Biomedical subjects

B Wehrle-Haller

Publications and source records attributed to B Wehrle-Haller.

17 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

Analysis of melanocyte precursors in Nf1 mutants reveals that MGF/KIT signaling promotes directed cell migration independent of its function in cell survival.

Neural crest-derived melanocyte precursors (MPs) in avian and murine embryos emerge from the dorsal neural tube into a migration staging area (MSA). MPs subsequently migrate from the MSA on a dorsolateral pathway between the dermamyotome and the overlying epithelium. In mouse embryos, MPs express the receptor tyrosine kinase, KIT, and require its cognate ligand, Mast cell growth factor (MGF), for survival and differentiation. Prior to the onset of MP migration, MGF is expressed on the dorsolateral pathway at some distance from cells in the MSA and appears to be required for normal MP development. To learn if MGF is required solely for MP survival on this pathway, or if it also provides directional cues for migration, we uncoupled survival from chemoattractive or motogenic functions of this ligand using mice that carry a targeted mutation at the Neurofibromin (Nf1) locus and consequently lack RAS-GAP function. We show that Nf1-mutant MPs survive in the absence of MGF in vitro and in vivo and that Nf1-mutant MPs disperse normally on the lateral migration pathway in the presence of MGF. In contrast, Nf1-mutant MPs persist in the location of the MSA but are not observed on the lateral migration pathway in double-mutant mice that also lack MGF. We conclude that MGF/KIT function provides a signal required for directed migration of the MPs on the lateral pathway in vivo, independent of its function in survival. We further suggest that the MGF mediates MP migration through a signaling pathway that does not involve RAS.

Animals↗

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↗

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↗

PubMed1999

Diminished matrix metalloproteinase 2 (MMP-2) in ectomesenchyme-derived tissues of the Patch mutant mouse: regulation of MMP-2 by PDGF and effects on mesenchymal cell migration.

Platelet-derived growth factors (PDGF) regulate cell proliferation, survival, morphology, and migration, as well as deposition and turnover of the extracellular matrix. Important roles for the A form of PDGF (PDGF-A) during connective tissue morphogenesis have been highlighted by the murine Patch mutation, which includes a deletion of the alpha subunit of the PDGF receptor. Homozygous (Ph/Ph) embryos exhibit multiple connective tissue defects including cleft face (involving the first branchial arch and frontonasal processes), incomplete heart septation, and heart valve abnormalities before they die in utero. Analyses of the cell biology underlying the defects in Ph/Ph embryos have revealed a deficit in a matrix metalloproteinase (MMP-2) and one of its activators (MT-MMP) that are likely to be involved in cell migration and tissue remodeling, two processes necessary for normal cardiac and craniofacial development. Morphogenesis of these structures requires infiltration of ectomesenchymal precursors and their subsequent deposition and remodeling of extracellular matrix components. First branchial arch and heart tissue from E10.5 embryos were examined by gelatin zymography and RT-PCR in order to characterize the expression of MMPs in these tissues. Of the MMPs examined, only MMP-2 and one of its activators, MT-MMP, were expressed in the first arch and heart at this stage of development. Tissues from Ph/Ph embryos exhibited a significant decrease in both MMP-2 and MT-MMP compared to tissues from normal embryos of the same developmental stage. In order to assess whether this decrease affects the motile activity of mesenchymal cells, cell migration from Ph/Ph branchial arch explants was compared to migration from normal arch tissue and found to be significantly less. In addition, the migratory ability of branchial arch cells from normal explants could be reduced in a similar manner using a specific MMP inhibitor. Although it is still unclear whether the MMP-2 reduction is a direct result of the absence of response of Ph/Ph cells to PDGF-A treatment of normal branchial arch cells in vitro with recombinant PDGF-AA significantly upregulated MMP-2 protein. Together, these results suggest that PDGF-A regulates MMP-2 expression and activation during normal development and that faulty proteinase expression may be at least partially responsible for the developmental defects exhibited by Ph/Ph embryos.

Animals↗

PubMed1999

Altered cell-surface targeting of stem cell factor causes loss of melanocyte precursors in Steel17H mutant mice.

The normal products of the murine Steel (Sl) and Dominant white spotting (W) genes are essential for the development of melanocyte precursors, germ cells, and hematopoietic cells. The Sl locus encodes stem cell factor (SCF), which is the ligand of c-kit, a receptor tyrosine kinase encoded by the W locus. One allele of the Sl mutation, Sl17H, exhibits minor hematopoietic defects, sterility only in males, and a complete absence of coat pigmentation. The Sl17H gene encodes SCF protein which exhibits an altered cytoplasmic domain due to a splicing defect. In this paper we analyzed the mechanism by which the pigmentation phenotype in Sl17H mutant mice occurs. We show that in embryos homozygous for Sl17H the number of melanocyte precursors is severely reduced on the lateral neural crest migration pathway by e11.5 and can no longer be detected by e13.5 when they would enter the epidermis in wildtype embryos. The reduced number of dispersing melanocyte precursors correlates with a reduction of SCF immunoreactivity in mutant embryos in all tissues examined. Regardless of the reduced amount, functional SCF is present at the cell surface of fibroblasts transfected with Sl17H mutant SCF cDNA. Since SCF immunoreactivity normally accumulates in basolateral compartments of SCF-expressing embryonic epithelial tissues, we analyzed the localization of wildtype and Sl17H mutant SCF protein in transfected epithelial (MDCK) cells in vitro. As expected, wildtype forms of SCF localize to and are secreted from the basolateral compartment. In contrast, mutant forms of SCF, which either lack a membrane anchor or exhibit the Sl17H altered cytoplasmic tail, localize to and are secreted from the apical compartment of the cultured epithelium. We suggest, therefore, that the loss of melanocyte precursors prior to epidermal invasion, and the loss of germ cells from mature testis, can be explained by the inability of Sl17H mutant SCF to be targeted to the basolateral compartment of polarized epithelial keratinocytes and Sertoli cells, respectively.

Animals↗

PubMed1998

Actin dynamics in living mammalian cells.

The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human &bgr ;-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured 'actin clouds' were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

3T3 Cells↗

PubMed1997

Autocrine regulation of neural crest cell development by steel factor.

Steel factor (SLF) and its cognate receptor, c-kit, have been implicated in the generation of melanocytes from migrating neural crest (NC) cells during early vertebrate embryogenesis. However, the source of SLF in the early avian embryo and its precise role in melanogenesis are unclear. We report here that NC cells themselves express and release SLF protein, which in turn acts as an autocrine factor to induce melanogenesis in nearby NC cells. These results indicate that NC cell subpopulations play an active role in the determination of their cell fate and suggest a different developmental role for the embryonic microenvironment than what has been previously proposed.

Animals↗

PubMed1997

Receptor tyrosine kinase-dependent neural crest migration in response to differentially localized growth factors.

How different neural crest derivatives differentiate in distinct embryonic locations in the vertebrate embryo is an intriguing issue. Many attempts have been made to understand the underlying mechanism of specific pathway choices made by migrating neural crest cells. In this speculative review we suggest a new mechanism for the regulation of neural crest cell migration patterns in avian and mammalian embryos, based on recent progress in understanding the expression and activity of receptor tyrosine kinases during embryogenesis. Distinct subpopulations of crestderived cells express specific receptor tyrosine kinases while residing in a migration staging area. We postulate that the differential expression of receptor tyrosine kinases by specific subpopulations of neural crest cells allows them to respond to localized growth factor ligand activity in the embryo. Thus, the migration pathway taken by neural crest subpopulations is determined by their receptor tyrosine kinase response to the differential localization of their cognate ligand.

Animals↗

PubMed1996

Ectopic c-kit expression affects the fate of melanocyte precursors in Patch mutant embryos.

The Patch (Ph) mutation in the mouse, a deletion that includes the gene for PDGFR alpha, is a recessive lethal that exhibits a dominant pigment phenotype in heterozygotes. To assess whether the Ph mutation acts cell-autonomously or non-autonomously on melanocyte development, we have examined the melanogenic potential of neural crest populations from normal and mutant crest cells in vitro and the pattern of dispersal and survival of melanocyte precursors (MPs) in vivo. We report that trunk neural crest cells from homozygous Ph embryos give rise to pigmented melanocytes in vitro in response to Steel factor (SlF). In vivo, homozygous Ph embryos contain a subpopulation of crest-derived cells that express c-kit and tyrosinase-related protein-2 characteristic of MPs. These cells begin to migrate normally on the lateral crest migration pathway, but then fail to disperse in the dermal mesenchyme and subsequently disappear. Although dermal mesenchyme is adversely affected in Ph homozygotes, SlF mRNA expression by the cells of the dermatome is normal in Ph embryos when neural crest-derived MPs start to migrate on the lateral pathway. In contrast, mRNA for the SlF receptor, c-kit, was observed to be ectopically expressed in somites and lateral mesenchyme in embryos carrying the Ph mutation. Based on this ectopic expression of c-kit in Ph mutant embryos, and the observed distribution of SlF protein in normal and mutant embryos, we suggest that competition for limited amounts of SlF localized on the lateral neural crest migration pathway alters melanocyte dispersal and survival.

Animals↗

PubMed1995

Soluble and cell-bound forms of steel factor activity play distinct roles in melanocyte precursor dispersal and survival on the lateral neural crest migration pathway.

Trunk neural crest cells segregate from the neuroepithelium and enter a 'migration staging area' lateral to the embryonic neural tube. After some crest cells in the migration staging area have begun to migrate on a medial pathway, a subpopulation of crest-derived cells remaining in the migration staging area expresses mRNAs for the receptor tyrosine kinase, c-kit, and tyrosinase-related protein-2, both of which are characteristic of melanocyte precursors. These putative melanocyte precursors are subsequently observed on the lateral crest migration pathway between the dermatome and overlying epithelium, and then dispersed in nascent dermal mesenchyme. Melanocyte precursors transiently require the c-kit ligand, Steel factor for survival. Although Steel factor mRNA is transiently expressed in the dorsal dermatome before the onset of trunk neural crest cell dispersal on the lateral pathway, it is no longer produced by dermatomal cells when melanocyte precursors have dispersed in the dermal mesenchyme. To assess the role of Steel factor in migration of melanocyte precursors on the lateral pathway, we analyzed melanocyte precursor dispersal and fate on the lateral pathway of two different Sl mutants, Sl, a null allele, and Sld, which lacks cell surface-associated Steel factor but produces a soluble form. No melanocyte precursors were detected in the dermatome of embryos homozygous for the Sl allele or in W mutants that lack functional c-kit. In contrast, in embryos homozygous for the Sld allele, melanocyte precursors appeared on the lateral pathway, but subsequently disappear from the dermis. These results suggest that soluble Steel factor is required for melanocyte precursor dispersal on the lateral pathway, or for their initial survival in the migration staging area. In contrast, membrane-bound Steel factor appears to promote melanocyte precursor survival in the dermis.

Animals↗

PubMed1994

Tenascin-C in peripheral nerve morphogenesis.

The extracellular matrix (ECM) molecule tenascin/cytotactin (TN-C) is expressed at a high level by satellite (glial precursor) cells in developing peripheral nerves of the chick embryo; synthesis of its mRNA peaks at the time period when axonal growth is maximal. When offered as a substrate in vitro, TN-C mediates neurite outgrowth by both motor and sensory neurons. The ability to grow neurites on TN-C is developmentally regulated: sensory neurons from 4-day chick embryos (the stage at which peripheral nerves start to develop) grow immediately and rapidly, whereas neurons from older embryos respond with a long delay. A TN-C domain responsible for this activity is located within the C-terminal (distal) portion of TN-C subunits. Integrin receptors seem to be involved on peripheral neurites because their growth on TN-C is completely blocked by antibodies to beta 1 integrins. In striking contrast to neuronal processes, nerve satellite cells can attach to a TN-C substrate but are completely inhibited in their migratory activity. Artificial substrate borders between tenascin and fibronectin or laminin act as selective barriers that allow neurites to pass while holding up satellite cells. The repulsive action of TN-C on satellite cells is similar to that observed for other cell types and is likely to be mediated by additional TN-C domains. In view of these data, it is surprising that mice seem to develop normally without a functional TN-C gene. TN-C is likely to be redundant, that is, its dual action on cell adhesion is shared by other molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Dual function of tenascin: simultaneous promotion of neurite growth and inhibition of glial migration.

The extracellular matrix molecule tenascin is expressed within the developing peripheral nervous system, first by migrating neural crest cells and later by satellite (Schwann precursor) cells at the growing tips of peripheral nerves. Here we found that the neurite promoting activity of tenascin for sensory neurons is developmentally regulated: very young sensory ganglia of stage 23 (4 days old) embryos grew neurites on tenascin as fast as on laminin and fibronectin. The growth response of older (day 7 and 9) ganglia on laminin and fibronectin was similar to that of 4-day-old ganglia, while on tenascin neurite growth occurred only after a lag phase and at a slower rate. Neurite growth on tenascin was inhibited by antibodies to beta 1 integrin and by heparin. While tenascin promotes neurite outgrowth of peripheral neurons, we found that it does not allow satellite cell migration when it is present on the substratum, and it inhibits migration of satellite cells on fibronectin when added in soluble form. In contrast, soluble tenascin did not significantly alter the rate of neurite growth on tenascin, fibronectin or laminin substrata, although neurites were straighter and less attached. When isolated satellite cells were added to neurites grown on tenascin, they preferentially adhered to and elongated along neurite surfaces. Using patterned substrata of tenascin versus fibronectin or laminin confirmed that tenascin borders allow neurites to pass but act as barriers to migrating satellite cells. We postulate that tenascin or related molecules with dual functions in cell adhesion are important for peripheral nerve morphogenesis. Tenascin allows axonal growth, but may restrict random satellite cell migration into the fibronectin-rich mesenchyme, thereby inducing the compaction of nerve fascicles.

Animals↗

PubMed1991

Epithelial synthesis of tenascin at tips of growing bronchi and graded accumulation in basement membrane and mesenchyme.

The extracellular matrix protein, tenascin, has been proposed as mediator in epithelial-mesenchymal interactions because of its characteristic distribution during embryogenesis. Here we compared the accumulation of tenascin and laminin in the early chicken lung bud. Laminin is deposited in the basement membrane, starting at the tips and increasing along the shafts of growing primary and secondary bronchi. In contrast, tenascin accumulation is highest in basement membranes and mesenchyme at sites where new bronchial branches are formed. By in situ hybridization, tenascin mRNA was found to be produced exclusively by the epithelium at sites of active growth of bronchial tubes.

Animals↗

PubMed1991

Chick laminin: isolation by monoclonal antibodies and differential distribution of variants in the embryo.

In order to study the expression and function of laminin variants during chick embryonic development, we have generated monoclonal antibodies against chick heart laminin. One monoclonal antibody (mAb), called 9/F-10, could be used to purify chick laminin to homogeneity. By rotary shadowing, cross-shaped and T-shaped laminin particles as well as aggregates of two laminin molecules crosslinked via their short arms could be observed in this preparation. Purified chick laminin was very potent in mediating neurite growth by chick embryonic neurons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced chick heart laminin revealed a complex pattern of polypeptides which are immunologically related to several mammalian laminin chains. The two mAbs, 9/F-10 and 3/E-8, recognize two different types of chick laminin subunits. By immunofluorescence, antibody 3/E-8 labels basement membranes, aortic smooth muscle, and mesenchyme of 6-day-old chick embryos. In contrast, staining by mAb 9/F-10 is confined to basement membranes. Therefore, the two antibodies seem to distinguish between two different chick laminin isoforms.

Animals↗

PubMed1991

Nerve-dependent and -independent tenascin expression in the developing chick limb bud.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Tenascin accumulates along developing peripheral nerves as they leave the spinal cord and enter the limb mesenchyme (Wehrle and Chiquet, Development 110, 401-415, 1990). Here we found that most but not all tenascin deposited along growing nerves is of glial origin. By in situ hybridization with a tenascin cDNA probe, we determined the site of tenascin mRNA accumulation both in normal and nerve-free limbs. In normal wing buds, tenascin mRNA was first detected within the developing limb nerves. Vinculin-positive glial precursor cells, which comigrate with the axons, are the likely source of this tenascin message. In nerveless wing grafts, tenascin was first expressed in tendon primordia in the absence, and thus independently, from innervation. In contrast to normal limbs, grafted wing buds neither contained vinculin-positive glial precursor cells, nor expressed tenascin in regions proximal to tendon primordia. In normal wing buds, tenascin deposited by tendon primordia transiently parallels and surrounds certain developing nerves. After the major nerve pattern is established, tenascin mRNA disappears from nerves in the upper limb, but is expressed in perichondrium and tendons. We propose that glial tenascin facilitates the penetration of axons into the limb bud and is important for nerve fasciculation. In some places, early tendon primordia might help to guide the migration of axons and glial precursor cells towards their target.

Animals↗