The actin cytoskeleton organization and disorganization properties of the photosynthetic dinoflagellate Symbiodinium kawagutii in culture

2014 ◽  
Vol 60 (11) ◽  
pp. 767-775 ◽  
Author(s):  
Marco A. Villanueva ◽  
Georgina Arzápalo-Castañeda ◽  
Raúl Eduardo Castillo-Medina
Keyword(s):  
Actin Cytoskeleton ◽  
Freeze Fracture ◽  
Fluorescein Isothiocyanate ◽  
Cytoskeleton Organization ◽  
Reticular Pattern ◽  
Thick Filaments ◽  
Actin Cytoskeleton Organization ◽  
Butanedione Monoxime ◽  
Marine Dinoflagellates

The actin cytoskeleton organization in symbiotic marine dinoflagellates is largely undescribed; most likely, due to their intense pigment autofluorescence and cell walls that block fluorescent probe access. Using a freeze–fracture and fixation procedure, we observed the actin cytoskeleton of Symbiodinium kawagutii cultured in vitro with fluorescently labeled phalloidin and by indirect immunofluorescence with monoclonal antibodies specific for actin. The cytoskeleton appeared as an organized network with interconnected cortical and cytoplasmic thick filaments, along with some intertwined fine filaments. It showed a grid-type, reticular pattern organized in a lattice-like structure within the cell and throughout the cytoplasm. This organization was similar when the observations were done with either fluorescein isothiocyanate (FITC)–phalloidin or anti-actin, although the latter showed a more evenly distributed fluorescence characteristic of nonpolymerized actin. The network organization collapsed upon treatment with latrunculin, resulting in bright foci and diffuse fluorescence. A similar effect was obtained upon butanedione monoxime treatment, except that no bright foci were observed. We have been able to successfully visualize the actin cytoskeleton of S. kawagutii cells using fluorescence-based procedures. This is the first report on the visualization of the organization of the actin cytoskeleton under various conditions in these walled, highly autofluorescent cells.

scholarly journals Regulation of Yeast Actin Cytoskeleton-Regulatory Complex Pan1p/Sla1p/End3p by Serine/Threonine Kinase Prk1p

2001 ◽  
Vol 12 (12) ◽  
pp. 3759-3772 ◽  
Author(s):  
Guisheng Zeng ◽  
Xianwen Yu ◽  
Mingjie Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Regulatory Protein ◽  
Strong Support ◽  
Stable Complex ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization

The serine/threonine kinase Prk1p is known to be involved in the regulation of the actin cytoskeleton organization in budding yeast. One possible function of Prk1p is the negative regulation of Pan1p, an actin patch regulatory protein that forms a complex in vivo with at least two other proteins, Sla1p and End3p. In this report, we identified Sla1p as another substrate for Prk1p. The phosphorylation of Sla1p by Prk1p was established in vitro with the use of immunoprecipitated Prk1p and in vivo with the use ofPRK1 overexpression, and was further supported by the finding that immunoprecipitated Sla1p contained PRK1- and ARK1-dependent kinase activities. Stable complex formation between Prk1p and Sla1p/Pan1p in vivo could be observed once the phosphorylation reaction was blocked by mutation in the catalytic site of Prk1p. Elevation of Prk1p activities in wild-type cells resulted in a number of deficiencies, including those in colocalization of Pan1p and Sla1p, endocytosis, and cell wall morphogenesis, likely attributable to a disintegration of the Pan1p/Sla1p/End3p complex. These results lend a strong support to the model that the phosphorylation of the Pan1p/Sla1p/End3p complex by Prk1p is one of the important mechanisms by which the organization and functions of the actin cytoskeleton are regulated.

scholarly journals Regulation of the Actin Cytoskeleton Organization in Yeast by a Novel Serine/Threonine Kinase Prk1p

1999 ◽  
Vol 144 (1) ◽  
pp. 71-82 ◽  
Author(s):  
Guisheng Zeng ◽  
Mingjie Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Asymmetric Distribution ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization ◽  
Related Proteins ◽  
Regulation Of Actin Cytoskeleton

Normal actin cytoskeleton organization in budding yeast requires the function of the Pan1p/ End3p complex. Mutations in PAN1 and END3 cause defects in the organization of actin cytoskeleton and endocytosis. By screening for mutations that can suppress the temperature sensitivity of a pan1 mutant (pan1-4), a novel serine/threonine kinase Prk1p is now identified as a new factor regulating the actin cytoskeleton organization in yeast. The suppression of pan1-4 by prk1 requires the presence of mutant Pan1p. Although viable, the prk1 mutant is unable to maintain an asymmetric distribution of the actin cytoskeleton at 37°C. Consistent with its role in the regulation of actin cytoskeleton, Prk1p localizes to the regions of cell growth and coincides with the polarized actin patches. Overexpression of the PRK1 gene in wild-type cells leads to lethality and actin cytoskeleton abnormalities similar to those exhibited by the pan1 and end3 mutants. In vitro phosphorylation assays demonstrate that Prk1p is able to phosphorylate regions of Pan1p containing the LxxQxTG repeats, including the region responsible for binding to End3p. Based on these findings, we propose that the Prk1 protein kinase regulates the actin cytoskeleton organization by modulating the activities of some actin cytoskeleton-related proteins such as Pan1p/End3p.

scholarly journals MiRNA-155 targets myosin light chain kinase and modulates actin cytoskeleton organization in endothelial cells

2014 ◽  
Vol 306 (8) ◽  
pp. H1192-H1203 ◽  
Author(s):  
Martina Weber ◽  
Sinae Kim ◽  
Nicole Patterson ◽  
Kimberly Rooney ◽  
Charles D. Searles
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Actin Cytoskeleton ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization ◽  
Mouse Aorta

Previously, we identified a microRNA (miRNA) signature for endothelial cells (ECs) subjected to unidirectional shear stress (USS). MiR-155, a multifunctional miRNA that has been implicated in atherosclerosis, was among the shear stress-responsive miRNAs. Here, we examined the role of miR-155 in modulating EC phenotype and function. In vitro, increased miR-155 levels in human ECs induced changes in morphology and filamentous (F)-actin organization. In addition, ECs transfected with miR-155 mimic were less migratory and less proliferative and had less apoptosis compared with control ECs. In mouse aorta, miR-155 expression was increased in the intima of thoracic aorta, where blood flow produces steady and unidirectional shear stress, compared with the intima of the lower curvature of the aortic arch, which is associated with oscillatory and low shear stress. These differences in miR-155 expression were associated with distinct changes in EC morphology and F-actin. The effects of miR-155 in vitro were mediated through suppression of two key regulators of the EC cytoskeleton organization: RhoA and myosin light chain kinase (MYLK). A novel direct interaction between miR-155 and the MYLK 3′UTR was verified by luciferase-MYLK 3′UTR reporter assays. Furthermore, the intensity of immunofluorescence staining for RhoA and MYLK in mouse aorta correlated inversely with miR-155 expression. In conclusion, a prominent effect of the multifunctional miR-155 in ECs is modulation of phenotype through alterations in RhoA, MYLK expression, and actin cytoskeleton organization.

SH3 domain-containing proteins and the actin cytoskeleton in yeast

2005 ◽  
Vol 33 (6) ◽  
pp. 1247-1249 ◽  
Author(s):  
G. Mirey ◽  
A. Soulard ◽  
C. Orange ◽  
S. Friant ◽  
B. Winsor
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Interactions ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sh3 Domains ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization

SH3 (Src homology-3) domains are involved in protein–protein interactions through proline-rich domains. Many SH3-containing proteins are implicated in actin cytoskeleton organization. The aim of our ongoing work is to study the functions of the SH3-containing proteins in actin cytoskeleton regulation. The yeast Saccharomyces cerevisiae proteome includes 29 SH3 domains distributed in 25 proteins. We have examined the direct involvement of these SH3 domains in actin polymerization using an in vitro polymerization assay on GST (glutathione S-transferase)–SH3-coated beads. As expected, not all SH3 domains show polymerization activity, and many recruit distinct partners as assessed by microscopy and pull-down experiments. One such partner, Las17p, the yeast homologue of WASP (Wiskott–Aldrich syndrome protein), was assayed because it stimulates actin nucleation via the Arp2/3 (actin-related protein 2/3) complex. Ultimately, proteins involved in specific biological processes, such as membrane trafficking, may also be recruited by some of these SH3 domains, shedding light on the SH3-containing proteins and actin cytoskeleton functions in these processes.

scholarly journals Pan1p, End3p, and Sla1p, Three Yeast Proteins Required for Normal Cortical Actin Cytoskeleton Organization, Associate with Each Other and Play Essential Roles in Cell Wall Morphogenesis

2000 ◽  
Vol 20 (1) ◽  
pp. 12-25 ◽  
Author(s):  
Hsin-Yao Tang ◽  
Jing Xu ◽  
Mingjie Cai
Keyword(s):  
Cell Wall ◽  
Actin Cytoskeleton ◽  
Normal Cell ◽  
Cell Cortex ◽  
Repeat Region ◽  
Cortical Actin ◽  
Cytoskeleton Organization ◽  
Eh Domain ◽  
Actin Cytoskeleton Organization ◽  
Cell Wall Morphogenesis

ABSTRACT The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Δ, andend3Δ mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast.

scholarly journals The Fibrinogen Globe of Tenascin-C Promotes Basic Fibroblast Growth Factor-induced Endothelial Cell Elongation

1999 ◽  
Vol 10 (9) ◽  
pp. 2933-2943 ◽  
Author(s):  
Susanne Schenk ◽  
Ruth Chiquet-Ehrismann ◽  
Edouard J. Battegay
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Fibroblast Growth ◽  
Aortic Endothelial Cells ◽  
Tenascin C ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization

To investigate the potential role of tenascin-C (TN-C) on endothelial sprouting we used bovine aortic endothelial cells (BAECs) as an in vitro model of angiogenesis. We found that TN-C is specifically expressed by sprouting and cord-forming BAECs but not by nonsprouting BAECs. To test whether TN-C alone or in combination with basic fibroblast growth factor (bFGF) can enhance endothelial sprouting or cord formation, we used BAECs that normally do not sprout and, fittingly, do not express TN-C. In the presence of bFGF, exogenous TN-C but not fibronectin induced an elongated phenotype in nonsprouting BAECs. This phenotype was due to altered actin cytoskeleton organization. The fibrinogen globe of the TN-C molecule was the active domain promoting the elongated phenotype in response to bFGF. Furthermore, we found that the fibrinogen globe was responsible for reduced cell adhesion of BAECs on TN-C substrates. We conclude that bFGF-stimulated endothelial cells can be switched to a sprouting phenotype by the decreased adhesive strength of TN-C, mediated by the fibrinogen globe.

Sign in / Sign up

Export Citation Format

Share Document