identified 92 hits that inhibited cargo uptake and/or altered the morphology of clathrin-coated structures. The hits include components of four functional complexes: coat proteins, V-ATPase subunits, spliceosome-associated proteins, and acetyltransferase subunits. Electron microscopy revealed that V-ATPase depletion caused the cell to form aberrant non-constricted clathrin-coated structures at the plasma membrane. The V- ATPase knockdown phenotype was rescued by addition of exogenous cholesterol, indicating that the knockdown blocks clathrin-mediated endocytosis by preventing cholesterol from recycling from endosomes back to the plasma membrane. The microarray analysis was performed to test whether the siRNA targets are expressed in the cell lines used in the screen. For the microarray analysis of gene expression, the two cell lines used in the study were analyzed in duplicate: HeLa-YXXΦ and HeLa-FXNPXY (Hela-M cells expressing a CD8-YXXΦ and CD8-FXNPXY constructs respectively). YXXΦ and FXNPXY are motifs for clathrin-mediated endocytosis.
2012-12-01 | E-GEOD-39954 | biostudies-arrayexpress
Project description:Clathrin-mediated endocytosis is essential for a wide range of cellular functions. We used a multi- step siRNA-based screening strategy to identify novel regulators of the first step of clathrin- mediated endocytosis, formation of clathrin-coated vesicles (CCVs) at the plasma membrane. A primary genome-wide screen identified 334 hits that caused accumulation of CCV cargo on the cell surface. A secondary screen identified 92 hits that inhibited cargo uptake and/or altered the morphology of clathrin-coated structures. The hits include components of four functional complexes: coat proteins, V-ATPase subunits, spliceosome-associated proteins, and acetyltransferase subunits. Electron microscopy revealed that V-ATPase depletion caused the cell to form aberrant non-constricted clathrin-coated structures at the plasma membrane. The V- ATPase knockdown phenotype was rescued by addition of exogenous cholesterol, indicating that the knockdown blocks clathrin-mediated endocytosis by preventing cholesterol from recycling from endosomes back to the plasma membrane. The microarray analysis was performed to test whether the siRNA targets are expressed in the cell lines used in the screen.
2012-12-01 | GSE39954 | GEO
Project description:As the unique cell type in articular cartilage, chondrocyte senescence is a crucial cellular event contributing to osteoarthritis development. Here we show that clathrin-mediated endocytosis and activation of Notch signaling promotes chondrocyte senescence and osteoarthritis development, which is negatively regulated by myosin light chain 3. Myosin light chain 3 (MYL3) protein levels decline sharply in senescent chondrocytes of cartilages from model mice and osteoarthritis (OA) patients. Conditional deletion of Myl3 in chondrocytes significantly promoted, whereas intra-articular injection of adeno-associated virus overexpressing MYL3 delayed, OA progression in male mice. MYL3 deficiency led to enhanced clathrin-mediated endocytosis by promoting the interaction between myosin VI and clathrin, further inducing the internalization of Notch and resulting in activation of Notch signaling in chondrocytes. Pharmacologic blockade of clathrin-mediated endocytosis-Notch signaling prevented MYL3 loss-induced chondrocyte senescence and alleviated OA progression in mice. Our results establish a previously unknown mechanism essential for cellular senescence and provide a potential therapeutic direction for OA.
2023-09-07 | PXD044684 | Pride
Project description:Commensal bacteria influence host physiology, including immune responses, without invading host tissues. We show that proteins from segmented filamentous bacteria (SFB), which are immunomodulatory commensal microbes, are transferred into intestinal epithelial cells by adhesion-directed endocytosis that is distinct from the clathrin-dependent endocytosis of invasive pathogens. SFB transfer microbial cell wall-associated proteins, including an antigen that stimulates mucosal Th17 cell differentiation, into the cytosol of epithelial cells. Removal of CDC42 activity in vivo led to disruption of endocytosis induced by SFB, decreased epithelial antigen acquisition with consequent loss of immune modulation by SFB-specific CD4 T cells and mucosal Th17 cells. Our findings indicate direct communication between a resident gut microbe and the host and show that intestinal epithelial cells acquire antigens from commensal bacteria for generation of T-cell responses to the resident microbiota.
2019-01-10 | GSE124848 | GEO
Project description:T follicular helper (Tfh) cell migration into germinal centers (GC) is essential for the generation of GC B cells and antibody responses to T dependent (TD) antigens. This process requires interactions between LFA-1 on Tfh cells and ICAMs on B cells. The mechanisms underlying defective antibody responses to TD antigens in DOCK8 deficiency are incompletely understood. We show that mice selectively lacking DOCK8 in T cells have impaired IgG antibody responses to TD antigens, decreased GC size, and reduced numbers of GC B cells. However, they develop normal numbers of Tfh cells with intact capacity for driving B cell differentiation into a GC phenotype in vitro. Notably, migration of DOCK8 deficient T cells into GCs is defective. Following TCR/CD3 ligation, DOCK8 deficient T cells have impaired LFA-1 activation and reduced binding to ICAM-1. DOCK8 is important for LFA1-dependent positioning of Tfh cells in GCs, and thereby the generation of GC B cells and IgG antibody responses to TD antigen.
2020-07-23 | GSE154897 | GEO
Project description:The clathrin and dynamin-independent endocytic pathway (CLIC-GEEC) is the major route of internalization of a large fraction of glycosylphosphatidylinositol-anchored proteins (GPI-APs) and other cell surface receptors. Unlike the clathrin-dependent endocytic pathway, where adaptors recruited to the endocytic pit provide the clues to recruit specific cargo, the CLIC-GEEC pathway is considered to take up portions of the membrane without any specificity. Here, we identified actin-binding protein swiprosin1 as the first cargo adaptor for the CLIC-GEEC pathway. We found that swiprosin1 interacts with active Rab21, a known regulator of integrin endocytosis, and couples Rab21 and the integrin cargo to the CLIC-GEEC endocytic machinery. Our work also identified the previously unknown pathway by which Rab21 mediates integrin endocytosis and linked the CLIC-GEEC pathway to integrin-dependent cancer cell invasion.
2021-08-18 | PXD016478 | Pride
Project description:Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here, we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation, but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.
2023-03-11 | PXD035444 | Pride
Project description:Toll-like receptor 4 (TLR4) plays a pivotal role in the host response to lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. Here we elucidated whether the endocytic adaptor protein Disabled-2 (Dab2) that is abundantly expressed in the macrophages plays a role in LPS-stimulated TLR4 signaling and trafficking. Molecular analysis and transcriptome profiling of the RAW264.7 macrophage-like cells expressing short-hairpin RNA of Dab2 revealed that Dab2 mainly regulated LPS-stimulated TRIF-dependent, but not MyD88-dependent TLR4 signaling. Consequently, knockdown of Dab2 augmented TRIF-dependent interferon regulatory factor 3 activation and the expression for the subsets of inflammatory cytokines and interferon-inducible genes. We further delineated that Dab2 acted as a “clathrin sponge” and sequestered clathrin from TLR4/MD2 complex in the resting stage of macrophages. Clathrin was released from Dab2 and associated with TLR4 to facilitate the internalization of TLR4/MD2 complex together with the bound ligand from the cell surface upon LPS stimulation. Dab2 thereby functions as a negative immune regulator of TLR4 endocytosis and signaling, supporting a novel role of Dab2-associated regulatory circuit in the control of inflammatory response of macrophage to endotoxin.
2017-02-09 | GSE81088 | GEO