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H J Geuze

Publications and source records attributed to H J Geuze.

At least 37 records · Page 2Linked to original sources
PubMed1999

The FcgammaRIa (CD64) ligand binding chain triggers major histocompatibility complex class II antigen presentation independently of its associated FcR gamma-chain.

Within multi-subunit Ig receptors, the FcR gamma-chain immunoreceptor tyrosine-based activation motif (ITAM) plays a crucial role in enabling antigen presentation. This process involves antigen-capture and targeting to specific degradation and major histocompatibility complex (MHC) class II loading compartments. Antigenic epitopes are then presented by MHC class II molecules to specific T cells. The high-affinity receptor for IgG, hFcgammaRIa, is exclusively expressed on myeloid lineage cells and depends on the FcR gamma-chain for surface expression, efficient ligand binding, and most phagocytic effector functions. However, we show in this report, using the IIA1.6 cell model, that hFcgammaRIa can potentiate MHC class II antigen presentation, independently of a functional FcR gamma-chain ITAM. Immunoelectron microscopic analyses documented hFcgammaRIa alpha-chain/rabbit IgG-Ovalbumin complexes to be internalized and to migrate via sorting endosomes to MHC class II-containing late endosomes. Radical deletion of the hFcgammaRIa alpha-chain cytoplasmic tail did not affect internalization of rabbit IgG-Ovalbumin complexes. Importantly, however, this resulted in diversion of receptor-ligand complexes to the recycling pathway and decreased antigen presentation. These results show the hFcgammaRIa cytoplasmic tail to contain autonomous targeting information for intracellular trafficking of receptor-antigen complexes, although deficient in canonical tyrosine- or dileucine-targeting motifs. This is the first documentation of autonomous targeting by a member of the multichain FcR family that may critically impact the immunoregulatory role proposed for hFcgammaRIa (CD64).

Amino Acid Sequence↗
PubMed1999

Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles.

We have determined the concentrations of the secretory proteins amylase and chymotrypsinogen and the membrane proteins KDELr and rBet1 in COPII- and COPI-coated pre-Golgi compartments of pancreatic cells by quantitative immunoelectron microscopy. COPII was confined to ER membrane buds and adjacent vesicles. COPI occurred on vesicular tubular clusters (VTCs), Golgi cisternae, the trans-Golgi network, and immature secretory granules. Both secretory proteins exhibited a first, significant concentration step in noncoated segments of VTC tubules and were excluded from COPI-coated tips. By contrast, KDELr and rBet1 showed a first, significant concentration in COPII-coated ER buds and vesicles and were prominently present in COPI-coated tips of VTC tubules. These data suggest an important role of VTCs in soluble cargo concentration by exclusion from COPI-coated domains.

Amylases↗
PubMed1999

Peptide loading in the endoplasmic reticulum accelerates trafficking of peptide:MHC class II complexes in B cells.

In a combination of biochemical and immunoelectron-microscopical approaches we studied intracellular trafficking and localization of the endoplasmic-reticulum (ER)-formed complexes of murine MHC class II molecule I-Ab and an antigenic peptide Ealpha52-68 covalently linked to its beta-chain. The association with the peptide in the ER leads to sharp acceleration of the intracellular trafficking of the complexes to the plasma membrane. Within the cells, Ealpha52-68:I-Ab complexes accumulate in the multivesicular MHC class II compartment (MIIC), but not in denser multilaminar or intermediate type MIICs. The changes in the trafficking of ER-formed complexes result solely from the presence of the tethered peptide, since wild-type class II molecules traffic similarly in bare lymphocyte syndrome cells and in wild-type antigen-presenting cells.

Animals↗
PubMed1999

Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells.

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.

Animals↗
PubMed1998

Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation.

Newly synthesized procollagen type I (PC) assembles into 300 nm rigid, rod-like triple helices in the lumen of the endoplasmic reticulum. This oligomeric complex moves to the Golgi and forms large electron-dense aggregates. We have monitored the transport of PC along the secretory pathway. We show that PC moves across the Golgi stacks without ever leaving the lumen of the Golgi cisternae. During transport from the endoplasmic reticulum to the Golgi, PC is found within tubular-saccular structures greater than 300 nm in length. Thus, supermolecular cargoes such as PC do not utilize the conventional vesicle-mediated transport to traverse the Golgi stacks. Our results imply that PC moves in the anterograde direction across the Golgi complex by a process involving progressive maturation of Golgi cisternae.

2,2'-Dipyridyl↗
PubMed1998

The tetraspan protein CD82 is a resident of MHC class II compartments where it associates with HLA-DR, -DM, and -DO molecules.

In specialized APCs, MHC class II molecules are synthesized in the endoplasmic reticulum and transported through the Golgi apparatus to organelles of the endocytic pathway collectively called MHC class II compartments (MIICs). There, the class II-associated invariant chain is degraded, and peptides derived from internalized Ag bind to empty class II in a reaction that is facilitated by the class II-like molecule HLA-DM. An mAb raised to highly purified, immunoisolated MIICs from human B lymphoblastoid cells recognized CD82, a member of the tetraspan family of integral membrane proteins. Subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy showed that CD82 is highly enriched in MIICs, particularly in their internal membranes. Coprecipitation analysis showed that CD82 associates in MIICs with class II, DM, and HLA-DO (an inhibitor of peptide loading that binds DM). Similar experiments showed CD63, another tetraspan protein found in MIICs, also associates with these molecules in the compartment and that CD82 and CD63 associate with each other. Preclearing experiments demonstrated that both CD82 and CD63 form complexes with DM-associated class II and DM-associated DO. The ability of CD82 and CD63 to form complexes with class II, DM, and DO in MIICs suggests that the tetraspan proteins may play an important role in the late stages of MHC class II maturation.

Animals↗
PubMed1998

Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes.

Association of major histocompatibility complex (MHC) class II molecules with peptides occurs in a series of endocytic vacuoles, termed MHC class II-enriched compartments (MIICs). Morphological criteria have defined several types of MIICs, including multivesicular MIICs, which are composed of 50-60-nm vesicles surrounded by a limiting membrane. Multivesicular MIICs can fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular space. The externalized vesicles, termed exosomes, carry MHC class II and can stimulate T-cells in vitro. In this study, we show that exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82. Interestingly, subcellular localization of these molecules revealed that they were concentrated on the internal membranes of multivesicular MIICs. In contrast to the tetraspans, other membrane proteins of MIICs, such as HLA-DM, Lamp-1, and Lamp-2, were mainly localized to the limiting membrane and were hardly detectable on the internal membranes of MIICs nor on exosomes. Because internal vesicles of multivesicular MIICs are thought to originate from inward budding of the limiting membrane, the differential distribution of membrane proteins on the internal and limiting membranes of MIICs has to be driven by active protein sorting.

Antigens, CD↗
PubMed1998

Mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles.

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic beta cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6-phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by approximately 90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in beta cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595-608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261-1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR-ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

Multivesicular bodies are an intermediate stage in the formation of platelet alpha-granules.

We have used ultrathin cryosectioning and immunogold cytochemistry to study the position of alpha-granules in the endocytic and biosynthetic pathways in megakaryocytes and platelets. Morphologically, we distinguished three types of granules; so-called multivesicular bodies type I (MVB I) with internal vesicles only, granules with internal vesicles and an electron dense matrix (MVB II), and the alpha-granules with mainly a dense content and often internal membrane vesicles at their periphery. The MVBs were prominent in cultured megakaryocytes and the megakaryoblastic cell line CHRF-288, but were less numerous in bone marrow megakaryocytes and platelets, whereas alpha-granules were most prominent in mature bone marrow megakaryocytes and in platelets. The internalization kinetics of bovine serum albumin-gold particles and of fibrinogen positioned the MVB subtypes and alpha-granules sequentially in the endocytic pathway. MVBs contained the secretory proteins von Willebrand factor (vWF) and beta-thromboglobulin (beta-TG), the platelet-specific membrane protein P-selectin, and the lysosomal membrane protein CD63. Within the MVBs, endocytosed fibrinogen and endogenous beta-TG were restricted to the matrix, while vWF was predominantly associated with internal vesicles. CD63 was also observed in association with internal membrane vesicles in the alpha-granules. These observations, and the gradual morphologic transition from granules containing vesicles to granules containing predominantly dense material, suggest that MVBs represent a developmental stage in alpha-granule maturation.

Animals↗
PubMed1997

Major histocompatibility complex class II compartments in human and mouse B lymphoblasts represent conventional endocytic compartments.

In most human and mouse antigen-presenting cells, the majority of intracellular major histocompatibility complex (MHC) class II molecules resides in late endocytic MHC class II compartments (MIICs), thought to function in antigen processing and peptide loading. However, in mouse A20 B cells, early endocytic class II-containing vesicles (CIIVs) have been reported to contain most of the intracellular MHC class II molecules and have also been implicated in formation of MHC class II-peptide complexes. To address this discrepancy, we have studied in great detail the endocytic pathways of both a human (6H5.DM) and a mouse (A20.Ab) B cell line. Using quantitative immunoelectron microscopy on cryosections of cells that had been pulse-chased with transferrin-HRP or BSA-gold as endocytic tracers, we have identified up to six endocytic subcompartments including an early MIIC type enriched in invariant chain, suggesting that it serves as an important entrance to the endocytic pathway for newly synthesized MHC class II/invariant chain complexes. In addition, early MIICs represented the earliest endocytic compartment containing MHC class II- peptide complexes, as shown by using an antibody against an abundant endogenous class II-peptide complex. The early MIIC exhibited several though not all of the characteristics reported for the CIIV and was situated just downstream of early endosomes. We have not encountered any special class II-containing endocytic structures besides those normally present in nonantigen-presenting cells. Our results therefore suggest that B cells use conventional endocytic compartments rather than having developed a unique compartment to accomplish MHC class II presentation.

Animals↗
PubMed1997

Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells.

Immature dendritic cells (DC) use both macropinocytosis and mannose receptor-mediated endocytosis to internalize soluble Ags efficiently. These Ags are ultimately presented to T cells after DC maturation and migration into the lymph nodes. We have previously described the immortalized myeloid cell line FSDC as displaying the characteristics of early DC precursors that efficiently internalize soluble Ags. To describe the different routes of Ag uptake and to identify the Ag retention compartments in FSDC, we followed the intracellular fate of FITC-coupled OVA, dextran (DX), transferrin, and Lucifer Yellow using flow cytometry, confocal microscopy, and immunoelectron microscopy. OVA and DX gained access into macropinosomes and early endosomes. DX was preferentially sorted into endosomal compartments, while most of the OVA entered macropinosomes via fluid phase uptake. We found a long-lasting retention of DX and OVA of up to 24 h. After 6 h of chase, these two molecules were concentrated in common vesicular compartments. These retention compartments were distinct from endosomes and lysosomes; they were much larger, only mildly acidic, and lacked the small GTP binding protein rab7. However, they were positive for lysosome-associated membrane protein-1, the protease cathepsin D, and MHC class II molecules, thus representing matured macropinosomes. These data suggest that the activity of vacuolar proteases is reduced at the mildly acidic pH of these vesicles, which explains their specific retention of an Ag. The retention compartments might be used by nonlymphoid tissue DC to store peripheral Ags during their migration to the lymph node.

Animals↗
PubMed1997

The autophagic and endocytic pathways converge at the nascent autophagic vacuoles.

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV--initial (AVi), intermediate (AVi/d), and degradative (AVd)--were defined by morphological criteria and immunogold labeling characteristics of marker enzymes. The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15-45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.

Animals↗
PubMed1997

Assembly of an abundant endogenous major histocompatibility complex class II/peptide complex in class II compartments.

To identify the intracellular site(s) of formation of an endogenous class II/peptide complex in a human B cell line, we employed kinetic pulse-chase labeling experiments followed by subcellular fractionation by Percoll density gradient centrifugation and immunogold labeling on ultrathin cryosections. For direct demonstration of assembly of such complexes, we used the monoclonal antibody YAe, which detects an endogenous complex of the mouse class II molecule I-Ab with a 17-amino acid peptide derived from the alpha chain of HLA-DR (DR alpha52-68). We show that in human B lymphocytes, these class II/peptide complexes assemble and transiently accumulate in major histocompatibility complex class II-enriched compartments before reaching the cell surface.

Animals↗
PubMed1997

ERD2 proteins mediate ER retention of the HNEL signal of LRP's receptor-associated protein (RAP).

The 39 kDa receptor-associated protein (RAP) is a receptor antagonist that interacts with several members of the low density lipoprotein (LDL) receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. Our recent studies have demonstrated that RAP is an endoplasmic reticulum (ER) resident protein and an intracellular chaperone for the LDL receptor-related protein (LRP). The HNEL sequence at the carboxyl terminus of RAP represents a novel ER retention signal that shares homology with the well-characterized KDEL signal. In the present study, using immunoelectron microscopy we demonstrate that cells stably transfected with human growth hormone (GH) tagged with either KDEL (GH + KDEL) or HNEL (GH + HNEL) signals exhibit ER and cis-Golgi localization typical of ER-retained proteins. Overexpression of not only GH + HNEL but also GH + KDEL cDNA in transfected cells results in saturation of ER retention receptors and secretion of endogenous RAP indicating that the two signals interact with the same ER retention receptor(s). The role of RAP in the maturation of LRP is further supported by the observation that functional LRP is reduced about 60% as a result of decreased intracellular RAP. Pulse-chase labeling and immunolocalization studies of ERD2.1 and ERD2.2 proteins in transfected cells demonstrate a long half-life and Golgi localization for both receptors. Finally, overexpression of either ERD2.1 or ERD2.2 proteins significantly increases the capacity of cells to retain both KDEL and HNEL-containing proteins. Taken together, our results thus demonstrate that ERD2 proteins are capable of retaining the novel ER retention signal associated with RAP.

Carrier Proteins↗
PubMed1997

Caveolae can be alternative endocytotic structures in elicited macrophages.

The aim of the present study was to identify omega-shaped plasma membrane invaginations, abundantly present in the plasma membrane of elicited macrophages, as caveolae. We have used an antibody against the major component of the caveolar coat (VIP21/caveolin), and the omega-shaped vesicles were found to be labeled with anti-VIP21 providing evidence that these structures were really caveolae. Filipin that had been shown to affect caveolae was used to investigate the possible endocytotic role of these structures in elicited macrophages. When caveolae were selectively inhibited by filipin, the rate of both fluid-phase and receptor-mediated endocytosis has been decreased. These data together with our results obtained from serial sectioning support that in elicited macrophages caveolae can pinch off from the plasma membrane and can function as alternative carriers in the endocytotic processes of these cells.

Animals↗