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B A Imhof

Publications and source records attributed to B A Imhof.

At least 19 recordsLinked to original sources
PubMed2001

Marching at the front and dragging behind: differential alphaVbeta3-integrin turnover regulates focal adhesion behavior.

Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain. At the cell front, adhesion sites containing alphaVbeta3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.

Cells, Cultured↗
PubMed2001

Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members.

Endothelial cells are linked to each other through intercellular junctional complexes that regulate the barrier and fence function of the vascular wall. The nature of these intercellular contacts varies with the need for permeability: For example, in brain the impervious blood-brain barrier is maintained by "tight" contacts between endothelial cells. By contrast, in high endothelial venules (HEVs), where lymphocytes continuously exit the bloodstream, the contacts are generally leaky. The precise molecular components that define the type of junction remain to be characterized. An immunoglobulin superfamily molecule named JAM-2, specifically expressed in lymphatic endothelial cells and HEVs, was recently identified. JAM-3 was cloned and characterized in the current study, and JAM-1, -2, and -3 were shown to form a novel protein family belonging to the larger cortical thymocyte Xenopus (CTX) molecular family. Using antibodies specific for each of the 3 family members, their specific participation in different types of cell-cell contact in vivo and their specific and differential localization in lateral contacts or tight junctions were demonstrated. Furthermore, it was shown that JAM-1 and JAM-2 differentially regulate paracellular permeability, suggesting that the presence of JAM-1, -2, or -3 in vascular junctions may play a role in regulating vascular function in vivo.

Amino Acid Sequence↗
PubMed2001

Stem cell factor presentation to c-Kit. Identification of a basolateral targeting domain.

Stem cell factor (also known as mast cell growth factor and kit-ligand) is a transmembrane growth factor with a highly conserved cytoplasmic domain. Basolateral membrane expression in epithelia and persistent cell surface exposure of stem cell factor are required for complete biological activity in pigmentation, fertility, learning, and hematopoiesis. Here we show by site-directed mutagenesis that the cytoplasmic domain of stem cell factor contains a monomeric leucine-dependent basolateral targeting signal. N-terminal to this motif, a cluster of acidic amino acids serves to increase the efficiency of basolateral sorting mediated by the leucine residue. Hence, basolateral targeting of stem cell factor requires a mono-leucine determinant assisted by a cluster of acidic amino acids. This mono-leucine determinant is functionally conserved in colony-stimulating factor-1, a transmembrane growth factor related to stem cell factor. Furthermore, this leucine motif is not capable of inducing endocytosis, allowing for persistent cell surface expression of stem cell factor. In contrast, the mutated cytoplasmic tail found in the stem cell factor mutant Mgf(Sl17H) induces constitutive endocytosis by a motif that is related to signals for endocytosis and lysosomal targeting. Our findings therefore present mono-leucines as a novel type of protein sorting motif for transmembrane growth factors.

Amino Acid Sequence↗
PubMed2001

MHC class II beta-chain and alphaIIbbeta3 integrin are expressed on T-cell progenitors in embryonic bone marrow.

The RR5 monoclonal antibody (mAb) was obtained after immunization of mice with hemopoietic cells from chicken embryos. The cDNA encoding the protein recognized by RR5 was cloned using COS-7 cells transfected with an embryonic bone marrow (BM) cDNA library. The epitope recognized by the RR5 mAb was located on the non-polymorphic MHC class II beta-chain molecule. In the embryonic BM, RR5 labeled 50% of the c-kit expressing cells. Previous experiments have shown that the T-cell progenitors are present in the MHC class II(+)/c-kit(+) BM population along with myeloid progenitors and that T-cell and myeloid progenitors also express the integrin alphaIIbbeta3. In this study, using intrathymic cell transfer experiments in chicks, we have tested the T-cell differentiation potential of MHC class II/alphaIIbbeta3 double positive cells. It proved to be similar to that of the c-kit/MHC class II positive cells. However, injection of triple positive cells resulted in a selection of cells with an increased T-cell potential. Most of the MHC class II positive cells which do not express c-kit are prone to apoptosis, indicating that these progenitors might need a survival signal via c-kit. Interestingly, the MHC class II positive progenitors lose this expression after intrathymic transfer. Taken together our data suggest that the presence of the MHC class II beta-chain molecule on the surface of BM progenitor cells could be implicated in differentiation toward myeloid and lymphoid lineages.

Amino Acid Sequence↗
PubMed2001

Integrin alpha(v)beta(3) expression in colon carcinoma correlates with survival.

Integrin alpha(v)beta(3) is expressed by newly formed blood vessels in diseased and neoplastic tissue and can therefore be used as a marker for angiogenesis. We investigated its expression on the vasculature of 40 colon carcinomas using the anti-alpha(v)beta(3)-specific monoclonal antibody LM609. The average relapse-free interval and overall survival in patients suffering from colon carcinomas with high vascular expression of alpha(v)beta(3) integrin was significantly reduced compared with that in patients with low alpha(v)beta(3) integrin expressing tumor vasculature. Moreover, the expression level of alpha(v)beta(3) integrin correlated with the presence of liver metastases. In conclusion, we propose vascular expression of alpha(v)beta(3) integrin as a prognostic indicator for colon carcinoma.

Aged↗
PubMed2001

Current concepts in lymphocyte homing and recirculation.

The immune system consists of a complex collection of leukocytes and dendritic cells that surveys most tissues in the body for the appearance of foreign antigens. For an efficient immune response, the interaction and co-localization of antigen-presenting cells, costimulatory helper cells and effector cells are crucial parameters. Therefore, the migration routes of antigen-presenting cells and potential antigen-specific lymphocytes merge in secondary lymphoid organs in order to increase the likelihood and speed of a lymphocyte finding its cognate antigen. Additionally, antigen-primed effector cells are directed to the tissue where they are most likely to encounter their cognate antigen. This highly organized and efficient antigen encounter is based on a continuous recirculation of antigen-specific lymphocytes between blood, peripheral tissue, and secondary lymphoid organs. Moreover, the efficacy of the immune system is further increased by the ability of different lymphocyte subsets to recirculate only through distinct tissues. The scope of this review is to outline the concept and mechanisms of lymphocyte homing and recirculation and to discuss the significance for the immune defense. Current models in leukocyte homing and recirculation and the underlying molecular functions of implicated cell adhesion molecules, chemokines, and chemokine receptors are discussed.

Animals↗
PubMed2001

HEMCAM/CD146 downregulates cell surface expression of beta1 integrins.

HEMCAM/gicerin, an immunoglobulin superfamily protein, is involved in homophilic and heterophilic adhesion. It interacts with NOF (neurite outgrowth factor), a molecule of the laminin family. Alternative splicing leads to mRNAs coding for HEMCAM with a short (HEMCAM-s) or a long cytoplasmic tail (HEMCAM-l). To investigate the cellular function of these two variants, we stably transfected murine fibroblasts with either form of HEMCAM. Expression of each isoform of this protein in L cells delayed proliferation and modified their adhesion properties to purified extracellular matrix proteins. Expression of either HEMCAM-s or HEMCAM-l inhibited integrin-dependent adhesion and spreading of fibroblasts to laminin 1, showing that this phenomenon did not depend on the cytoplasmic region. By contrast, L-cell adhesion and spreading to fibronectin depended on the HEMCAM isoform expressed. Flow cytometry and immunoprecipitation studies revealed that the expression of HEMCAM downregulated expression of the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1, and fibronectin receptor alpha5beta1 from the cell surface. Semi-quantitative PCR and northern blot experiments showed that the expression of alpha6beta1 integrin modified by HEMCAM occurred at a translation or maturation level. Thus, our data demonstrate that HEMCAM regulates fibroblast adhesion by controlling beta1 integrin expression.

Animals↗
PubMed2001

Superactivation of integrin alphavbeta3 by low antagonist concentrations.

Integrins are implicated in cell adhesion, migration and homeostasis. An important feature is their ability to adopt different affinity states that can be regulated by a variety of intra- and extracellular factors. To study affinity modulation of the integrin ectodomain by extracellular factors, we produced a soluble recombinant form of mouse integrin alphavbeta3 in a mammalian expression system and isolated it to purity. We show that the two transmembrane truncated integrin subunits stably associate to form a functional receptor, soluble recombinant alphavbeta3. The affinity of this receptor for its ligands vitronectin, fibronectin and fibrinogen can be modulated by the divalent cations magnesium, calcium and manganese. Most importantly, we found that a cyclic RGD-peptide has a biphasic effect on rsalphavbeta3and native purified alphavbeta3, with an antagonistic phase at high concentrations, and an agonistic phase at low concentrations. This integrin superactivation by low antagonist concentrations is shown in binding of sralphavbeta3 to immobilized ligands by ELISA, and in adhesion of cells that express the chimaeric integrin ligand KISS31 to immobilized rsalphavbeta3 and native purified alphavbeta3. Our results indicate that low concentrations of the ligand mimetic cyclo-RGD can result in superactivation of the extracellular domain of integrin alphavbeta3 to a comparable level as activation by manganese.

Cell Adhesion↗
PubMed2000

Intestinal CD8 alpha alpha and CD8 alpha beta intraepithelial lymphocytes are thymus derived and exhibit subtle differences in TCR beta repertoires.

Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the first line of cellular defense against enteric pathogens. Therefore, determining the origin of these cells has important implications for the mechanisms of T cell maturation and repertoire selection. Recent evidence suggests that murine CD8 alpha alpha intestinal IELs (iIELs) can mature and undergo selection in the absence of a thymus. We analyzed IEL origin by cell transfer, using two congenic chicken strains. Embryonic day 14 and adult thymocytes did not contain any detectable CD8 alpha alpha T cells. However, when TCR(+) thymocytes were injected into congenic animals, they migrated to the gut and developed into CD8alphaalpha iIELs, while TCR(-) T cell progenitors did not. The TCR V beta 1 repertoire of CD8 alpha alpha(+) TCR V beta 1(+) iIELs contained only part of the TCR V beta 1 repertoire of total iIELs, and it exhibited no new members compared with CD8(+) T cells in the thymus. This indicated that these T cells emigrated from the thymus at an early stage in their developmental process. In conclusion, we show that while CD8 alpha alpha iIELs originate in the thymus, T cells acquire the expression of CD8 alpha alpha homodimers in the gut microenvironment.

Animals↗
PubMed2000

MHC class II and c-kit expression allows rapid enrichment of T-cell progenitors from total bone marrow cells.

T-cell progenitors in the embryonic bone marrow express the tyrosine kinase receptor c-kit. RR5, an anti-MHC class II beta chain monoclonal antibody, subdivides this c-kit positive population. Intrathymic transfer experiments showed that most of the T-cell progenitors belong to the MHC class II(+)/c-kit(+) bone marrow population in the embryo and young adult. On transplantation, these bone marrow progenitors lose this expression and differentiate into CD4 CD8 T lymphocytes. In contrast, erythroid progenitors are restricted to the MHC class II(-)/c-kit(+) population. The MHC class II(+)/c-kit(+) pro-T cells are metabolically active, because they stain brightly with rhodamin 123. Their cyclin A and B expression level suggests that they are in the mitotic phase of the cell cycle. Thus, we define an easy sorting protocol, which allows enrichment of T-cell progenitors from total bone marrow hemopoietic cells. (Blood. 2000;96:3988-3990)

Animals↗
PubMed2000

JAM-2, a novel immunoglobulin superfamily molecule, expressed by endothelial and lymphatic cells.

Cell-cell contacts are essential for morphogenesis and tissue function and play a vital role in mediating endothelial cohesion within the vascular system during vessel growth and organization. We identified a novel junctional adhesion molecule, named JAM-2, by a selective RNA display method, which allowed identification of transcripts encoding immunoglobulin superfamily molecules regulated during coculture of endothelial cells with tumor cells. The JAM-2 transcript is highly expressed during embryogenesis and is detected in lymph node and Peyer's patches RNA of adult mice. Accordingly, antibodies specific for JAM-2 stain high endothelial venules and lymphatic vessels in lymphoid organs, and vascular structures in the kidney. Using real time video microscopy, we show that JAM-2 is localized within minutes to the newly formed cell-cell contact. The role of the protein in the sealing of cell-cell contact is further suggested by the reduced paracellular permeability of cell monolayer transfected with JAM-2 cDNA, and by the localization of JAM-2 to tight junctional complexes of polarized cells. Taken together, our results suggest that JAM-2 is a novel vascular molecule, which participates in interendothelial junctional complexes.

Amino Acid Sequence↗
PubMed2000

The role of cell traffic in the emergence of the T lymphoid system.

The role of the thymus is to ensure the differentiation and selection of T lymphocytes, which are one of the major players in the immune system. Recent studies show that the establishment of the T lymphoid system requires a complex cell traffic. In this field, avian embryos yield particularly informative developmental models because they are amenable to many experimental approaches during the phases of morphogenesis, and, in addition, the immune system resembles that of mammals.

Cell Differentiation↗
PubMed2000

In vivo veritas.

Explore the source record for details and available documents.

Animals↗
PubMed2000

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration.

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein-transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.

Actins↗
PubMed2000

PECAM-1/CD31 trans-homophilic binding at the intercellular junctions is independent of its cytoplasmic domain; evidence for heterophilic interaction with integrin alphavbeta3 in Cis.

PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin alphavbeta3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin alphavbeta3 associates with PECAM-1 on the same cell surface as in a cis manner.

Alternative Splicing↗