Integrin and ligand-independent PDGFr signaling synergistically contribute to directional migration of Xenopus mesendoderm

Both PDGF signaling and adhesion to fibronectin (FN) matrix have been implicated in the directional collective migration of Xenopus mesendoderm cells at gastrulation. However, mesendoderm explants cultured on FN-coated substrates migrate directionally even in the absence of a source of PDGF. Integrin adhesion has been reported to up-regulate PDGF ligand-independent signaling through the PDGF receptor (PDGFr) in cultured mammalian cells. In order to address whether a similar mechanism stimulates PDGFr signaling in the absence of PDGF-A ligand in amphibian mesendoderm, isolated cells were cultured on bacterial fusion proteins containing the Type-III repeats 9-11 of FN (GST-9.11). Type III9-11 contains the RGD and “synergy” (PPSRN) sites required for integrin α5β1 adhesion and activation but lacks the PDGF-A ligand-binding site present in the full-length FN protein. In order to ensure mesendoderm was not exposed to PDGF in vivo prior to removal and culture in vitro, antisense morpholinos were used to inhibit normal expression of PDGF-A ligand in embryos. P-Akt levels were reduced two-fold when either the PDGFr-α was knocked down or when cells were plated on GST-9.11a, which contains a point mutation (PPSRN>PPSAN) that prevents both full activation of integrin α5β1 and cell spreading. Reduced expression of PDGFr-α was accompanied by perturbations in tissue migration, cytoskeletal organization, polarity of cell protrusions, and focal adhesion area. Mesendoderm cells became rounded, and the actin and cytokeratin filaments appeared collapsed and often colocalized near the cell center. Taken together, these findings suggest that integrin adhesion to FN, acting in synergy with PDGFr-α, is sufficient to elevate PI3K-Akt signaling in the mesendoderm even in the absence of the PDGF-A ligand, and to promote forward-directed protrusions and directional tissue migration.

Desmosomes: interconnected calcium-dependent structures of remarkable stability with significant integral membrane protein turnover

Desmosomes are prominent cell adhesion structures that are major stabilizing elements, together with the attached cytoskeletal intermediate filament network, of the cytokeratin type in epithelial tissues. To examine desmosome dynamics in tightly coupled cells and in situations of decreased adhesion, fluorescent desmosomal cadherin desmocollin 2a (Dsc2a) chimeras were stably expressed in human hepatocellular carcinoma-derived PLC cells (clone PDc-13) and in Madin-Darby canine kidney cells (clone MDc-2) for the continuous monitoring of desmosomes in living cells. The hybrid polypeptides integrated specifically and without disturbance into normal-appearing desmosomes that occurred in association with typical cytokeratin filament bundles. Tracking of labeled adhesion sites throughout the cell cycle by time-lapse fluorescence microscopy revealed that they were immobile and that they maintained their structural integrity for long periods of time. Time-space diagrams further showed that desmosomal positioning was tightly controlled, even during pronounced cell shape changes, although the desmosomal arrays extended and contracted, suggesting that they were interconnected by a flexible system with intrinsic elasticity. Double-fluorescence microscopy detecting Dsc2a chimeras together with fluorescent cytokeratin 18 chimeras revealed the association and synchronous movement of labeled desmosomes and fluorescent cytokeratin filaments. Only a minor destabilization of desmosomes was observed during mitosis, demonstrated by increased diffuse plasma membrane fluorescence and the fusion of desmosomes into larger structures. Desmosomes did not disappear completely at any time in any cell, and residual cytokeratin filaments remained in association with adhesion sites throughout cell division. On the other hand, a rapid loss of desmosomes was observed upon calcium depletion, with irreversible uptake of some desmosomal particles. Simultaneously, diffusely distributed desmosomal cadherins were detected in the plasma membrane that retained the competence to nucleate the reformation of desmosomes after the cells were returned to a standard calcium-containing medium. To examine the molecular stability of desmosomes, exchange rates of fluorescent chimeras were determined by fluorescence recovery after photobleaching, thereby identifying considerable Dsc2a turnover with different rates of fluorescence recovery for PDc-13 cells (36±17% recovery after 30 minutes) and MDc-2 cells (60±20% recovery after 30 minutes). Taken together, our observations suggest that desmosomes are pliable structures capable of fine adjustment to functional demands despite their overall structural stability and relative immobility.

RNA anchoring in the vegetal cortex of the Xenopus oocyte

The body plan of the embryo is established by a polarized source of developmental information in the oocyte. The Xenopus laevis oocyte creates polarity by anchoring mRNAs in the vegetal cortex, including Vg1 and Xwnt-11, which might function in body plan specification, and Xcat-2, which might function in germ cell development. To identify components of the RNA anchoring mechanism, we used the manually isolated vegetal cortex (IVC) to assay loss or change in spatial arrangement of mRNAs caused by disruption of cortical elements. The role of cytoskeleton in mRNA anchoring was tested by treating oocytes with inhibitors that selectively disrupted actin microfilaments and cytokeratin filaments. Treatment of oocytes with cytochalasin B caused clumping of Vg1 and Xwnt-11 as revealed by in situ hybridization of the IVC, but did not cause their release, as confirmed by RT-PCR analysis. These mRNA clumps did not match the distribution of actin microfilament clumps, but were distributed similarly to the remnant cytokeratin filaments. Treatment of oocytes with monoclonal anti-cytokeratin antibody C11 released these mRNAs from the cortex. C11 altered the texture of the cytokeratin network, but did not affect the actin meshwork. These results show that Vg1 and Xwnt-11 are retained by a cytokeratin filament-dependent mechanism, and that organization of the cytokeratin network depend on an intact actin meshwork. Colcemid did not disrupt Vg1 and Xwnt-11 retention in the IVC, so anchoring of these mRNAs are independent of microtubules. Membrane disruption in the IVC by Triton X-100 decreased Vg1 and Xwnt-11. Loss of these mRNAs was due mainly to ribonuclease activity released from membrane components. However, when ribonuclease activity was suppressed under cold temperature, a higher amount of Vg1 and Xwnt-11 was recovered in the supernatant. This result suggested that a fraction of these mRNAs required membranes to be retained in the cortex. By contrast, Xcat-2 mRNA was neither released nor degraded following treatments with cytochalasin B, C11, colcemid and Triton X-100 under cold temperature, so no cortical element could be implicated in its anchoring.

Linking Integrin α6β4-based Cell Adhesion to the Intermediate Filament Cytoskeleton: Direct Interaction between the β4 Subunit and Plectin at Multiple Molecular Sites

Recent studies with patients suffering from epidermolysis bullosa simplex associated with muscular dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin's interactions with hemidesmosomal proteins on the molecular level, we studied its interaction with the uniquely long cytoplasmic tail domain of the β4 subunit of the basement membrane laminin receptor integrin α6β4 that has been implicated in connecting the transmembrane integrin complex with hemidesmosome-anchored cytokeratin filaments. In vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains. Furthermore, we show in vitro self-interaction of integrin β4 cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of hemidesmosome-associated endogenous plectin upon ectopic overexpression of this domain in PtK2 and/or 804G cells. The close association of plectin molecules with hemidesmosomal structures and their apparent random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our data support a model in which plectin stabilizes hemidesmosomes, via directly interlinking integrin β4 subunits and cytokeratin filaments.

Malignant Rhabdoid Tumor in the Gastric Wall of an Aged Orangutan (Pongo pygmaeus)

A 34-year-old female orangutan ( Pongo pygmaeus) developed renal failure and became uremic. At necropsy, large gastric masses were present around the cardia and in the corpus. Abdominal metastases occurred in the liver, pancreas, and right ovary. Light microscopic examination of the tumor revealed polygonal cells with vesicular nuclei and prominent nucleoli. The growth pattern was predominantly solid. Focal areas contained excentric cytoplasmic intermediate filament inclusions, as identified by immunohistochemistry and electron microscopy. Immunohisiochemical procedures demonstrated mainly the vimentin type of intermediate filaments. Except for occasional cytokeratin, other intermediate filament markers and neural, lymphocytic, and histiocytic markers stained negative. The morphologic and ultrastructural characteristics are typical for a malignant rhabdoid tumor, a term used in human pathology to describe a rare and extremely aggressive malignancy of uncertain histogenesis. Although usually located in the infant kidney, a few reports have documented the occurrence of similar lesions in extrarenal sites of adults. In human tumors, vimentin is often combined with the expression of cytokeratins. The sparsity of the cytokeratin filaments in this case might be due to species-specific variations and/or may reflect the hypothesis of a phenotypic concept encompassing a spectrum of histogenetic diversity.

Cultured microvascular endothelial cells (MVEC) differ in cytoskeleton, expression of cadherins and fibronectin matrix. A study under the influence of interferon-gamma

Endothelial cells are known to undergo transitions in cell shape during long-term culture. Thus, the assumption that the separate phenotypes of microvascular endothelial cells (MVEC) recently isolated from bovine corpus luteum represent constitutively different cell strains cannot automatically be made. For this reason, particular morphological qualities from four of five reported MVEC types were studied. Confluent cultures of MVEC types 1, 3, 4 and 5 were either left untreated or exposed to recombinant bovine interferon-gamma (IFN-gamma; 200 units/0.5 ml culture medium) for 3 days. Paraformaldehyde-fixed monolayers were permeabilized with Triton X-100 prior to the detection of filamentous actin, using phalloidin-FITC. Vimentin filaments, cytokeratin filaments, microtubules, E- and N-cadherins as molecules of cell adhesion plaques, and fibronectin filaments were localized by the application of specific antibodies in combination with epifluorescence microscopy. Cells from untreated single cultures uniformly and reproducibly showed an actin cytoskeleton that distinguished the particular MVEC type. MVEC type 1 presented a circular band of fine actin filaments. MVEC type 3 preferentially had developed a starburst-like actin pattern. MVEC type 4 mainly exhibited a polygonal network. MVEC type 5 showed a prominent circular band of thick microfilament bundles from which short filaments radiated. Cytokeratin filaments were noted in MVEC type 1 only. Vimentin filaments occurred as a dense network constricted to the central area in MVEC type 1, while they were spread out in MVEC types 3 and 4. A wavy path comparable to the course of microtubules was apparent in MVEC type 5. Fibronectin assembled into two differently shaped layers at the basal cell side of each MVEC type. Under IFN-gamma treatment, cytoskeletal diversities were maintained between the MVEC types, yet each MVEC type showed specific modulations to its cytoskeleton and to its fibronectin matrix. Upregulation of anti-E-cadherin labelling was detected in MVEC type 1, showing a fluorescent cell border of linear contour. The upregulation of E-cadherin by IFN-gamma treatment could also be demonstrated by western blotting, which revealed a 135 kDa full-sized molecule and a 95 kDa tryptic fragment characteristic of cadherins. Anti-N-cadherin labelling was evident for MVEC type 5, giving rise to a fluorescent punctate cell margin. Our investigations support the existence of truly separate MVEC types.