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PubMed2004

Altered glucose metabolism and proteolysis in pancreatic cancer cell conditioned myoblasts: searching for a gene expression pattern with a microarray analysis of 5000 skeletal muscle genes.

BACKGROUND AND AIMS: We verified whether conditioned media (CM) from pancreatic cancer cell lines (MIAPaCa2, CAPAN-1, PANC-1, BxPC3) alter glucose metabolism and gene expression profiles (microarray experiment with a platform of 5000 skeletal muscle cDNA) in mice myoblasts. METHODS: Myoblasts were incubated with control or pancreatic cancer CM for 24 and 48 hours. RESULTS: Lactate significantly increased in CM compared with non-conditioned myoblasts. No variations in expression levels of the main genes involved in glycolysis were found in CM myoblasts. Propionyl coenzyme A carboxylase and isocitrate dehydrogenase 3 beta genes, which encode enzymes of the tricarboxylic acid cycle, were overexpressed, while IGFIIR and VAMP5 genes were underexpressed in CM myoblasts. PAFAH1B1 and BCL-2 genes (intracellular signal transduction) and the serine protease cathepsin G (proteolysis), were overexpressed in CM myoblasts. Tyrosine accumulation in CM myoblasts suggested that proteolysis overcomes protein synthesis. Sorcin, actin alpha, troponin T1, and filamin A were underexpressed in CM myoblasts. CONCLUSIONS: Our findings demonstrate that pancreatic cancer cell conditioned media enhanced lactate production and induced proteolysis, possibly by altering expression levels of a large number of genes, not only those involved in protein biosynthesis and degradation or glucose metabolism, but also those involved in the tricarboxylic acid cycle and in vesicle traffic.

Aged↗
PubMed1987

Colonic pericrypt sheath cells: characterisation of cell type with new monoclonal antibody.

A new monoclonal antibody, PR 2D3, was raised against a crude homogenate of normal colorectal mucosa and found to react with the cells in the pericrypt sheath. It also reacted with smooth muscle throughout the body and, in specific sites, with those mesenchymal cells known as myofibroblasts. It did not react with cardiac or skeletal muscle, nor with fibroblasts. PR 2D3 is an IgG1 antibody and identifies a membrane component of about 140 K molecular weight. The pericrypt cells have been described as fibroblasts, but in view of the specificity of PR 2D3 for smooth muscle, and its selective staining of the colonic pericrypt cells, this cell type was re-examined for other smooth muscle properties. Ultrastructurally, the cells had many characteristics in common with smooth muscle and were identical with the myofibroblasts of the umbilical cord. On immunocytochemical examination they were found to contain desmin, myosin, and filamin. The confirmation that the pericrypt cells are myofibroblasts suggest that they have both contractile and secretory roles.

Animals↗
PubMed2006

Continuous membrane-cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics.

The plasma membrane of most animal cells conforms to the cytoskeleton and only occasionally separates to form blebs. Previous studies indicated that many weak interactions between cytoskeleton and the lipid bilayer kept the surfaces together to counteract the normal outward pressure of cytoplasm. Either the loss of adhesion strength or the formation of gaps in the cytoskeleton enables the pressure to form blebs. Membrane-associated cytoskeleton proteins, such as spectrin and filamin, can control the movement and aggregation of membrane proteins and lipids, e.g., phosphoinositol phospholipids (PIPs), as well as blebbing. At the same time, lipids (particularly PIPs) and membrane proteins affect cytoskeleton and signaling dynamics. We consider here the roles of the major phosphatidylinositol-4,5-diphosphate (PIP2) binding protein, MARCKS, and PIP2 levels in controlling cytoskeleton dynamics. Further understanding of dynamics will provide important clues about how membrane-cytoskeleton adhesion rapidly adjusts to cytoskeleton and membrane dynamics.

Animals↗
PubMed2006

Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids.

The Ca2+-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca2+ or Mg2+ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca2+ (Ca2+(i)) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G(q/11) or G(i/o) signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca2+(i) mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists.

Actins↗
PubMed1996

Nuclear ion channel activity is regulated by actin filaments.

Actin filaments are novel second messengers involved in ion channel regulation. Because cytoskeletal components interact with the nuclear envelope, the actin cytoskeleton may also control nuclear membrane function. In this report, the patch-clamp technique was applied to isolated nuclei from amphibian A6 epithelial cells to assess the role of actin filaments on nuclear ion channel activity under nucleus-attached or -excised conditions. The most prevalent spontaneous nuclear ion channel species, 76% (n = 46), was cation selective and had a maximal single-channel conductance of approximately 420 pS. Nuclear ion channels also displayed multiple subconductance states, including channel activity of 26 pS that was frequently observed. Nuclear ion channel activity on otherwise quiescent patches was induced by either addition of the actin cytoskeleton disrupter cytochalasin D (CD; 5 micrograms/ml, 60%, 3 of 5 patches) or actin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches (59%, 13 of 22 patches). Actin also induced ion channel activity in quiescent excised inside-out patches from the nuclear envelope (80%, 4 of 5 patches). In contrast, addition of bovine serum albumin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches was without effect on nuclear ion channel activity (5 of 5 patches). The monoclonal antibody MAb414, specific for nuclear pore complex proteins, completely prevented either spontaneous or cytosolic actin-induced nuclear ion channels under nucleus-attached conditions (4 of 4 patches) but not intranuclear actin-induced nuclear ion channels under excised inside-out conditions (3 of 3 patches). In nucleus-attached patches, channel activity was readily activated by addition of the G-actin-binding protein deoxyribonuclease I to nucleus-attached patches (56%, 5 of 9 patches) or further addition of the actin-cross-linker filamin in the presence of actin (57%, 4 of 7 patches). The data indicate that dynamic changes in actin filament organization may represent a novel mechanism to control nuclear function.

Actins↗
PubMed2005

Glycoprotein VI is associated with GPIb-IX-V on the membrane of resting and activated platelets.

The platelet collagen receptor, glycoprotein (GP)VI, initiates platelet aggregation at low shear stress while GPIb-IX-V, which binds von Willebrand factor, elicits platelet aggregation under high shear conditions. To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface, we first ascertained that aggregation induced by a GPVI-specific agonist, collagen-related peptide, like collagen, is markedly cross-blocked by a GPIb alpha-specific monoclonal antibody, SZ2. Immunoprecipitation of GPIb-IX with anti-GPIb alpha from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblotting with anti-GPVI demonstrated association between GPIb-IX and GPVI. This association was maintained when platelets were activated by thrombin. Pre-treatment of platelets with methyl-beta-cyclodextrin to disrupt lipid rafts did not affect association in resting platelets under these conditions of detergent lysis. The association is also independent of cytoskeletal attachment, since it was unaffected by treatment with N-ethylmaleimide or DNaseI, which dissociate GPIb-IX from filamin and the actin-containing cytoskeleton, respectively. Finally, the association involves an interaction between the ectodomains of GPIb alpha and GPVI, since soluble fragments of GPIb alpha (glycocalicin) and GPVI are co-precipitated from the platelet supernatant under conditions where GPVI is shed. A contribution of GPIb-IX-V to GPVI-induced platelet responses, and vice versa, therefore warrants further investigation.

Binding Sites↗
PubMed1986

Myosin isozymes in rabbit and human smooth muscles.

Although multiple forms of myosin in cardiac and skeletal muscles have been identified, it has not been firmly established that myosin isozymes are present in adult smooth muscle. Myosin, extracted from human thoracic aorta and lower saphenous vein and rabbit aorta and uterus, was analyzed by pyrophosphate gel electrophoresis to determine if myosin isozymes are present in these tissues. In all smooth muscle tissues studied, two myosin isozymes were detected and labelled as smooth muscle 1 and smooth muscle 2, smooth muscle 2 being the faster migrating isozyme. Bovine cultured smooth muscle cells from the media of thoracic aorta also contained two forms of myosin. However, cultured fibroblasts contained only one form of myosin. Extracting myosin from either relaxed or contracting rabbit aortic smooth muscle did not influence the mobilities of smooth muscle 1 and smooth muscle 2 on pyrophosphate gels, suggesting that the degree of light chain phosphorylation did not significantly alter the electrophoretic mobility under our conditions. Smooth muscle 1 and smooth muscle 2 myosins each contain heavy chains (200,000 daltons) and light chains (20,000 and 17,000 daltons) in addition to filamin (235,000 daltons), which is closely associated with the native protein. Myosin peptide maps of rabbit aorta and uterus revealed areas of substantially different banding patterns between smooth muscle 1 and smooth muscle 2 from the same tissue. Similar peptide maps of smooth muscle 1 bands were produced from the different tissues, but the smooth muscle 2 maps were dissimilar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Collagen-stimulated human platelet aggregation is mediated by endogenous calcium-activated neutral protease.

To clarify the physiological role of calcium-activated neutral protease (CANP) in human platelets, we loaded the platelets with a Ca2+ -sensitive fluorescent dye, fura-2, and measured the degree of aggregation, cytosolic calcium ion concentration [( Ca2+]i), and proteolysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At physiological concentration of Ca2+ (1 mM) in the incubation medium, [Ca2+]i was below 0.5 microM and platelet aggregation was not shown. Ionomycin (0.15 microM) or collagen (50 micrograms/ml), but not ADP (10 microM), sharply enhanced the [Ca2+]i to near 1 microM and caused the aggregation. A calcium entry blocker, verapamil, completely abolished both the [Ca2+]i rise and the aggregation. NCO-700, a membrane permeable inhibitor against cysteine proteases (including CANP), dose-dependently blocked the aggregation but did not change the [Ca2+]i transient. SDS-PAGE revealed that filamin, talin, and 70 kDa protein were specifically degraded when platelets were aggregated by ionomycin or collagen and that the proteolysis was not observed when the aggregation was blocked by verapamil or NCO-700. These data provided evidence that Ca2+ entry exceeding 0.5 microM is essential, but not sufficient per se, and that activation of cysteine protease, most likely CANP, is involved in the platelet aggregation by collagen or calcium ionophore.

Blood Platelets↗
PubMed2005

Gene profiling changes in cytoskeletal proteins during clinical recovery after left ventricular-assist device support.

BACKGROUND: After left ventricular-assist device (LVAD) support, a proportion of patients recover sufficient ventricular function to enable explantation of the device. The exact molecular mechanisms involved in myocardial recovery remain unknown. Cytoskeletal proteins are essential for the structure and function of the cardiac myocyte and might play a major role. METHODS AND RESULTS: A total of 15 patients with nonischemic cardiomyopathy who required LVAD implantation were studied; 6 recovered sufficiently to allow explantation of the device compared with 9 who did not recover and required transplantation. LV myocardial samples were collected at implantation and explantation/transplantation. Affymetrix microarray analysis was performed on the paired samples and analyzed with reference to sarcomeric and nonsarcomeric cytoskeletal proteins. In the recovery group, of the nonsarcomeric proteins, lamin A/C increased 1.5-fold (P<0.05) and spectrin 1.6-fold (P<0.05) between the times of implantation and explantation. Integrins beta1, beta6, and alpha7 decreased 1.7-fold (P<0.05), 2.4-fold (P<0.05), and 1.5-fold (P<0.05), respectively, but integrins alpha5 and beta5 increased 2.3-fold (P<0.01) and 1.2-fold (P<0.01) at explantation. The following sarcomeric proteins changed in the recovered group only: beta-actin increased 1.4-fold (P<0.05); alpha-tropomyosin, 1.3-fold (P<0.05); alpha1-actinin, 1.8-fold (P<0.01); and alpha-filamin A, 1.6-fold (P<0.05). Both troponin T3 and alpha2-actinin decreased by 1.6-fold at the time of explantation (P<0.05). Vinculin decreased 1.7-fold (P=0.001) in the recovered group but increased by 1.7-fold (P<0.05) in the nonrecovered group. Vinculin protein levels decreased 4.1-fold in the recovered group. CONCLUSIONS: Myocardial recovery was associated with a specific pattern of changes in sarcomeric, nonsarcomeric, and membrane-associated proteins, which could have important implications in understanding the mechanisms involved.

Adrenergic beta-2 Receptor Agonists↗
PubMed1992

Early maturation of force production in pig tracheal smooth muscle during fetal development.

The contractility of airway smooth muscle is fully established at late term at birth but its responsiveness during fetal life has not been defined. In this study, the contractile force of airway smooth muscle to acetylcholine (ACh), K+ depolarizing solution, and electrical field stimulation (EFS) was measured in tracheas from small fetal pigs. Contraction to either agonist and to EFS was detectable in fetuses of as low as 9 g body weight, which corresponds to approximately 36 days of gestation. Isometric force increased progressively with age, reaching 4.1 +/- 0.4 mN for K+ and 5.8 +/- 0.5 mN for ACh (10(-4) M) at 600 g fetal weight (90 days). However, when normalized for cross sectional area of smooth muscle, the stress was essentially the same from 17- to 600-g fetuses. (K+: 17 g = 74.4 +/- 10.6 mN/mm2, 600 g = 89.3 +/- 13.0 mN/mm2; ACh [10(-4) M]: 17 g = 76.3 +/- 16.0 mN/mm2, 600 g = 127.0 +/- 13.0 mN/mm2). The sensitivities of the various groups to ACh were not significantly different (e.g., EC50: 30 g = 4.0 +/- 0.2 x 10(-6) M, 600 g = 3.7 +/- 1.1 x 10(-6) M). EFS produced frequency-dependent contractile responses in all groups. With increasing fetal size, there was a corresponding increase in force. When this force was normalized to a maximum ACh response (10(-4) M), there was no significant difference between groups of fetuses. Histologic examination showed the major tissue components of the trachea were present in fetuses above 7 g. Immunocytochemistry detected myosin, caldesmon, and filamin in the smooth muscle from fetuses of 7 g and above, showing that contractile and actin-binding proteins were present from a very early age. It is concluded that smooth muscle contractile function is well developed very early in fetal life in pigs.

Acetylcholine↗
PubMed2000

Molecular genetic classification of central nervous system malformations.

Traditional schemes of classifying nervous system malformations are based on descriptive morphogenesis of anatomic processes of ontogenesis, such as neurulation, neuroblast migration, and axonal pathfinding. This proposal is a first attempt to incorporate the recent molecular genetic data that explain programming of development etiologically. A scheme based purely on genetic mutations would not be practical, in part because only in a few dysgeneses are the specific defects known, but also because several genes might be involved sequentially and many genes inhibit or augment the expression of others. The same genes serve different functions at different stages and are involved in multiple organ systems. Some complex malformations, such as holoprosencephaly, result from several unrelated defective genes. Finally, a pure genetic classification would be too inflexible to incorporate some anatomic criteria. The basis for the proposed scheme is, therefore, disturbances in patterns of genetic expression; polarity gradients of the axes of the neural tube (eg, upregulation or downregulation of genetic influences); segmentation (eg, deletions of specific neuromeres, ectopic expression); mutations that cause change in cell lineage (eg, dysplastic gangliocytoma of cerebellum, myofiber differentiation within brain); and specific genes or molecules that mediate neuroblast migration in its early (eg, filamin-1), middle (eg, LIS1, double-cortin), or late course (eg, reelin, L1-CAM). The proposed scheme undoubtedly will undergo many future revisions, but it provides a starting point using currently available data.

Central Nervous System↗
PubMed2002

Amelioration of the macrothrombocytopenia associated with the murine Bernard-Soulier syndrome.

An absent platelet glycoprotein (GP) Ib-IX receptor results in the Bernard-Soulier syndrome and is characterized by severe bleeding and the laboratory presentation of macrothrombocytopenia. Although the macrothrombocytopenic phenotype is directly linked to an absent GP Ib-IX complex, the disrupted molecular mechanisms that produce the macrothrombocytopenia are unknown. We have utilized a mouse model of the Bernard-Soulier syndrome to engineer platelets expressing an alpha-subunit of GP Ib (GP Ibalpha) in which most of the extracytoplasmic sequence has been replaced by an isolated domain of the alpha-subunit of the human interleukin-4 receptor (IL-4Ralpha). The IL-4Ralpha/GP Ibalpha fusion is membrane expressed in Chinese hamster ovary (CHO) cells, and its expression is facilitated by the presence of human GP IX and the beta-subunit of GP Ib. Transgenic animals expressing a chimeric receptor were generated and bred into the murine Bernard-Soulier syndrome-producing animals devoid of mouse GP Ibalpha but expressing the IL-4Ralpha/GP Ibalpha fusion sequence. The characterization of these mice revealed a 2-fold increase in circulating platelet count and a 50% reduction in platelet size when compared with platelets from the mouse model of the Bernard-Soulier syndrome. Immunoprecipitation confirmed that the IL-4Ralpha/GP Ibalpha subunit interacts with filamin-1 and 14-3-3zeta, known binding proteins to the GP Ibalpha cytoplasmic tail. Mice expressing the chimeric receptor retain a severe bleeding phenotype, confirming a critical role for the GP Ibalpha extracytoplasmic domain in hemostasis. These results provide in vivo insights into the structural elements of the GP Ibalpha subunit that contribute to normal megakaryocyte maturation and thrombopoiesis.

Animals↗
PubMed2006

Cognitive dysfunction in NF1 knock-out mice may result from altered vesicular trafficking of APP/DRD3 complex.

BACKGROUND: It has been estimated that more than 50% of patients with Neurofibromatosis type 1 (NF1) have neurobehavioral impairments which include attention deficit/hyperactivity disorder, visual/spatial learning disabilities, and a myriad of other cognitive developmental problems. The biological mechanisms by which NF1 gene mutations lead to such cognitive deficits are not well understood, although excessive Ras signaling and increased GABA mediated inhibition have been implicated. It is proposed that the cognitive deficits in NF1 are the result of dysfunctional cellular trafficking and localization of molecules downstream of the primary gene defect. RESULTS: To elucidate genes involved in the pathogenic process, gene expression analysis was performed comparing the expression profiles in various brain regions for control and Nf1+/- heterozygous mice. Gene expression analysis was performed for hippocampal samples dissected from postnatal day 10, 15, and 20 mice utilizing the Affymetrix Mouse Genome chip (Murine 430 2.0). Analysis of expression profiles between Nf1+/- and wild-type animals was focused on the hippocampus because of previous studies demonstrating alterations in hippocampal LTP in the Nf1+/- mice, and the region's importance in visual/spatial learning. Network analysis identified links between neurofibromin and kinesin genes, which were down regulated in the Nf1+/- mice at postnatal days 15 and 20. CONCLUSION: Through this analysis, it is proposed that neurofibromin forms a binding complex with amyloid precursor protein (APP) and through filamin proteins interacts with a dopamine receptor (Drd3). Though the effects of these interactions are not yet known, this information may provide novel ideas about the pathogenesis of cognitive defects in NF1 and may facilitate the development of novel targeted therapeutic interventions.

Amyloid beta-Protein Precursor↗
PubMed1982

Partial biochemical maturation of aneurally cultured human skeletal muscle.

We studied cultures of human skeletal muscle in vitro and established standards for biochemical markers of cellular differentiation. DNA synthesis ceased at the time of fusion, implying the absence of fibroblasts. Myosin heavy-chain synthesis, creatine and pyruvate kinase activities, and isoenzymes of creatine kinase were measured serially over 36 days. Filamin and fibronectin proteins were identified in these cultures. Compared to chick muscle in culture, human skeletal muscle cells remained relatively immature. These data provide a basis for the study of diseased human muscle cells in culture.

Animals↗
PubMed1989

Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones.

Actin filaments and their interactions with cell surface molecules have key roles in tissue cell behaviour. Axonal pathfinding during embryogenesis, an especially complex cell behaviour, is based on the migration of nerve growth cones. We have used fluorescence immunocytochemistry to examine the distribution in growth cones, their filopodia and lamellipodia of several actin-associated proteins and nerve cell adhesion molecules. The leading margins of chick dorsal root ganglion nerve growth cones and their protrusions stain strongly for f-actin, filamin, alpha-actinin, myosin, tropomyosin, talin and vinculin. MAP2 is absent from DRG growth cones, and staining for spectrin fodrin extends into growth cones, but not along filopodia. Thus, organization of the leading margins of growth cones may strongly resemble the leading lamella of migrating fibroblasts. The adhesion-mediating molecules integrin, L1, N-CAM and A-CAM are all found on DRG neurites and growth cones. However, filopodia stain relatively more strongly for integrin and L1 than for A-CAM or N-CAM. In fact, the 180 X 10(3) Mr form of N-CAM may be absent from most of the length of filopodia. DRG neurones cultured in cytochalasin B display differences in immunofluorescence staining which further emphasize that these adhesion molecules interact differentially with the actin filament system of migrating growth cones. Several models for neuronal morphogenesis emphasize the importance of regulation of the expression of adhesion molecules. Our results support hypotheses that cellular distribution and transmembrane interactions are key elements in the functions of these adhesion molecules during axonal pathfinding.

Animals↗
PubMed1997

An activity-dependent network of interactions links the Rel protein Dorsal with its cytoplasmic regulators.

A signaling pathway active on the ventral side of the Drosophila embryo defines dorsoventral polarity. A cell surface signal relayed by Toll, Tube and Pelle releases the Rel-related protein Dorsal from its cytoplasmic inhibitor Cactus; free Dorsal translocates into nuclei and directs expression of ventral fates. Using the yeast two-hybrid system and immunoprecipitation experiments, we define scaffolding and anchoring interactions among the pathway components. We show that Dorsal binds specifically to Tube, Pelle and Cactus, and that the protein kinase activity of Pelle differentially regulates its interactions with Dorsal and Tube. We also identify Drosophila Filamin as a potential adaptor linking the interaction network, via Tube, to the transmembrane receptor Toll.

Animals↗
PubMed1993

Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions.

Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the alpha 5 beta 1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of beta 1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the beta 1 integrin subunit, actin, filamin and alpha-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the beta 1 integrin subunit or by a combination of adhesion blocking antibodies recognising the alpha subunits that associate with beta 1 (alpha 2, alpha 3 and alpha 5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte beta 1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.

Actins↗
PubMed1994

Calponin is localised in both the contractile apparatus and the cytoskeleton of smooth muscle cells.

Calponin and caldesmon are two thin filament-binding proteins found in smooth muscle that have both been attributed a role in modulating the interaction of actin and myosin. Using high-resolution dual-label immunocytochemistry we have determined the distribution of calponin relative to the contractile and cytoskeletal compartments of the smooth muscle cell. We show, using chicken gizzard smooth muscle, that calponin occurs in the cytoskeleton, with beta-cytoplasmic actin, filamin and desmin, as well as in the contractile apparatus, with myosin and caldesmon. According to the observed labelling intensities, calponin was more concentrated in the cytoskeleton and it was additionally localised in the cytoplasmic dense bodies as well as in the adhesion plaques at the cell surface, which both harbour the beta-cytoplasmic isoform of actin. It is probable that these results explain earlier conflicting reports on the composition of smooth muscle thin filaments and suggest that calponin, together with a Ca(2+)-receptor protein, could just as likely serve a role in the cytoskeleton of smooth muscle as in the contractile apparatus.

Animals↗