Skeletal muscle LIM protein 1 regulates integrin-mediated myoblast adhesion, spreading, and migration

2003 ◽  
Vol 284 (3) ◽  
pp. C681-C695 ◽  
Author(s):  
Paul A. Robinson ◽  
Susan Brown ◽  
Meagan J. McGrath ◽  
Imogen D. Coghill ◽  
Rajendra Gurung ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Dependent Manner ◽  
Lim Protein ◽  
Muscle Lim Protein ◽  
And Migration ◽  
Green Fluorescent

The skeletal muscle LIM protein 1 (SLIM1) is highly expressed in skeletal and cardiac muscle, and its expression is downregulated significantly in dilated human cardiomyopathy. However, the function of SLIM1 is unknown. In this study, we investigated the intracellular localization of SLIM1. Endogenous and recombinant SLIM1 localized to the nucleus, stress fibers, and focal adhesions in skeletal myoblasts plated on fibronectin, collagen, or laminin. However, after inhibition of integrin signaling either by plating on poly-l-lysine or by soluble RGD peptide, SLIM1 localized diffusely in the cytosol, with decreased nuclear expression. Disruption of the actin cytoskeleton by cytochalasin D did not inhibit nuclear localization of SLIM1 in integrin-activated cells. Green fluorescent protein-tagged SLIM1 shuttled in the nucleus of untransfected NIH 3T3 cells, in a heterokaryon fusion assay. Overexpression of SLIM1 in Sol8 myoblasts inhibited cell adhesion and promoted cell spreading and migration. These studies show SLIM1 localizes in an integrin-dependent manner to the nucleus and focal adhesions where it functions downstream of integrin activation to promote cell spreading and migration.

scholarly journals Arfophilins Are Dual Arf/Rab 11 Binding Proteins That Regulate Recycling Endosome Distribution and Are Related to Drosophila Nuclear Fallout

2003 ◽  
Vol 14 (7) ◽  
pp. 2908-2920 ◽  
Author(s):  
Gilles R.X. Hickson ◽  
Johanne Matheson ◽  
Blake Riggs ◽  
Valerie H. Maier ◽  
Andrew B. Fielding ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Binding Capacity ◽  
Binding Protein ◽  
Focal Adhesions ◽  
Dependent Manner ◽  
Recycling Endosome ◽  
Nuclear Fallout ◽  
Endosomal Recycling ◽  
Green Fluorescent

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.

PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs

2001 ◽  
Vol 281 (2) ◽  
pp. C709-C718 ◽  
Author(s):  
Ilia A. Yamboliev ◽  
Jennifer Chen ◽  
William T. Gerthoffer
Keyword(s):  
Tyrosine Kinases ◽  
Src Kinase ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Src Tyrosine Kinases ◽  
Intracellular Signal ◽  
Pulmonary Artery Smooth Muscle ◽  
And Migration

Pulmonary artery smooth muscle cell (PASMC) adhesion, spreading, and migration depend on matrix-stimulated reorganization of focal adhesions. Platelet-derived growth factor (PDGF) activates intracellular signal transduction cascades that also regulate adhesion, spreading, and migration, but the signaling molecules involved in these events are poorly defined. We hypothesized that phosphatidylinositol (PI) 3-kinases and Src tyrosine kinases translate matrix and PDGF-initiated signals into cell motility. In experiments with cultured canine PASMCs, inhibition of PI 3-kinases with wortmannin (0.3 μM) and LY-294002 (50 μM) and of Src kinase with PP1 (30 μM) did not decrease spontaneous (nonstimulated) or PDGF-stimulated (10 ng/ml) adhesion onto collagen. PI 3-kinase and Src kinase activities, however, were necessary for cell spreading: PP1 inhibited cell spreading and Src Tyr-418 phosphorylation in a concentration-dependent manner. Inhibition of PI 3-kinase and Src partially reduced cell migration, while at 10 and 30 μM, PP1 eliminated migration, likely due to inhibition of PDGF receptors. In conclusion, both PI 3-kinases and Src tyrosine kinases are components of pathways that mediate spreading and migration of cultured PASMCs on collagen.

scholarly journals Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us about the Physical Nature of Propagons

2020 ◽  
Vol 22 (1) ◽  
pp. 90
Author(s):  
Mehdi Kabani
Keyword(s):  
Protein Quality ◽  
Fluorescent Protein ◽  
Physical Nature ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Prions ◽  
Daughter Cells ◽  
Cytoplasmic Proteins ◽  
Green Fluorescent ◽  
Functionally Diverse

The yeast Saccharomyces cerevisiae hosts an ensemble of protein-based heritable traits, most of which result from the conversion of structurally and functionally diverse cytoplasmic proteins into prion forms. Among these, [PSI+], [URE3] and [PIN+] are the most well-documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Yeast prions propagate by molecular chaperone-mediated fragmentation of these aggregates, which generates small self-templating seeds, or propagons. The exact molecular nature of propagons and how they are faithfully transmitted from mother to daughter cells despite spatial protein quality control are not fully understood. In [PSI+] cells, Sup35p forms detergent-resistant assemblies detectable on agarose gels under semi-denaturant conditions and cytosolic fluorescent puncta when the protein is fused to green fluorescent protein (GFP); yet, these macroscopic manifestations of [PSI+] do not fully correlate with the infectivity measured during growth by the mean of protein infection assays. We also discovered that significant amounts of infectious Sup35p particles are exported via extracellular (EV) and periplasmic (PV) vesicles in a growth phase and glucose-dependent manner. In the present review, I discuss how these vesicles may be a source of actual propagons and a suitable vehicle for their transmission to the bud.

scholarly journals Microinjection of antibodies against talin inhibits the spreading and migration of fibroblasts

1992 ◽  
Vol 102 (4) ◽  
pp. 753-762
Author(s):  
G.H. Nuckolls ◽  
L.H. Romer ◽  
K. Burridge
Keyword(s):  
Extracellular Matrix ◽  
Tissue Culture ◽  
Actin Filaments ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
And Migration ◽  
Extracellular Matrix Receptors

Talin is believed to be one of the key proteins involved in linking actin filaments to extracellular matrix receptors in focal adhesions. Our strategy for studying the function of talin has been to inactivate talin in living fibroblasts in tissue culture through the microinjection of affinity-purified, polyclonal anti-talin antibodies. The effect of the injected anti-talin antibodies on cell spreading was found to depend on how recently the cells had been plated. Cells that were in the process of spreading on a fibronectin substratum, and which had newly developed focal adhesions, were induced to round up and to disassemble many of the adhesions. However, if fibroblasts were allowed to spread completely before they were microinjected with the anti-talin antibody, focal adhesions remained intact and the flat morphology of the cells was unaffected. The percentage of cells that were able to maintain a spread morphology despite the injection of anti-talin antibodies increased during the first few hours after plating on fibronectin substrata. Fibroblasts that were allowed to spread completely before microinjection with the anti-talin antibody retained both intact focal adhesions and a flat, well-spread morphology, but failed to migrate effectively. Our experiments do not directly address the role of talin in mature focal adhesions, but they indicate that talin is essential for the spreading and migration of fibroblasts on fibronectin as well as for the development and initial maintenance of focal adhesions on this substratum.

scholarly journals RACK1 Regulates Integrin-mediated Adhesion, Protrusion, and Chemotactic Cell Migration via Its Src-binding Site

2003 ◽  
Vol 14 (2) ◽  
pp. 658-669 ◽  
Author(s):  
Elisabeth A. Cox ◽  
David Bennin ◽  
Ashley T. Doan ◽  
Timothy O'Toole ◽  
Anna Huttenlocher
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Site ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Expression Cloning ◽  
Chemotactic Migration ◽  
Cell Protrusion ◽  
Kinase C ◽  
Green Fluorescent

Mammalian cDNA expression cloning was used to identify novel regulators of integrin-mediated cell-substratum adhesions. Using a focal adhesion morphology screen, we identified a cDNA with homology to a receptor for activated protein kinase C (RACK1) that induced a loss of central focal adhesions and stress fibers in CHO-K1 cells. The identified cDNA was a C-terminal truncated form of RACK1 that had one of the putative protein kinase C binding sites but lacked the region proposed to bind the β integrin cytoplasmic domain and the tyrosine kinase Src. To investigate the role of RACK1 during cell spreading and migration, we tagged RACK1, a C-terminal truncated RACK1 and a point mutant that does not bind Src (RACK Y246F) with green fluorescent protein and expressed them in CHO-K1 cells. We found that RACK1 regulates the organization of focal adhesions and that it localizes to a subset of nascent focal complexes in areas of protrusion that contain paxillin but not vinculin. We also found that RACK1 regulates cell protrusion and chemotactic migration through its Src binding site. Together, these findings suggest that RACK1 regulates adhesion, protrusion, and chemotactic migration through its interaction with Src.

Intracellular localization and in vivo trafficking of p24A and p23

1999 ◽  
Vol 112 (4) ◽  
pp. 537-548 ◽  
Author(s):  
R. Blum ◽  
F. Pfeiffer ◽  
P. Feick ◽  
W. Nastainczyk ◽  
B. Kohler ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Maximum Speed ◽  
Heterotrimeric G Proteins ◽  
P24 Protein ◽  
Intermediate Compartment ◽  
Copi Vesicle ◽  
Green Fluorescent ◽  
Golgi Network

Recently, p24A and p23 (also termed Tmp21), two members of the p24 protein family, have been proposed to function as integral receptors for the COPI-vesicle coat. This study describes the intracellular localization and trafficking of p24A in comparison to p23. For immunolocalization of p24A and p23, strong reduction and denaturation conditions were necessary to allow antibody interaction. Both p24A and p23 cycle continuously between intermediate compartment (IC) elements and the cis-Golgi network. In vivo trafficking of p24A and p23 tagged to green fluorescent protein (GFP) revealed that both proteins travel by large (up to 1 micrometer in length) microtubule-dependent pre-Golgi carriers with a maximum speed of up to 1.6 micrometer s-1 from the IC to the Golgi cisternae. Aluminum fluoride, a general activator of heterotrimeric G-proteins, blocked peripheral pre-Golgi movements of GFP-p24A/p23 and inhibited fluorescence recovery after photobleaching in the perinuclear Golgi area. p24A and p23 are predominantly colocalized. Overexpression of GFP-p24A, to an extent which did not destroy the Golgi complex, induced delocalization of part of the proteins into ER elements. This study therefore gives new insights into the localization and trafficking behavior of the two COPI-binding proteins p24A and p23.

scholarly journals Characterization of the Roles of Vimentin in Regulating the Proliferation and Migration of HSCs during Hepatic Fibrogenesis

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1184 ◽  
Author(s):  
Pei-Wen Wang ◽  
Tung-Ho Wu ◽  
Tung-Yi Lin ◽  
Mu-Hong Chen ◽  
Chau-Ting Yeh ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Messenger Rna ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Nuclear Antigen ◽  
Dependent Manner ◽  
Hepatic Fibrogenesis ◽  
And Migration ◽  
Proliferation And Migration

The activation of hepatic stellate cells (HSCs) manifested as proliferation and migration is the pivotal event involved in liver fibrogenesis. The vimentin network, an intermediate filament (IF) system, is one of the critical cascades by which the cell morphology, growth, and motility are modulated. However, the vimentin-mediated cytoskeletal cross talk, as well as the signaling transduction, which further coordinates the cellular responses during hepatic fibrogenesis, is poorly understood. In the current study, both messenger RNA (mRNA) and the vimentin protein were significantly increased in a time-dependent manner in the dimethylnitrosamine (DMN)-exposed liver. In particular, vimentin was highly expressed in the activated HSCs. Again, the overexpressed vimentin was observed in the plasma samples derived from patients with hepatic fibrosis/cirrhosis, suggesting that vimentin may be a key factor in regulating the progression of liver fibrosis. Meanwhile, vimentin knockdown suppressed the migratory propensity, provoked morphological changes, and disturbed the focal adhesions in the HSCs due to the breakdown of associated cytoskeletal proteins. Western blotting showed that vimentin deletion inhibited proliferating cell nuclear antigen (PCNA) and arrested the Rho GTPase family, thereby impairing the HSCs’ growth as well as motility. The phosphorylated extracellular-signal regulated kinase (ERK) and AKT signals were also notably reduced in response to the silence of vimentin. Inhibitors of selected signaling pathways suppressed the migration and differentiation of activated HSCs by regulating specific serine phosphorylated sites on vimentin. Taken together, these findings revealed a novel mechanism of vimentin through which various signaling pathways controlled the proliferation, differentiation, and movement of the HSCs via the ERK/AKT and Rho cascades.

scholarly journals The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

2005 ◽  
Vol 387 (3) ◽  
pp. 573-584 ◽  
Author(s):  
Sandra MILASTA ◽  
Nicholas A. EVANS ◽  
Laura ORMISTON ◽  
Shelagh WILSON ◽  
Robert J. LEFKOWITZ ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Weak Interactions ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Mapk ◽  
Hydroxy Amino ◽  
C Terminus ◽  
Green Fluorescent ◽  
Kinetics Of

The orexin-1 receptor interacts with β-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with β-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of β-arrestin-2–GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with β-arrestin-2, studies in wild-type and β-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a β-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with β-arrestin-2, and indicate an important role of β-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.

scholarly journals Activation of Neurokinin 3 Receptors Stimulates GnRH Release in a Location-Dependent but Kisspeptin-Independent Manner in Adult Mice

Endocrinology ◽  
2013 ◽  
Vol 154 (11) ◽  
pp. 3984-3989 ◽  
Author(s):  
Garrett T. Gaskins ◽  
Katarzyna M. Glanowska ◽  
Suzanne M. Moenter
Keyword(s):  
Green Fluorescent Protein ◽  
Carbon Fiber ◽  
Fluorescent Protein ◽  
Hypogonadotropic Hypogonadism ◽  
Brain Slices ◽  
Dependent Manner ◽  
Gnrh Secretion ◽  
Gnrh Release ◽  
Carbon Fiber Microelectrodes ◽  
Green Fluorescent

GnRH neurons form the final common pathway for the central control of reproduction. GnRH release occurs from terminals in the external layer of the median eminence (ME) for neuroendocrine control of the pituitary, and near GnRH-GnRH fiber appositions within the preoptic area (POA). Whether or not control of GnRH secretion by neuromodulators is different in these 2 areas is unknown. Mutations in neurokinin B (NKB) or the neurokinin-3 receptor (NK3R) are linked to hypogonadotropic hypogonadism in humans, suggesting that NKB may regulate GnRH secretion. Using fast scan cyclic voltammetry through carbon-fiber microelectrodes, we examined real-time GnRH release in response to the NK3R agonist senktide in the ME and POA. Coronal brain slices were acutely prepared from adult gonad-intact GnRH-green fluorescent protein male mice, and carbon-fiber microelectrodes were placed either within green fluorescent protein-positive terminal fields of the ME or near GnRH-GnRH fiber appositions in the POA. Senktide induced GnRH release consistently in the ME but not the POA, indicating that GnRH release is differentially regulated by NKB in a location-dependent manner. Senktide also induced GnRH secretion in the ME of kisspeptin-knockout (Kiss1 knockout) mice. Interestingly, release amplitude was lower compared with wild-type mice. These data indicate regulation of GnRH release by NK3R agonists is site specific and suggest that kisspeptin is not a required mediator between NK3R activation and GnRH secretion in the ME. This information will be useful for informing future models of afferent regulation of GnRH release.

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