Late Endocytic Structures Research Articles

Effects of Na +/H + exchanger inhibitors on subcellular localisation of endocytic organelles and intracellular dynamics of protein transduction domains HIV–TAT peptide and octaarginine

Protein transduction domains such as those derived from the HIV protein TAT have great potential as vectors for delivery of therapeutic entities such as genes and proteins into cells. Extensive studies have shown that a major fraction of the most studied variants enters cells via an endocytic mechanism. However, controversy surrounds the exact uptake mechanism and whether a specific pathway is utilised. Studies showing inhibition of uptake of protein transduction domains in the presence of ion-transport inhibitors such as amiloride and its more potent analogue 5-( N-ethyl- N-isopropyl) amiloride (EIPA) suggest a link between peptide internalisation and macropinocytosis. In this study, using immunolabelling of early and late components of the endocytic pathway, we show that treatment of cells with EIPA and to a lesser extent amiloride affects the morphology and subcellular location of early, late endosomes and lysosomes. Enlarged early and late endocytic structures were observed in EIPA-treated cells, and these organelles accumulated in a perinuclear region. Results from experiments investigating the effects of EIPA on distribution of fluorescent octaarginine were in agreement with the immunolocalisation studies. Treatment of the CD34 + leukaemia cell line KG1a with EIPA in the presence of fluorescent conjugates of HIV–TAT peptide and octaarginine showed distinct vesicular staining in agreement with untreated cells but EIPA-treated cells were additionally characterized by increased localization of the peptides in the cytosol. At levels previously shown to inhibit uptake of HIV–TAT peptide and octaarginine in other cell lines, EIPA was without major effect on uptake of both peptides in KG1a cells.

Spatial Separation of HLA-DM/HLA-DR Interactions within MIIC and Phagosome-Induced Immune Escape

Major Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading. To follow this process in living cells, we have generated cells containing HLA-DR3/Cyan Fluorescent Protein (CFP), HLA-DM/Yellow Fluorescent Protein (YFP), and invariant chain. HLA-DR/DM interactions were observed by Fluorescence Resonance Energy Transfer (FRET). These interactions were pH insensitive, yet occurred only in internal structures and not at the limiting membrane of MIIC. In a cellular model of infection, phagosomes formed a limiting membrane surrounding internalized Salmonella. HLA-DR and HLA-DM did not interact in Salmonella-induced vacuoles, and HLA-DR was not loaded with antigens. The absence of HLA-DR/DM interactions at the limiting membrane prevents local loading of MHC class II molecules in phagosomes. This may allow these bacteria to successfully evade the immune system.

Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization

Mucolipin-1 is a 65-kDa membrane protein encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), a rare neurodegenerative lysosomal storage disorder. We studied the subcellular localization of wild-type and three different mutant forms (T232P, F408del and F465L) of mucolipin by expressing Myc-tagged proteins in HeLa cells. The overexpressed wild-type mucolipin colocalizes to late endocytic structures and induces an aberrant distribution of these compartments. F408del and F465L MLIV mutant proteins show a distribution similar to the wild-type protein, whereas T232P is retained in the endoplasmic reticulum. Among the mutants, only F408del induces a redistribution of the late endocytic compartment. These findings suggest that the overexpression of the mucolipin cation channel influences the dynamic equilibrium of late endocytic compartments.

Active PIKfyve Associates with and Promotes the Membrane Attachment of the Late Endosome-to-trans-Golgi Network Transport Factor Rab9 Effector p40

PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function. Enzymatically inactive PIKfyve mutants elicit enormous dilation of late endocytic structures, suggesting a role for PIKfyve in endosome-to-trans-Golgi network (TGN) membrane retrieval. Here we report that p40, a Rab9 effector reported previously to bind Rab9-GTP and stimulate endosome-to-TGN transport, interacts with PIKfyve as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells. The interaction engages the PIKfyve chaperonin domain and four out of the six C-terminally positioned kelch repeats in p40. Differential centrifugation in a HEK293 cell line, stably expressing PIKfyveWT, showed the membrane-associated immunoreactive p40 co-sedimenting with PIKfyve in the high speed pellet (HSP) fraction. Remarkably, similar analysis in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKfyveK1831E demonstrated a marked depletion of p40 from the HSP fraction. GST-p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5-P and PtdIns 3,5-P2 in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity. A band with the p40 electrophoretic mobility was found to react with a phosphoserine-specific antibody mainly in the PIKfyveWT-containing fractions obtained by density gradient sedimentation of total membranes from PIKfyveWT-expressing HEK293 cells. Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome-to-TGN transport.

T-cell engagement of dendritic cells rapidly rearranges MHC class II transport.

Assembly of major histocompatibility complex (MHC) molecules, which present antigen in the form of short peptides to T lymphocytes, occurs in the endoplasmic reticulum; once assembled, these molecules travel from the endoplasmic reticulum to their final destination. MHC class II molecules follow a route that takes them by means of the endocytic pathway, where they acquire peptide, to the cell surface. The transport of MHC class II molecules in 'professional' antigen-presenting cells (APCs) is subject to tight control and responds to inflammatory stimuli such as lipopolysaccharide. To study class II transport in live APCs, we replaced the mouse MHC class II gene with a version that codes for a class II molecule tagged with enhanced green fluorescent protein (EGFP). The resulting mice are immunologically indistinguishable from wild type. In bone-marrow-derived dendritic cells, we observed class II molecules in late endocytic structures with transport patterns similar to those in Langerhans cells observed in situ. We show that tubular endosomes extend intracellularly and polarize towards the interacting T cell, but only when antigen-laden dendritic cells encounter T cells of the appropriate specificity. We propose that such tubulation serves to facilitate the ensuing T-cell response.

The HPV16 E5 oncogene inhibits endocytic trafficking.

The small hydrophobic E5 protein of Human Papillomavirus type 16 (HPV16) binds to the 16-kDa subunit of the V-H+-ATPase. This binding has been suggested to interfere with acidification of late endocytic structures. We here used video microscopy, ratio imaging and confocal microscopy of living C127 fibroblasts to study the effects of E5. Various endocytic markers including the pH-sensitive probe DM-NERF coupled to dextran, TransFluoSpheres and TRITC-concanavalin A, were applied. In E5-transfected cells, none of these markers colocalized with the membrane permeable probe LysoTracker Red, which accumulates in acidic, late endocytic structures, or with a green fluorescent version of the small GTPase Rab7 labeling late endocytic structures. Importantly, however, late endocytic structures accumulating LysoTracker were still present in the E5-transfected cells. It is therefore concluded that HPV16 E5 perturbs trafficking from early to late endocytic structures rather than acidification.

Loss of cation-independent mannose 6-phosphate receptor expression promotes the accumulation of lysobisphosphatidic acid in multilamellar bodies.

A number of recent studies have highlighted the importance of lipid domains within endocytic organelles in the sorting and movement of integral membrane proteins. In particular, considerable attention has become focussed upon the role of the unusual phospholipid lysobisphosphatidic acid (LBPA). This lipid appears to be directly involved in the trafficking of cholesterol and glycosphingolipids, and accumulates in a number of lysosomal storage disorders. Antibody-mediated disruption of LBPA function also leads to mis-sorting of cation-independent mannose 6-phosphate receptors. We now report that the converse is also true, and that spontaneous loss of cation-independent mannose 6-phosphate receptors from a rat fibroblast cell line led to the formation of aberrant late endocytic structures enriched in LBPA. Accumulation of LBPA was directly dependent upon the loss of the receptors, and could be reversed by expression of bovine cation-independent mannose 6-phosphate receptors in the mutant cell line. Ultrastructural analysis indicated that the abnormal organelles were electron-dense, had a multi-lamellar structure, accumulated endocytosed probes, and were distinct from dense-core lysosomes present within the same cells. The late endocytic structures present at steady state within any particular cell likely reflect the balance of membrane traffic through the endocytic pathway of that cell, and the rate of maturation of individual endocytic organelles. Moreover, there is considerable evidence which suggests that cargo receptors also play a direct mechanistic role in membrane trafficking events. Therefore, loss of such a protein may disturb the overall equilibrium of the pathway, and hence cause the accumulation of aberrant organelles. We propose that this mechanism underlies the phenotype of the mutant cell line, and that the formation of inclusion bodies in many lysosomal storage diseases is also due to an imbalance in membrane trafficking within the endocytic pathway.

Rab7: a key to lysosome biogenesis.

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.

Kinesin-mediated organelle translocation revealed by specific cellular manipulations.

The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)-positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi-associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin-membrane interactions may be of key importance for determining the intracellular distribution of selected organelles.

Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus.

We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.

Characterization of early and late endocytic compartments of the transferrin cycle. Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome.

We describe a detailed morphological characterization of the endocytic pathway in differentiating chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (AEV). These cells express high levels of transferrin receptors (TfR) when induced to differentiate at 42 degrees C. Biochemical analysis showed that most (approximately 90%) of the internalized 125I-Tf recycled within approximately 30 min while a smaller fraction of 125I-Tf required up to 2 h for recycling. By immunocytochemistry, the bulk of Tf and TfR was localized at the plasma membrane and in tubuloreticular early endosomes. This structure contained coated buds that labelled with an antibody specific for the clathrin light chain. Decreasing amounts of both Tf and TfR were detected in two distal compartments, spherical endosome vesicles resembling multivesicular bodies and the prelysosomal compartment (PLC) enriched in cation-independent mannose 6-phosphate receptor. As shown by fluorescent (FITC-Tf) labelling of living cells, the movement of Tf/TfR complex into these late structures was accompanied by a significant drop in pH from about 6, the value displayed by early endosomes, to values below pH 5.0. Since no detectable 125I-Tf degradation was observed during a 4 h period we believe that the Tf/TfR detected in these late endocytic structures avoids degradation and recycles back to the cell surface. The addition of an anti-TfR monoclonal antibody to the culture medium of these cells blocks their differentiation. Under this condition the antibody-TfR complex was trapped in an early endosome compartment that enlarged to more than twice its normal size. However, this condition did not affect the transport kinetics of horseradish peroxidase from the medium to the PLC.

Axonal and dendritic endocytic pathways in cultured neurons

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially.

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells.

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.

PH-induced microtubule-dependent redistribution of late endosomes in neuronal and epithelial cells.

The interaction between late endocytic structures and microtubules in polarized cells was studied using a procedure previously shown to cause microtubule-dependent redistribution of lysosomes in fibroblasts and macrophages (Heuser, J. 1989. J. Cell Biol. 108:855-864). In cultured rat hippocampal neurons, low cytoplasmic pH caused cation-independent mannose-6-phosphate receptor-enriched structures to move out of the cell body and into the processes. In filter grown MDCK cells lowering the cytosolic pH to approximately 6.5 caused late endosomes to move to the base of the cell and this process was shown to be microtubule dependent. Alkalinization caused a shift in distribution towards the apical pole of the cell. The results are consistent with low pH causing the redistribution of late endosomes towards the plus ends of the microtubules. In MDCK cells the microtubules orientated vertically in the cell may play a role in this process. The shape changes that accompanied the redistribution of the late endosomes in MDCK cells were examined by electron microscopy. On low pH treatment fragmentation of the late endosomes was observed whereas after microtubule depolymerization individual late endosomal structures appeared to fuse together. The late endosomes of the MDCK cell appear to be highly pleomorphic and dependent on microtubules for their form and distribution in the cell.