Podosome-type adhesions and focal adhesions, so alike yet so different

Cell-matrix adhesions are essential for cell migration, tissue organization and differentiation, therefore playing central roles in embryonic development, remodeling and homeostasis of tissues and organs. Matrix adhesion-dependent signals cooperate with other pathways to regulate biological functions such as cell survival, cell proliferation, wound healing, and tumorigenesis. Cell migration and invasion are integrated processes requiring the continuous, coordinated assembly and disassembly of integrin-mediated adhesions. An understanding of how integrins regulate cell migration and invasiveness through the dynamic regulation of adhesions is fundamental to both physiological and pathological situations. A variety of cell-matrix adhesions has been identified, namely, focal complexes, focal adhesions, fibrillar adhesions, podosomes, and invadopodia (podosome-type adhesions). These adhesion sites contain integrin clusters able to develop specialized structures, which are different in their architecture and dynamics although they share almost the same proteins. Here we compare recent advances and developments in the elucidation of the organization and dynamics of focal adhesions and podosome-type adhesions, in order to understand how such subcellular sites - though closely related in their composition - can be structurally and functionally different. The underlying question is how their respective physiological or pathological roles are related to their distinct organization.     

START-GAP3/DLC3 is a GAP for RhoA and Cdc42 and is localized in focal adhesions regulating cell morphology

In the human genome there are three genes encoding RhoGAPs that contain the START (steroidogenic acute regulatory protein (StAR)-related lipid transfer)—domain. START-GAP3/DLC3 is a tumor suppressor gene similar to two other human START-GAPs known as DLC1 or DLC2. Although expression of START-GAP3/DLC3 inhibits the proliferation of cancer cells, its molecular function is not well understood. In this study we carried out biochemical characterization of START-GAP3/DLC3, and explored the effects of its expression on cell morphology and intracellular localization. We found that START-GAP3/DLC3 serves as a stimulator of PLCδ1 and as a GAP for both RhoA and Cdc42 in vitro. Moreover, we found that the GAP activity is responsible for morphological changes. The intracellular localization of endogenous START-GAP3/DLC3 was explored by immunocytochemistry and was revealed in focal adhesions. These results indicate that START-GAP3/DLC3 has characteristics similar to other START-GAPs and the START-GAP family seems to share common characteristics.     

Polycystins, focal adhesions and extracellular matrix interactions

Polycystic kidney disease is the most common heritable disease in humans. In addition to epithelial cysts in the kidney, liver and pancreas, patients with autosomal dominant polycystic kidney disease (ADPKD) also suffer from abdominal hernia, intracranial aneurysm, gastrointestinal cysts, and cardiac valvular defects, conditions often associated with altered extracellular matrix production or integrity. Despite more than a decade of work on the principal ADPKD genes, PKD1 and PKD2, questions remain about the basis of cystic disease and the role of extracellular matrix in ADPKD pathology. This review explores the links between polycystins, focal adhesions, and extracellular matrix gene expression. These relationships suggest roles for polycystins in cell-matrix mechanosensory signaling that control matrix production and morphogenesis. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

A computational model of cell migration coupling the growth of focal adhesions with oscillatory cell protrusions

Cell migration is a highly integrated process where actin turnover, actomyosin contractility, and adhesion dynamics are all closely linked. In this paper, we propose a computational model investigating the coupling of these fundamental processes within the context of spontaneous (i.e. unstimulated) cell migration. In the unstimulated cell, membrane oscillations originating from the interaction between passive hydrostatic pressure and contractility are sufficient to lead to the formation of adhesion spots. Cell contractility then leads to the maturation of these adhesion spots into focal adhesions. Due to active actin polymerization, which reinforces protrusion at the leading edge, the traction force required for cell translocation can be generated. Computational simulations first show that the model hypotheses allow one to reproduce the main features of fibroblast cell migration and established results on the biphasic aspect of the cell speed as a function of adhesion strength. The model also demonstrates that certain temporal parameters, such as the adhesion proteins recycling time and adhesion lifetimes, influence cell motion patterns, particularly cell speed and persistence of the direction of migration. This study provides some elements, which allow a better understanding of spontaneous cell migration and enables a first glance at how an individual cell would potentially react once exposed to a stimulus.     

PDMP,a ceramide analogue,acts as an inhibitor of mTORC1 by inducing its translocation from lysosome to endoplasmic reticulum

Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism, and cell differentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a well known glycosphingolipid synthesis inhibitor, also affects the structures and functions of various organelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates the mTORC1 activity through its effects on organellar behavior. PDMP induced the translocation of mTORC1 from late endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells. Surprisingly, we found mTORC1 translocation to the ER upon PDMP treatment. This effect of PDMP was independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor activity, showed essentially the same effect. We confirmed that PDMP inhibits the mTORC1 activity based on the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase in LC3 puncta, reflecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation and differentiation of MC3T3-E1 cells. Accordingly, the present results reveal a novel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR from lysosomes to the ER.     

CPT1c is localized in endoplasmic reticulum of neurons and has carnitine palmitoyltransferase activity

CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.     

Integrating actin dynamics,mechanotransduction and integrin activation: The multiple functions of actin binding proteins in focal adhesions

Focal adhesions are clusters of integrin transmembrane receptors that mechanically couple the extracellular matrix to the actin cytoskeleton during cell migration. Focal adhesions sense and respond to variations in force transmission along a chain of protein-protein interactions linking successively actin filaments, actin binding proteins, integrins and the extracellular matrix to adapt cell-matrix adhesion to the composition and mechanical properties of the extracellular matrix. This review focuses on the molecular mechanisms by which actin binding proteins integrate actin dynamics, mechanotransduction and integrin activation to control force transmission in focal adhesions.     

ADAM12-mediated focal adhesion formation is differently regulated by beta1 and beta3 integrins

ADAM12, adisintegrin and metalloprotease, has been demonstrated to be upregulated in human malignant tumors and to accelerate the malignant phenotype in a mouse model for breast cancer. ADAM12 is a substrate for beta1 integrins and may affect tumor and stromal cell behavior through its binding to beta1 integrins. Here, we report that cells deficient in beta1 integrin or overexpressing beta3 integrin can bind to recombinant full-length human ADAM12 via beta3 integrin. Furthermore, cell binding to ADAM12 via beta3 integrin results in the formation of focal adhesions, which are not formed upon beta1 integrin-mediated cell attachment. We also show that RhoA is involved in beta3 integrin-mediated focal adhesion formation.     

Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions

Integrin-mediated cell adhesion often results in cell spreading and the formation of focal adhesions. We exploited the capacity of recombinant human alpha IIb beta 3 integrin to endow heterologous cells with the ability to adhere and spread on fibrinogen to study the role of integrin cytoplasmic domains in initiation of cell spreading and focal adhesions. The same constructs were also used to analyze the role of the cytoplasmic domains in maintenance of the fidelity of the integrin repertoire at focal adhesions. Truncation mutants of the cytoplasmic domain of alpha IIb did not interfere with the ability of alpha IIb beta 3 to initiate cell spreading and form focal adhesions. Nevertheless, deletion of the alpha IIb cytoplasmic domain allowed indiscriminate recruitment of alpha IIb beta 3 to focal adhesions formed by other integrins. Truncation of the beta 3 subunit cytoplasmic domain abolished cell spreading mediated by alpha IIb beta 3 and also abrogated recruitment of alpha IIb beta 3 to focal adhesions. This truncation also dramatically impaired the ability of alpha IIb beta 3 to mediate the contraction of fibrin gels. In contrast, the beta 3 subunit cytoplasmic truncation did not reduce the fibrinogen binding affinity of alpha IIb beta 3. Thus, the integrin beta 3 subunit cytoplasmic domain is necessary and sufficient for initiation of cell spreading and focal adhesion formation. Further, the beta 3 cytoplasmic domain is required for the transmission of intracellular contractile forces to fibrin gels. The alpha subunit cytoplasmic domain maintains the fidelity of recruitment of the integrins to focal adhesions and thus regulates their repertoire of integrins.     

Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions

Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures.     

Membrane-type 1 matrix metalloproteinase modulates focal adhesion stability and cell migration

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in extracellular matrix-induced cell migration and the activation of extracellular signal-regulated kinase (ERK). We showed here that transfection of the MT1-MMP gene into HeLa cells promoted fibronectin-induced cell migration, which was accompanied by fibronectin degradation and reduction of stable focal adhesions, which function as anchors for actin-stress fibers. MT1-MMP expression attenuated integrin clustering that was induced by adhesion of cells to fibronectin. The attenuation of integrin clustering was abrogated by MT1-MMP inhibition with a synthetic MMP inhibitor, BB94. When cultured on fibronectin, HT1080 cells, which endogenously express MT1-MMP, showed so-called motile morphology with well-organized focal adhesion formation, well-oriented actin-stress fiber formation, and the lysis of fibronectin through trails of cell migration. Inhibition of endogenous MT1-MMP by BB94 treatment or expression of the MT1-MMP carboxyl-terminal domain, which negatively regulates MT1-MMP activity, resulted in the suppression of fibronectin lysis and cell migration. BB94 treatment promoted stable focal adhesion formation concomitant with enhanced phosphorylation of tyrosine 397 of focal adhesion kinase (FAK) and reduced ERK activation. These results suggest that lysis of the extracellular matrix by MT1-MMP promotes focal adhesion turnover and subsequent ERK activation, which in turn stimulates cell migration.     

Protein mislocalization: Mechanisms,functions and clinical applications in cancer

The changes from normal cells to cancer cells are primarily regulated by genome instability, which foster hallmark functions of cancer through multiple mechanisms including protein mislocalization. Mislocalization of these proteins, including oncoproteins, tumor suppressors, and other cancer-related proteins, can interfere with normal cellular function and cooperatively drive tumor development and metastasis. This review describes the cancer-related effects of protein subcellular mislocalization, the related mislocalization mechanisms, and the potential application of this knowledge to cancer diagnosis, prognosis, and therapy.     

Phosphorylation of SHP-2 regulates interactions between the endoplasmic reticulum and focal adhesions to restrict interleukin-1-induced Ca2+ signaling

Interleukin-1 (IL-1)-induced Ca2+ signaling in fibroblasts is constrained by focal adhesions. This process involves the proteintyrosine phosphatase SHP-2, which is critical for IL-1-induced phosphorylation of phospholipase Cgamma1, thereby enhancing IL-1-induced Ca2+ release and ERK activation. Currently, the mechanisms by which SHP-2 modulates Ca2+ release from the endoplasmic reticulum are not defined. We used immunoprecipitation and fluorescence protein-tagged SHP-2 or endoplasmic reticulum (ER)-protein expression vectors, and an ER-specific calcium indicator, to examine the functional relationships between SHP-2, focal adhesions, and IL-1-induced Ca2+ release from the ER. By total internal reflection fluorescence microscopy to image subplasma membrane compartments, SHP-2 co-localized with the ER-associated proteins calnexin and calreticulin at sites of focal adhesion formation in fibroblasts. IL-1beta promoted time-dependent recruitment of SHP-2 and ER proteins to focal adhesions; this process was blocked in cells treated with small interfering RNA for SHP-2 and in cells expressing a Y542F SHP-2 mutant. IL-1 stimulated inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the ER subjacent to the plasma membrane that was tightly localized around fibronectin-coated beads and was reduced 4-fold in cells expressing Tyr-542 SHP-2 mutant. In subcellular fractions enriched for ER proteins, immunoprecipitation demonstrated that IL-1-enhanced association of SHP-2 with the type 1 inositol 1,4,5-trisphosphate receptor was dependent on Tyr-542 of SHP-2. We conclude that Tyr-542 of SHP-2 modulates IL-1-induced Ca2+ signals and association of the ER with focal adhesions.     

Translocation of the IL-1 receptor to focal adhesions is regulated through the C-terminal end of the cytoplasmic domain

The mechanisms of translocation of the IL-1 receptor to focal adhesions were analysed using EGFP reporter constructs and confocal microscopy. In fibronectin-attached cells, the receptor fusion protein was present in distinct punctate areas, co-localizing with the transmembrane-linkage protein vinculin. In migrating cells, the two proteins co-localized at the leading edge in ruffle-like structures. Experiments using a series of receptor mutants revealed that translocation of the IL-1 receptor to focal adhesions was dependent on motifs conserved between members of the TIR family, in the C-terminal end of the receptor. Further, an enhanced level of expression of the wild-type receptor at attachment sites was shown to correlate with an increased severity of IL-1 induced structural effects. This report demonstrates that translocation of the IL-1 receptor to focal adhesions is dependent on conserved domains in the C-terminal end of the protein, and that receptor localization at these sites is important in structural regulation of IL-1 mediated responses.     

Analysis of the proteins,glycoproteins and glycosaminoglycans of fibroblast adhesions to substratum

The focal adhesion preparations which remain attached to a glass substratum when fibroblast bodies are removed by a gentle stream of buffer have been analysed by gel electrophoresis coupled with other selective methods of analysis. The results are consistent with the presence of three classes of macromolecular components. (i) Muscle and associated proteins amongst which actin was abundant with significant amounts of tropomyosin, some myosin and traces of α-actinin. Some vimentin was present but no vinculin. We detected a major new protein component, as yet unidentified, with a molecular weight in the region of 50 000–55 000 which is not desmin or tubulin and could have an important function at the focal adhesion. (ii) Glycoproteins which are a specialised subset of those in the whole plasma membrane and included a family which bind ricin and therefore contain β-galactose end groups, together with a series having carbohydrate chains which bound neither ricin nor concanavalin A. The relative proportion of ricin-binding glycoproteins compared to concanavalin A-binding glycoproteins was higher than in whole plasma membranes. (iii) Glycosaminoglycans, with hyaluronate identified as the major component by column chromatography and its susceptibility to Streptomyces hyaluronidase.     

CPEB4 is a cell survival protein retained in the nucleus upon ischemia or endoplasmic reticulum calcium depletion

The RNA binding protein CPEB (cytoplasmic polyadenylation element binding) regulates cytoplasmic polyadenylation and translation in germ cells and the brain. In neurons, CPEB is detected at postsynaptic sites, as well as in the cell body. The related CPEB3 protein also regulates translation in neurons, albeit probably not through polyadenylation; it, as well as CPEB4, is present in dendrites and the cell body. Here, we show that treatment of neurons with ionotropic glutamate receptor agonists causes CPEB4 to accumulate in the nucleus. All CPEB proteins are nucleus-cytoplasm shuttling proteins that are retained in the nucleus in response to calcium-mediated signaling and alpha-calcium/calmodulin-dependent kinase protein II (CaMKII) activity. CPEB2, -3, and -4 have conserved nuclear export signals that are not present in CPEB. CPEB4 is necessary for cell survival and becomes nuclear in response to focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose. Further analysis indicates that nuclear accumulation of CPEB4 is controlled by the depletion of calcium from the ER, specifically, through the inositol-1,4,5-triphosphate (IP3) receptor, indicating a communication between these organelles in redistributing proteins between subcellular compartments.     

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell–cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell–cell and/or cell–ECM contacts and anchorage-independent cell proliferation.     

Membrane contacts of the endoplasmic reticulum and their possible functions in the plant cell

Close contacts of the endoplasmic reticulum membrane and plasmalemma have been visualized inside plant cells by means of electron microscopy. The qualitative similarity of these contacts to high-permeable intercellular contacts in animals has been shown. New data confirming the hypothesis of the identity of stromules, i.e., dynamic tubular protuberances of the plastid membrane of the plant cell, and tubular elements of the endoplasmic reticulum have been presented. New possible functions of the contacts of the endoplasmic reticulum membrane with other membranes inside the cell have been discussed on the basis of this hypothesis.     

Effects of PPARgamma agonists on cell survival and focal adhesions in a Chinese thyroid carcinoma cell line

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists cause cell death in several types of cancer cells. The aim of this study was to examine the effects of two PPARgamma agonists, ciglitazone and 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2), on the survival of thyroid carcinoma CGTH W-2 cells. Both ciglitazone and 15dPGJ2 decreased cell viability in a time- and dose-dependent manner. Cell death was mainly due to apoptosis, with a minor contribution from necrosis. Increased levels of active caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and cytosolic cytochrome-c were noted. In addition, ciglitazone and 15dPGJ2 induced detachment of CGTH W-2 cells from the culture substratum. Both the protein levels and immunostaining signals of focal adhesion (FA) proteins, including vinculin, integrin beta1, focal adhesion kinase (FAK), and paxillin were decreased after PPARgamma agonist treatment. Meanwhile, reduced phosphorylation of FAK and paxillin was noted. Furthermore, PPARgamma agonists induced expression of protein tyrosine phosphatase-PEST (PTP-PEST), and of phosphatase and tensin homologue deleted on chromosome ten (PTEN). The upregulation of these phosphatases might contribute to the dephosphorylation of FAK and paxillin, since pre-treatment with orthovanadate prevented PPARgamma agonist-induced dephosphorylation of FAK and paxillin. Perturbation of CGTH W-2 cells with anti-integrin beta1 antibodies induced FA disruption and apoptosis in the same cells, thus the downregulation of integrin beta1 by PPARgamma agonists resulted in FA disassembly and might induce apoptosis via anoikis. Our results suggested the presence of crosstalk between apoptosis and integrin-FA signaling. Moreover, upregulation and activation of PTEN was correlated with reduced phosphorylation of Akt, and this consequence disfavored cell survival. In conclusion, PPARgamma agonists induced apoptosis of thyroid carcinoma cells via the cytochrome-c caspase 3 and PTEN-Akt pathways, and induced necrosis via the PARP pathway.     

Subcellular localization of adenosine kinase in mammalian cells: The long isoform of AdK is localized in the nucleus

Two isoforms of adenosine kinase (AdK) have been identified in mammalian organisms with the long isoform (AdK-long) containing extra 20-21 amino acids at the N-terminus (NTS). The subcellular localizations of these isoforms are not known and they contain no identifiable targeting sequence. Immunofluorescence labeling of mammalian cells expressing either only AdK-long or both isoforms with AdK-specific antibody showed only nuclear labeling or both nucleus and cytoplasmic labeling, respectively. The AdK-long and -short isoforms fused at the C-terminus with c-myc epitope also localized in the nucleus and cytoplasm, respectively. Fusion of the AdK-long NTS to green fluorescent protein also resulted in its nuclear localization. AdK-long NTS contains a cluster of conserved amino acids (PKPKKLKVE). Replacement of KK in this sequence with either AA or AD abolished its nuclear localization capability, indicating that this cluster likely serves as a nuclear localization signal. AdK in nucleus is likely required for sustaining methylation reactions.