Jonathan C. R. Jones

β4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium

Tumor cells can evade chemotherapy by acquiring resistance to apoptosis. We investigated the molecular mechanism whereby malignant and nonmalignant mammary epithelial cells become insensitive to apoptosis. We show that regardless of growth status, formation of polarized, three-dimensional structures driven by basement membrane confers protection to apoptosis in both nonmalignant and malignant mammary epithelial cells. By contrast, irrespective of their malignant status, nonpolarized structures are sensitive to induction of apoptosis. Resistance to apoptosis requires ligation of beta4 integrins, which regulates tissue polarity, hemidesmosome formation, and NFkappaB activation. Expression of beta4 integrin that lacks the hemidesmosome targeting domain interferes with tissue polarity and NFkappaB activation and permits apoptosis. These results indicate that integrin-induced polarity may drive tumor cell resistance to apoptosis-inducing agents via effects on NFkappaB.

Desmosomes and hemidesmosomes: structure and function of molecular components.

Desmosomes and hemidesmosomes are the major cell surface attachment sites for intermediate filaments at cell‐cell and cell‐substrate contacts, respectively. The transmembrane molecules of the desmosome belong to the cadherin family of calcium‐dependent adhesion molecules, whereas those in the hemidesmosome include the integrin class of cell matrix receptors. In each junction, the cytoplasmic domains of certain transmembrane junction components contain unusually long carboxy‐ter‐ minal tails not found in those family members involved in linkage of actin to the cell surface. These domains are thought to be important for the regulation of junction assembly and specific attachment of intermediate filaments via associated adapter proteins. Recent developments have suggested the exciting possibility that these junctions, in addition to playing an important structural function in tissue integrity, are both acceptors and affectors of cell signaling pathways. Many desmosomal and hemides‐ mosomal constituents are phosphoproteins and in certain cases the function of specific phosphorylation sites in regulating protein‐protein interactions is being uncovered. In addition, a more active role in transmitting signals that control morphogenesis during development and possibly even regulate cell growth and differentiation are being defined for cytoplasmic and membrane components of these junctions.—Green, K. J., Jones, J. C. R. Desmosomes and hemidesmosomes: structure and function of molecular components. FASEB J. 10,871‐881 (1996)

STRUCTURE AND ASSEMBLY OF HEMIDESMOSOMES

The hemidesmosome is a complex junction containing many proteins. The keratin cytoskeleton attaches to its cytoplasmic plaque, while its transmembrane elements interact with components of the extracellular matrix. Hemidesmosome assembly involves recruitment of α6β4 integrin heterodimers, as well as cytoskeletal elements and cytoskeleton‐associated proteins to the cell surface. In our cell culture models, these phenomena appear to be triggered by laminin‐5 in the extracellular matrix. Cell interaction with laminin‐5 apparently induces both phosphorylation and dephosphorylation of subunits of α6β4 integrin. There is emerging evidence that such events are necessary for subsequent cytoskeleton anchorage to the hemidesmosome cytoplasmic plaque. Once assembled, the hemidesmosome plays an essential role in maintaining firm epithelial adhesion to the basement membrane, with hemidesmosome disruption being a hallmark of certain devastating blistering diseases. However, the hemidesmosome is more than just a stable anchor, as it may also be the site of signal transduction, mediated by its α6β4 integrin component. This review discusses our current knowledge of the structure and assembly of the hemidesmosome. BioEssays 20:488–494, 1998.

The vimentin cytoskeleton regulates focal contact size and adhesion of endothelial cells subjected to shear stress

Recently, we reported that vimentin-type intermediate filaments, in addition to microfilaments, associate with αvβ3 integrin-positive focal contacts in endothelial cells. To gain insight into intermediate filament-focal contact interaction, we induced expression of yellow fluorescent protein (YFP)-integrin β3 and cyan fluorescent protein (CFP)-vimentin protein in endothelial cells. At least 50% of the YFP-β3 integrin-labeled focal contacts associated with CFP-labeled vimentin intermediate filaments in live cells. Moreover, focal contacts and intermediate filaments moved in concert in the plane of the membrane and assembling focal contacts were sites of vimentin filament assembly. When endothelial cells were subjected to flow, large focal contacts assembled and associated with thick vimentin bundles. These large focal contacts showed minimal dynamic activity. Cells in which vimentin expression had been inhibited by RNA interference assembled smaller than normal focal contacts. More dramatically, such cells showed decreased adhesion to the substratum. These data provide evidence that the vimentin cytoskeleton regulates focal contact size and helps stabilize cell-matrix adhesions in endothelial cells.

Intermediate Filaments: Possible Functions as Cytoskeletal Connecting Links Between the Nucleus and the Cell Surface

Intermediate filaments (IF), along with microtubules and microfilaments constitute the three major fibrous protein systems that have been defined by electron microscopical, biochemical, and immunological studies of a wide variety of cell types.’-’ In addition to the major proteinaceous subunits making up the backbone or core of these cytoplasmic fibers (e.g. tubulin in microtubules), there are also numerous “associated proteins” which appear to complex with their walls. These associated proteins are thought to be involved in various functions, including crosslinking and the regulation of polymerization. Numerous examples of such associated proteins can be found in the literature including many of the actin or microfilament associated proteins such as tropomyosin, a-actinin, filamin, etc.,’” and the microtubule-associated proteins (MAPS), both of which have been described extensively. Recently, IF-associated proteins (IFAPs) have also been described and activity in this area of study will undoubtedly increase in intensity in the near future.’.’ When considered together these three fiber systems and their associated proteins constitute a major proportion of total cell protein which collectively has become known as the cytoskeleton or the cytoskeletal system. Unfortunately, the term “cytoskeleton” when used in this fashion, is a misleading one, as it implies a lack of dynamic activity and further suggests that these three distinctive fibrous protein systems are similar with regard to their functions. This is certainly not the case and is contrary to well established facts that demonstrate that the three cytoplasmic fiber systems are distinctly different with regard to their subcellular organization, their relationship to different cell functions, their relative stabilities, and their biochemical and immunological properties.’-’ In spite of these considerations, numerous recent biochemical and molecular studies have emphasized associations of various cellular components (for example, nuclei, ribosomes, etc.) with nondescript “cytoskeletal elements” that are usually

Complex interactions between the laminin α4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo

The α4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins αvβ3, α3β1, and α6β1. An antibody against the G domain (residues 919-1207; G919–1207) of the α4 laminin subunit inhibits angiogenesis in a mouse–human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G919–1207 to support endothelial cell adhesion. In particular, endothelial cell adhesion to G919–1207 is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016–1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins αvβ3 and β1 and a combination of antibodies against α3 and α6 integrin subunits inhibit endothelial cell attachment to G919–1207. Moreover, both αvβ3 and α3β1 integrin bind with high affinity to G919–1207. Together, our studies demonstrate that the G domain of laminin α4 chain is a specific, high affinity ligand for the αvβ3 and α3β1 integrin heterodimers and that these integrins, together with α6β1, function cooperatively to mediate endothelial cell–α4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the αvβ3 integrin in angiogenesis.

Intermediate Filament Networks: Organization and Possible Functions of a Diverse Group of Cytoskeletal Elements

SUMMARY Immunofluorescence and electron microscopic observations demonstrate that intermediate filaments (IF) form cytoplasmic networks between the nucleus and cell surface in several types of cultured cells. Intermediate filaments interact with the nuclear surface, where they appear to terminate at the level of the nuclear envelope. From this region, they radiate towards the cell surface where they are closely associated with the plasma membrane. On the basis of these patterns of IF organization, we suggest that IF represent a cytoskeletal system interconnecting the cell surface with the nucleus. Furthermore, IF also appear to interact with other cytoskeletal components including microtubules and microfilaments. In the former case microtubule–IF interactions are seen in cytoplasmic regions between the nucleus and the cell membrane, whereas microfilament–IF interactions occur in the cortical cytoplasm. IF also appear to be cross-linked to each other; especially in the case of the IF bundles that occur in epithelial cells. In order to determine the molecular and biochemical bases of the organizational state of IF we have developed procedures for obtaining IF-enriched ‘cytoskeletons’ of cultured cells. In these preparations IF–nuclear and IF–cell surface associations are retained. Thus, these preparations have enabled us to begin to study various IF-associated structures (e.g. desmosomes) and associated proteins (IFAPs) using biochemical and immunological methodologies. To date, the results support the idea that IF and their associated proteins may comprise the cell type specific molecular infrastructure that is involved in transmitting and distributing information amongst the major cellular domains; the cell surface/extracellular matrix, the cytoplasm and the nuclear surface/nuclear matrix.

Integrin β4 Regulates Migratory Behavior of Keratinocytes by Determining Laminin-332 Organization

Whether α6β4 integrin regulates migration remains controversial. β4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express β4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type β4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with α6β4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the α6β4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.

Recruitment of vimentin to the cell surface by β3 integrin and plectin mediates adhesion strength

Much effort has been expended on analyzing how microfilament and microtubule cytoskeletons dictate the interaction of cells with matrix at adhesive sites called focal adhesions (FAs). However, vimentin intermediate filaments (IFs) also associate with the cell surface at FAs in endothelial cells. Here, we show that IF recruitment to FAs in endothelial cells requires β3 integrin, plectin and the microtubule cytoskeleton, and is dependent on microtubule motors. In CHO cells, which lack β3 integrin but contain vimentin, IFs appear to be collapsed around the nucleus, whereas in CHO cells expressing β3 integrin (CHOwtβ3), vimentin IFs extend to FAs at the cell periphery. This recruitment is regulated by tyrosine residues in the β3 integrin cytoplasmic tail. Moreover, CHOwtβ3 cells exhibit significantly greater adhesive strength than CHO or CHO cells expressing mutated β3 integrin proteins. These differences require an intact vimentin network. Therefore, vimentin IF recruitment to the cell surface is tightly regulated and modulates the strength of adhesion of cells to their substrate.

Laminin-5 coating enhances epithelial cell attachment, spreading, and hemidesmosome assembly on Ti-6Al-4V implant material in vitro

Enhancement of epithelial cell attachment to laminin-5-coated titanium alloy (Ti-6Al-4V) implant material was evaluated in vitro. Protein analysis showed that Ti-6Al-4V has a high affinity for laminin-5 and adsorbed significantly more laminin-5 than laminin-1. DNA analysis showed that laminin-5 enhanced attachment of normal human epidermal keratinocytes (NHEK) to Ti-6Al-4V significantly more than did laminin-1 or uncoated controls. The effect of passivation on laminin-5 adsorption and activity on Ti-6Al-4V also was evaluated. Passivation had no significant effect on the amount of protein adsorbed; however, AFM, ESCA, and ToF-SIMS analyses suggested that passivation affects the conformation of adsorbed laminin-5. Although laminin-5 coating significantly enhanced rapid attachment of epithelial cells to both passivated and unpassivated Ti-6Al-4V, surface area measurements showed that cells spread on laminin-5-coated passivated Ti-6Al-4V covered a significantly larger surface area than cells spread on laminin-5-coated unpassivated samples. TEM analysis showed that cells formed significantly more hemidesmosomes on the surface of laminin-5 coated passivated than on the surface of laminin-5 coated unpassivated titanium alloy. The enhancement of rapid cell attachment, spreading, and hemidesmosome assembly on laminin-5-coated passivated samples may reflect better integration between epithelial cells and titanium alloy and thus may be predictive of long-term implant stability.