Cell biology: the Golgi grows up.
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Animals↗
Biomedical subjects
Satyajit Mayor
Publications and source records attributed to Satyajit Mayor.
Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery.
In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.
ADP-Ribosylation Factor 6↗
A role for the Hsp90 molecular chaperone family in antigen presentation to T lymphocytes via major histocompatibility complex class II molecules.
The heat shock protein (HSP) Hsp90 is known to chaperone cytosolic peptides for MHC class I (MHCI)-restricted antigen presentation to T lymphocytes. We now demonstrate a role for Hsp90 activity in presentation of antigens on MHCII. Treatment of mouse antigen-presenting cells (APC) with the pharmacological Hsp90 inhibitor, geldanamycin, inhibited MHCII-mediated presentation of endocytosed and cytosolic proteins as well as synthetic peptides to specific T cells. Ectopic expression of human Hsp90 in APC enhanced MHCII-mediated antigen presentation. Further, pharmacological Hsp90 inhibition reduced, while retroviral Hsp90 overexpression enhanced, the levels of stable compact MHCII heterodimers correlating with the antigen presentation phenotype. Pharmacological inhibition of Hsp90 activity in IFN-gamma-treated APC resulted in severe abrogation of MHCII-restricted presentation of cytosolic antigen, but only partially inhibited exogenous antigen presentation. Our data suggest a major role for Hsp90 activity in MHCII-mediated antigen presentation pathways, and implicate IFN-gamma-inducible Hsp90-independent mechanisms.
Animals↗
The Nef protein of HIV-1 induces loss of cell surface costimulatory molecules CD80 and CD86 in APCs.
The Nef protein of HIV-1 is essential for its pathogenicity and is known to down-regulate MHC expression on infected cell surfaces. We now show that Nef also redistributes the costimulatory molecules CD80 and CD86 away from the cell surface in the human monocytic U937 cell line as well as in mouse macrophages and dendritic cells. Furthermore, HIV-1-infected U937 cells and human blood-derived macrophages show a similar loss of cell surface CD80 and CD86. Nef colocalizes with MHC class I (MHCI), CD80, and CD86 in intracellular compartments, and binds to both mouse and human CD80 and CD86. Some Nef mutants defective in MHCI down-modulation, including one from a clinical isolate, remain capable of down-modulating CD80 and CD86. Nef-mediated loss of surface CD80/CD86 is functionally significant, because it leads to compromised activation of naive T cells. This novel immunomodulatory role of Nef may be of potential importance in explaining the correlations of macrophage-tropism and Nef with HIV-1 pathogenicity and immune evasion.
Amino Acid Sequence↗
Use of Forster's resonance energy transfer microscopy to study lipid rafts.
Rafts in cell membranes have been a subject of much debate and many models have been proposed for their existence and functional significance. Recent studies using Forster's resonance energy transfer (FRET) microscopy have provided one of the first glimpses into the organization of putative raft components in living cell membranes. Here we discuss how and why FRET microscopy provides an appropriate non-invasive methodology to examine organization of raft components in cell membranes; a combination of homo and hetero-FRET microscopy in conjunction with detailed theoretical analyses are necessary for characterizing structures at nanometre scales. Implications of the physical characteristics of the organization of GPI-anchored proteins in cell membranes suggest new models of lipid-based assemblies in cell membranes based on active principles.
Animals↗
Coming home.
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Biological Science Disciplines↗
Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles.
Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1null (Cav1-/-) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1-/- MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveolae and to identify noncaveolar endocytic vehicles. In WT MEFs, a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2% per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.
ADP-Ribosylation Factor 6↗
The pathway for MHCII-mediated presentation of endogenous proteins involves peptide transport to the endo-lysosomal compartment.
Antigen-presenting cells (APCs) are expected to present peptides from endocytosed proteins via major histocompatibility complex (MHC) class II (MHCII) molecules to T cells. However, a large proportion of peptides purified from MHCII molecules are derived from cytosolic self-proteins making the pathway of cytosolic peptide loading onto MHCII of critical relevance in the regulation of immune self-tolerance. We show that peptides derived from cytoplasmic proteins either introduced or expressed in the cytoplasm are first detectable as MHCII-peptide complexes in LAMP-1(+) lysosomes, prior to their delivery to the cell surface. These peptide-MHC complexes are formed in a variety of APCs, including peritoneal macrophages, dendritic cells, and B cells, and are able to activate T cells. This process requires invariant chain (Ii)-dependent sorting of MHCII to the lysosome and the activity of the molecular chaperone H-2M. This pathway is independent of the ER resident peptide transporter complex TAP and does not take place by cross-presentation from neighbouring cells. In conjunction with our earlier results showing that these peptides are derived by cytosolic processing via the proteasome, these observations provide evidence for a ubiquitous route for peptide transport into the lysosome for the efficient presentation of endogenous and cytoplasmic proteins to CD4 T cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Folate receptor endocytosis and trafficking.
Folate absorption is primarily mediated by a membrane transporter with micro-molar affinities for folates. Paradoxically, folates are only present at low nanomolar concentrations in extracellular milieus; membrane folate receptors (FRs) with nanomolar affinities for folates are likely to substantially modulate folate availability for these transporters. Functional isoforms of FRs are anchored to the membrane by a glycolipid anchor, the glycosylphosphatidylinositol (GPI) anchor. In this chapter we discuss recent insights that have been obtained with regards to GPI-anchored protein trafficking in the context of folate transport via the GPI-anchored FR. Specifically, we focus on recent advances in elucidating the pathways and mechanisms of endocytic sorting of the GPI-anchored FR, and the connection with membrane rafts.
Animals↗
Nanoscale organization of multiple GPI-anchored proteins in living cell membranes.
Cholesterol and sphingolipid-enriched "rafts" have long been proposed as platforms for the sorting of specific membrane components including glycosyl-phosphatidylinositol-anchored proteins (GPI-APs), however, their existence and physical properties have been controversial. Here, we investigate the size of lipid-dependent organization of GPI-APs in live cells, using homo and hetero-FRET-based experiments, combined with theoretical modeling. These studies reveal an unexpected organization wherein cell surface GPI-APs are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters. These clusters are composed of at most four molecules and accommodate diverse GPI-AP species; crosslinking GPI-APs segregates them from preexisting GPI-AP clusters and prevents endocytosis of the crosslinked species via a GPI-AP-selective pinocytic pathway. In conjunction with an analysis of the statistical distribution of the clusters, these observations suggest a mechanism for functional lipid-dependent clustering of GPI-APs.
Animals↗
Sorting GPI-anchored proteins.
The study of glycosylphosphatidylinositol-anchored-protein sorting has led to some surprising new findings and concepts. Evidence is accumulating that, during their delivery to the surface, different types of plasma membrane protein might be sorted from each other early in this pathway, in the endoplasmic reticulum. Furthermore, membrane-lipid composition and microdomains might have a role in the process of protein sorting in both the secretory and endocytic pathways.
Dynamins↗
Rafts: scale-dependent, active lipid organization at the cell surface.
Rafts have been conceptualized as lateral heterogeneities in the organization of cholesterol and sphingolipids, endowed with sorting and signaling functions. In this review we critically examine evidence for the main tenet of the 'raft hypothesis', namely lipid-dependent segregation of specific membrane components in the plasma membrane. We suggest that conventional approaches to studying raft organization wherein membranes are treated as passive, thermally equilibrated systems are unlikely to provide an adequate framework to understand the mechanisms of raft-organization in vivo. An emerging view of raft organization is that it is spatio-temporally regulated at different scales by the cell. This argues that rafts must be defined by simultaneous observation of components involved in particular functions. Recent evidence from the study of glycosylphosphatidyl inositol-anchored proteins, a common raft-marker, supports this picture in which larger scale, more stable rafts are induced from preexisting small-scale lipid-dependent structures actively maintained by cellular processes.
Animals↗
The order of rafts. Conference on microdomains, lipid rafts and caveolae.
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1,2-Dipalmitoylphosphatidylcholine↗
deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.
Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.
Animals↗
Endocytosis of lipid rafts: an identity crisis.
Membrane rafts enriched in cholesterol and sphingolipids have been hypothesized to be key mediators of sorting and signaling functions of associated molecules. Apart from a limited number of biophysical studies in living cell membranes, raft-association has been defined by a simple biochemical criterion, namely the ability to partition with detergent-resistant membranes (DRMs). Here we examine the evidence for the specification of internalization mechanisms and endocytic pathways by rafts as defined by this criterion. We have surveyed the endocytic trafficking of a variety of molecules such as lipids, toxins, glycosylphosphatidylinositol (GPI)-anchored proteins, and DRM-associated transmembrane proteins.
Animals↗
GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway.
Endocytosis of cell-surface proteins via specific pathways is critical for their function. We show that multiple glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed to the recycling endosomal compartment but not to the Golgi via a nonclathrin, noncaveolae mediated pathway. GPI anchoring is a positive signal for internalization into rab5-independent tubular-vesicular endosomes also responsible for a major fraction of fluid-phase uptake; molecules merely lacking cytoplasmic extensions are not included. Unlike the internalization of detergent-resistant membrane (DRM)-associated interleukin 2 receptor, endocytosis of DRM-associated GPI-APs is unaffected by inhibition of RhoA or dynamin 2 activity. Inhibition of Rho family GTPase cdc42, but not Rac1, reduces fluid-phase uptake and redistributes GPI-APs to the clathrin-mediated pathway. These results describe a distinct constitutive pinocytic pathway, specifically regulated by cdc42.
Animals↗