The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo. (1/108)

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.  (+info)

Unique properties of lamp2a compared to other lamp2 isoforms. (2/108)

Lamp2a acts as a receptor in the lysosomal membrane for substrate proteins of chaperone-mediated autophagy. Using antibodies specific for the cytosolic tail of lamp2a and others recognizing all lamp2 isoforms, we found that in rat liver lamp2a represents 25% of lamp2s in the lysosome. We show that lamp2a levels in the lysosomal membrane in rat liver and fibroblasts in culture directly correlate with rates of chaperone-mediated autophagy in a variety of physiological and pathological conditions. The concentration of other lamp2s in the lysosomal membrane show no correlation under the same conditions. Furthermore, substrate proteins bind to lamp2a but not to other lamp2s. Four positively-charged amino acids uniquely present in the cytosolic tail of lamp2a are required for the binding of substrate proteins. Lamp2a also distributes to an unique subpopulation of perinuclear lysosomes in cultured fibroblasts in response to serum withdrawal, and lamp2a, more than other lamp2s, tends to multimerize. These characteristics may be important for lamp2a to act as a receptor for chaperone-mediated autophagy.  (+info)

From lysosomes to the plasma membrane: localization of vacuolar-type H+ -ATPase with the a3 isoform during osteoclast differentiation. (3/108)

Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification is carried out by vacuolar type H+-ATPase (V-ATPase) localized in the plasma membrane. We have shown that a3, one of the four subunit a isoforms (a1, a2, a3, and a4), is a component of the plasma membrane V-ATPase (Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760-8765). To establish the unique localization of V-ATPase, we have used a murine macrophage cell line, RAW 264.7, that can differentiate into multinuclear osteoclast-like cells on stimulation with RANKL (receptor activator of nuclear factor kappaB ligand). The V-ATPase with the a3 isoform was localized to late endosomes and lysosomes, whereas those with the a1 and a2 isoforms were localized to organelles other than lysosomes. After stimulation, the V-ATPase with the a3 isoform was immunochemically colocalized with lysosome marker lamp2 and was detected in acidic organelles. These organelles were also colocalized with microtubules, and the signals of lamp2 and a3 were dispersed by nocodazole, a microtubule depolymerizer. In RAW-derived osteoclasts cultured on mouse skull pieces, the a3 isoform was transported to the plasma membrane facing the bone and accumulated inside podosome rings. These findings indicate that V-ATPases with the a3 isoform localized in late endosomes/lysosomes are transported to the cell periphery during differentiation and finally assembled into the plasma membrane of mature osteoclasts.  (+info)

Role for Rab7 in maturation of late autophagic vacuoles. (4/108)

The small GTP binding protein Rab7 has a role in the late endocytic pathway and lysosome biogenesis. The role of mammalian Rab7 in autophagy is, however, unknown. We have addressed this by inhibiting Rab7 function with RNA interference and overexpression of dominant negative Rab7. We show here that Rab7 was needed for the formation of preferably perinuclear, large aggregates, where the autophagosome marker LC3 colocalised with Rab7 and late endosomal and lysosomal markers. By electron microscopy we showed that these large aggregates corresponded to autophagic vacuoles surrounding late endosomal or lysosomal vesicles. Our experiments with quantitative electron microscopy showed that Rab7 was not needed for the initial maturation of early autophagosomes to late autophagic vacuoles, but that it participated in the final maturation of late autophagic vacuoles. Finally, we showed that the recruitment of Rab7 to autophagic vacuoles was retarded in cells deficient in the lysosomal membrane proteins Lamp1 and Lamp2, which we have recently shown to accumulate late autophagic vacuoles during starvation. In conclusion, our results showed a role for Rab7 in the final maturation of late autophagic vacuoles.  (+info)

LAMP-1 and LAMP-2, but not LAMP-3, are reliable markers for activation-induced secretion of human mast cells. (5/108)

BACKGROUND: Mast cells are resident tissue cells that induce anaphylactic reactions by rapidly releasing mediators after antigen-mediated cross-linking of immunoglobulin E receptors. In the similarly active peripheral blood basophilic leukocyte, lysosome-associated membrane protein 3 (LAMP-3; CD63) has been described as an activation marker, but LAMPs have not been investigated in normal tissue mast cells. METHODS: Intra- and extracellular expressions of LAMP-1 (CD107a), LAMP-2 (CD107b), and LAMP-3 (CD63) were analysed by flow cytometry, immunocytochemistry, and functional assays in unstimulated and stimulated leukemic human mast cell line 1 (HMC-1) and skin mast cells. RESULTS: On flow cytometry, all mast cells expressed LAMP-3 at their cell membranes, whereas LAMP-1 and LAMP-2 were barely detectable (HMC-1 cells) or expressed at low levels (<10% of skin mast cells). After fixation and permeabilisation, high intracellular levels of all three LAMPs were noted in both cell types. After stimulation, a rapid translocation of intracellular LAMPs to the cell membrane, with an associated release of histamine, leukotriene C(4) and prostaglandin D(2), was ascertained in skin mast cells only. CONCLUSION: These results show that LAMP-1 and LAMP-2 are activation markers for normal mast cells. The lack of LAMP translocation after activation of leukemic mast cells may be related to maturation or malignancy-associated defects of these cells.  (+info)

The adaptor protein AP-4 as a component of the clathrin coat machinery: a morphological study. (6/108)

The four members of the AP (adaptor protein) family are heterotetrameric cytosolic complexes that are involved in the intracellular trafficking of cargo proteins between different organelles. They interact with motifs present in the cytoplasmic tails of their specific cargo proteins at different intracellular locations. While AP-1, AP-2 and AP-3 have been investigated extensively, very few studies have focused on the fourth member, AP-4. In the present study, we report on the intracellular localization of AP-4 in the MDCK (Madin-Darby canine kidney) and MelJuSo cell lines after immunogold labelling of ultrathin cryosections. We find that AP-4 is localized mainly in the Golgi complex, as well as on endosomes and transport vesicles. Interestingly, we show for the first time that AP-4 is localized with the clathrin coat machinery in the Golgi complex and in the endocytic pathway. Furthermore, we find that AP-4 is localized with the CI-MPR (cation-independent mannose 6-phosphate receptor), but not with the transferrin receptor, LAMP-2 (lysosomal-associated membrane protein-2) or invariant chain. The difference in morphology between CI-MPR/AP-4-positive vesicles and CI-MPR/AP-1-positive vesicles raises the possibility that AP-4 acts at a location different from that of AP-1 in the intracellular trafficking pathway of CI-MPR.  (+info)

Glycogen storage diseases presenting as hypertrophic cardiomyopathy. (7/108)

BACKGROUND: Unexplained left ventricular hypertrophy often prompts the diagnosis of hypertrophic cardiomyopathy, a sarcomere-protein gene disorder. Because mutations in the gene for AMP-activated protein kinase gamma2 (PRKAG2) cause an accumulation of cardiac glycogen and left ventricular hypertrophy that mimics hypertrophic cardiomyopathy, we hypothesized that hypertrophic cardiomyopathy might also be clinically misdiagnosed in patients with other mutations in genes regulating glycogen metabolism. METHODS: Genetic analyses performed in 75 consecutive unrelated patients with hypertrophic cardiomyopathy detected 40 sarcomere-protein mutations. In the remaining 35 patients, PRKAG2, lysosome-associated membrane protein 2 (LAMP2), alpha-galactosidase (GLA), and acid alpha-1,4-glucosidase (GAA) genes were studied. RESULTS: Gene defects causing Fabry's disease (GLA) and Pompe's disease (GAA) were not found, but two LAMP2 and one PRKAG2 mutations were identified in probands with prominent hypertrophy and electrophysiological abnormalities. These results prompted the study of two additional, independent series of patients. Genetic analyses of 20 subjects with massive hypertrophy (left ventricular wall thickness, > or =30 mm) but without electrophysiological abnormalities revealed mutations in neither LAMP2 nor PRKAG2. Genetic analyses of 24 subjects with increased left ventricular wall thickness and electrocardiograms suggesting ventricular preexcitation revealed four LAMP2 and seven PRKAG2 mutations. Clinical features associated with defects in LAMP2 included male sex, severe hypertrophy, early onset (at 8 to 17 years of age), ventricular preexcitation, and asymptomatic elevations of two serum proteins. CONCLUSIONS: LAMP2 mutations typically cause multisystem glycogen-storage disease (Danon's disease) but can also present as a primary cardiomyopathy. The glycogen-storage cardiomyopathy produced by LAMP2 or PRKAG2 mutations resembles hypertrophic cardiomyopathy but is distinguished by electrophysiological abnormalities, particularly ventricular preexcitation.  (+info)

Simultaneous reconstitution of multiple cytomegalovirus-specific CD8+ cell populations with divergent functionality in hematopoietic stem-cell transplant recipients. (8/108)

A panel of 7 human cytomegalovirus (CMV) epitope peptides and corresponding major histocompatibility class 1 tetramers was used to evaluate cellular immunity in healthy seropositive donors and in hematopoietic stem-cell transplant recipients. Broad CMV-specific T cell responses to epitopes were found within several CMV polypeptides and were restricted by multiple human leukocyte antigen alleles. Their cytotoxic functionality was evaluated by use of an assay that measures transient surface levels of lysosomal membrane proteins LAMP-1 (CD107a) and LAMP-2 (CD107b) after peptide stimulation. This assay can be combined with tetramer staining of antigen-specific CD8(+) T lymphocytes and has potential as a surrogate marker for cytotoxic function. CD8(+) T lymphocytes specific for epitopes within the pp65 or pp50 gene products exhibited significantly higher functionality, compared with populations recognizing CMV major immediate early-1 epitopes. These functional differences between T lymphocyte populations within the same individual may have implications for protection against CMV.  (+info)