Biomedical subjects

E van Donselaar

Publications and source records attributed to E van Donselaar.

12 recordsLinked to original sources

PubMed2000

Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity.

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

Amino Acid Sequence↗

PubMed2000

Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel critical to intestinal anion secretion. In addition to phosphorylation, vesicle traffic regulates CFTR in some epithelial cells. Studies of cultured intestinal cells are conflicting regarding the role of cAMP-dependent vesicle traffic in regulating chloride transport. Whether CFTR is present in vesicular compartments within chloride secretory cells in the intestine is unknown and the role of cAMP-dependent vesicle insertion in regulating CFTR and intestinal fluid secretion remains unclear. The purpose of this study was to: (1) examine and quantify the subcellular distribution for CFTR in rat intestine, (2) further define the ultrastructure of the previously identified CFTR High Expresser (CHE) cell, and (3) examine the cellular distribution of CFTR following cAMP stimulation in vivo. Using the sensitive techniques of cryoimmunogold electron microscopy we identified CFTR in subapical vesicles and on the apical plasma membrane in crypt, Brunner glands, and CHE cells. cAMP stimulation in rat proximal small intestine produced a fluid secretory response and was associated with an apical redistribution of CFTR, supporting a physiologic role for cAMP-dependent CFTR vesicle insertion in regulating CFTR in the intestine.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1999

EFA6, a sec7 domain-containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization.

We have identified a human cDNA encoding a novel protein, exchange factor for ARF6 (EFA6), which contains Sec7 and pleckstrin homology domains. EFA6 promotes efficient guanine nucleotide exchange on ARF6 and is distinct from the ARNO family of ARF1 exchange factors. The protein localizes to a dense matrix on the cytoplasmic face of plasma membrane invaginations, induced on its expression. We show that EFA6 regulates endosomal membrane recycling and promotes the redistribution of transferrin receptors to the cell surface. Furthermore, expression of EFA6 induces actin-based membrane ruffles that are inhibited by co-expression of dominant-inhibitory mutant forms of ARF6 or Rac1. Our results demonstrate that by catalyzing nucleotide exchange on ARF6 at the plasma membrane and by regulating Rac1 activation, EFA6 coordinates endocytosis with cytoskeletal rearrangements.

ADP-Ribosylation Factor 1↗

PubMed1999

Separate pathways for antigen presentation by CD1 molecules.

The ability to sample relevant intracellular compartments is necessary for effective antigen presentation. To detect peptide antigens, MHC class I and II molecules differentially sample cytosolic and endosomal compartments. CD1 constitutes another lineage of lipid antigen-presenting molecules. We show that CD1b traffics deeply into late endosomal compartments, while CD1a is excluded from these compartments and instead traffics independently in the recycling pathway of the early endocytic system. Further, CD1b but not CD1a antigen presentation is dependent upon vesicular acidification. Since lipids and various bacteria are known to traffic differentially, either penetrating deeply into the endocytic system or following the route of recycling endosomes, these findings elucidate efficient monitoring of distinct components of the endocytic compartment by CD1 lipid antigen-presenting molecules.

Antigen Presentation↗

PubMed1998

Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast.

The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1(+), which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis.

Amino Acid Sequence↗

PubMed1998

Modulation of intracellular transport by transported proteins: insight from regulation of COPI-mediated transport.

Intracellular transport is best understood for how proteins are shuttled among different compartments of the secretory pathway by membrane-bound transport carriers. However, it remains unclear whether regulation of this transport is modulated by the transported (cargo) proteins in the lumen of transport pathways. In the early secretory pathways that connect the endoplasmic reticulum (ER) and the Golgi complex, the small GTPase ADP-ribosylation factor 1 (ARF1) recruits a cytosolic coat protein complex named COPI onto membranes as a key step in the formation of transport vesicles. Transport of newly synthesized proteins that leave the ER includes a class of cargo proteins with a sequence motif of KDEL. When these KDEL proteins leave the ER to reach the Golgi complex, they are recognized by their receptor and transported retrograde in COPI-coated vesicles back to the ER. We now demonstrate that stimulation of the KDEL receptor by a KDEL protein enhances an interaction between the KDEL receptor and a GTPase-activating protein for ARF1. As a result, more cytosolic GTPase-activating protein is recruited to membranes to inactivate ARF1. Thus, the KDEL proteins are examples of luminal cargo proteins that regulate transport by activating their receptor. Most likely, this regulation affects retrograde transport from the Golgi complex to the ER, as activated KDEL receptor appears to reside only in retrograde COPI-coated vesicles.

ADP-Ribosylation Factor 1↗

PubMed1998

ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

ADP-Ribosylation Factor 6↗

PubMed1998

Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.

Mutations of the genes encoding APC or beta-catenin in colon carcinoma induce the constitutive formation of nuclear beta-catenin/Tcf-4 complexes, resulting in activated transcription of Tcf target genes. To study the physiological role of Tcf-4 (which is encoded by the Tcf7/2 gene), we disrupted Tcf7/2 by homologous recombination. Tcf7/2-/- mice die shortly after birth. A single histopathological abnormality was observed. An apparently normal transition of intestinal endoderm into epithelium occurred at approximately embryonic day (E) 14.5. However, no proliferative compartments were maintained in the prospective crypt regions between the villi. As a consequence, the neonatal epithelium was composed entirely of differentiated, non-dividing villus cells. We conclude that the genetic program controlled by Tcf-4 maintains the crypt stem cells of the small intestine. The constitutive activity of Tcf-4 in APC-deficient human epithelial cells may contribute to their malignant transformation by maintaining stem-cell characteristics.

Animals↗

PubMed1997

Cell-type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo.

Caveolae are microdomains of the plasma membrane that have been implicated in organizing and compartmentalizing signal transducing molecules. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membrane in vivo. Recently, we and other laboratories have identified a family of caveolin-related proteins; caveolin has been re-termed caveolin-1. Here, we examine the cell-type and tissue-specific expression of caveolin-2. For this purpose, we generated a novel mono-specific monoclonal antibody probe that recognizes only caveolin-2, but not caveolins-1 and -3. A survey of cell and tissue types demonstrates that the caveolin-2 protein is most abundantly expressed in endothelial cells, smooth muscle cells, skeletal myoblasts (L6, BC3H1, C2C12), fibroblasts, and 3T3-L1 cells differentiated to adipocytes. This pattern of caveolin-2 protein expression most closely resembles the cellular distribution of caveolin-1. In line with these observations, co-immunoprecipitation experiments with mono-specific antibodies directed against either caveolin-1 or caveolin-2 directly show that these molecules form a stable hetero-oligomeric complex. The in vivo relevance of this complex was further revealed by dual-labeling studies employing confocal laser scanning fluorescence microscopy. Our results indicate that caveolins 1 and 2 are strictly co-localized within the plasma membrane and other internal cellular membranes. Ultrastructurally, this pattern of caveolin-2 localization corresponds to caveolae membranes as seen by immunoelectron microscopy. Despite this strict co-localization, it appears that regulation of caveolin-2 expression occurs independently of the expression of either caveolin-1 or caveolin-3 as observed using two different model cell systems. Although caveolin-1 expression is down-regulated in response to oncogenic transformation of NIH 3T3 cells, caveolin-2 protein levels remain unchanged. Also, caveolin-2 protein levels remain unchanged during the differentiation of C2C12 cells from myoblasts to myotubes, while caveolin-3 levels are dramatically induced by this process. These results suggest that expression levels of caveolins 1, 2, and 3 can be independently up-regulated or down-regulated in response to a variety of distinct cellular cues.

3T3 Cells↗

PubMed1996

Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs.

CD1 proteins have been implicated as antigen-presenting molecules for T cell-mediated immune responses, but their intracellular localization and trafficking remain uncharacterized. CD1b, a member of this family that presents microbial lipid antigens of exogenous origin, was found to localize to endocytic compartments that included the same specialized subset of endosomes in which major histocompatibility complex (MHC) class II molecules are proposed to bind endocytosed antigens. Unlike MHC class II molecules, which traffic to antigen-loading endosomal compartments [MHC class II compartments (MIICs)] primarily as a consequence of their association with the invariant chain, localization of CD1b to these compartments was dependent on a tyrosine-based motif in its own cytoplasmic tail.

Amino Acid Sequence↗

PubMed1995

An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface.

The mammalian endopeptidase, furin, is predominantly localized to the trans-Golgi network (TGN) at steady state. The localization of furin to this compartment seems to be the result of a dynamic process in which the protein undergoes cycling between the TGN and the plasma membrane. Both TGN localization and internalization from the plasma membrane are mediated by targeting information contained within the cytoplasmic domain of furin. Here, we report the results of a mutagenesis analysis aimed at identifying the source(s) of targeting information within the furin cytoplasmic domain. Our studies show that there are at least two cytoplasmic determinants that contribute to the steady-state localization and trafficking of furin. The first determinant corresponds to a canonical tyrosine-based motif, YKGL (residues 758-761), that functions mainly as an internalization signal. The second determinant consists of a strongly hydrophilic sequence (residues 766-783) that contains a large cluster of acidic residues (E and D) and is devoid of any tyrosine-based or di-leucine-based motifs. This second determinant is capable of conferring localization to the TGN as well as mediating internalization from the plasma membrane. Thus, these observations establish the existence of a novel, autonomous determinant distinct from sorting signals described previously.

Amino Acid Sequence↗

PubMed1995

A lysosomal targeting signal in the cytoplasmic tail of the beta chain directs HLA-DM to MHC class II compartments.

In human B cells, class II molecules of the major histocompatibility complex (MHC-II) accumulate in an endosomal/lysosomal compartment, the MIIC, in which they may encounter and bind peptides. An additional molecule required for MHC-II peptide binding, HLA-DM (DM), has also been localized to the MIIC. Neither the relationship of the MIIC to the endosomal system nor the mechanisms by which DM localizes to the MIIC are understood. To address these issues, DM localization was analyzed in cells that do or do not express MHC-II. DM alpha beta heterodimers were localized in transfected MHC-II-negative HeLa and NRK cells, in the absence of the MHC-II-associated invariant chain, to a prelysosomal/lysosomal compartment by immunofluorescence microscopy. To identify a potential targeting determinant, we analyzed the localization of a chimeric protein, T-T-Mb, in which the cytoplasmic tail of murine DM beta (Mb) was appended to the lumenal and transmembrane domains of a cell surface protein, Tac. Like intact DM, T-T-Mb was localized to a lysosomal compartment in HeLa and NRK cells, as judged by immunofluorescence and immunoelectron microscopy. T-T-Mb was rapidly degraded in this compartment by a process that was blocked by inhibitors of lysosomal proteolysis. The DM beta cytoplasmic tail also mediated internalization of anti-Tac antibody from the cell surface and delivery to lysosomes. Deletion from the DM beta cytoplasmic tail of the tyrosine-based motif, YTPL, resulted in cell surface expression of T-T-Mb and a loss of both degradation and internalization; alanine scanning mutagenesis showed that the Y and L residues were critical for these functions. Similarly, mutation of the same Y residue within full-length DM beta resulted in cell surface expression of DM alpha beta heterodimers. Lastly, T-T-Mb was localized by immunoelectron microscopy to the MIIC in a human B lymphoblastoid cell line. Our results suggest that a motif, YTPL, in the cytoplasmic tail of the beta chain of DM is sufficient for targeting either to lysosomes or to the MIIC.

Animals↗