scholarly journals Dia- and Rok-dependent enrichment of capping proteins in a cortical region

2021 ◽  
Author(s):  
Anja Schmidt ◽  
Long Li ◽  
Zhiyi Lv ◽  
Shuling Yan ◽  
Jörg Großhans
Keyword(s):  
Myosin Ii ◽  
Rho Kinase ◽  
Cortical Region ◽  
Protein Enrichment ◽  
Cortical Actin ◽  
Capping Protein ◽  
Capping Proteins ◽  
Muscle Myosin ◽  
Drosophila Embryos

Rho signaling with its major targets the formin Dia, Rho kinase (Rok) and non-muscle myosin II control turnover, amount and contractility of actomyosin. Much less investigated has been a potential function for the distribution of F-actin plus and minus ends. In syncytial Drosophila embryos Rho1 signaling is high between actin caps, i. e. the cortical intercap region. Capping protein binds to free plus ends of F-actin to prevent elongation of the filament. Capping protein has served as a marker to visualize the distribution of F-actin plus ends in cells and in vitro. Here, we probed the distribution of plus ends with capping protein in syncytial Drosophila embryos. We found that Capping proteins are specifically enriched in the intercap region similar to Dia and MyoII but distinct from overall F-actin. The intercap enrichment of Capping protein was impaired in dia mutants and embryos, in which Rok and MyoII activation was inhibited. Our observations reveal that Dia and Rok/MyoII control Capping protein enrichment and support a model that Dia and Rok/MyoII control the organization of cortical actin cytoskeleton downstream of Rho1 signaling.

scholarly journals Drosophila non-muscle myosin II motor activity determines the rate of tissue folding

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Claudia G Vasquez ◽  
Sarah M Heissler ◽  
Neil Billington ◽  
James R Sellers ◽  
Adam C Martin
Keyword(s):  
Motor Activity ◽  
Molecular Motor ◽  
Myosin Ii ◽  
Cell Contractility ◽  
Myosin Motor ◽  
Muscle Myosin ◽  
Shape Changes ◽  
Drosophila Embryos

Non-muscle cell contractility is critical for tissues to adopt shape changes. Although, the non-muscle myosin II holoenzyme (myosin) is a molecular motor that powers contraction of actin cytoskeleton networks, recent studies have questioned the importance of myosin motor activity cell and tissue shape changes. Here, combining the biochemical analysis of enzymatic and motile properties for purified myosin mutants with in vivo measurements of apical constriction for the same mutants, we show that in vivo constriction rate scales with myosin motor activity. We show that so-called phosphomimetic mutants of the Drosophila regulatory light chain (RLC) do not mimic the phosphorylated RLC state in vitro. The defect in the myosin motor activity in these mutants is evident in developing Drosophila embryos where tissue recoil following laser ablation is decreased compared to wild-type tissue. Overall, our data highlights that myosin activity is required for rapid cell contraction and tissue folding in developing Drosophila embryos.

scholarly journals Pharmaco-genetic screen to uncover actin regulators targeted by prostaglandins during Drosophila oogenesis

2019 ◽  
Author(s):  
Andrew J. Spracklen ◽  
Maureen C. Lamb ◽  
Christopher M. Groen ◽  
Tina L. Tootle
Keyword(s):  
Genetic Interaction ◽  
Actin Dynamics ◽  
Cortical Actin ◽  
Cox Inhibitors ◽  
Capping Protein ◽  
Cytoskeletal Remodeling ◽  
Inflammatory Drugs ◽  
Muscle Myosin ◽  
Follicle Maturation

AbstractProstaglandins (PGs) are lipid signaling molecules with numerous physiologic functions, including pain/inflammation, fertility, and cancer. PGs are produced downstream of cyclooxygenase (COX) enzymes, the targets of non-steroidal anti-inflammatory drugs (NSAIDs). In numerous systems, PGs regulate actin cytoskeletal remodeling, however, their mechanisms of action remain largely unknown. To address this deficiency, we undertook a pharmaco-genetic interaction screen during late-stage Drosophila oogenesis. Drosophila oogenesis is as an established model for studying both actin dynamics and PGs. Indeed, during Stage 10B, cage-like arrays of actin bundles surround each nurse cell nucleus, and during Stage 11, the cortical actin contracts, squeezing the cytoplasmic contents into the oocyte. Both of these cytoskeletal properties are required for follicle development and fertility, and are regulated by PGs. Here we describe a pharmaco-genetic interaction screen that takes advantage of the facts that Stage 10B follicles will mature in culture and COX inhibitors, such as aspirin, block this in vitro follicle maturation. In the screen, aspirin was used at a concentration that blocks 50% of the wild-type follicles from maturing in culture. By combining this aspirin treatment with heterozygosity for mutations in actin regulators, we quantitatively identified enhancers and suppressors of COX inhibition. Here we present the screen results and initial follow-up studies on three strong enhancers – Enabled, Capping protein, and non-muscle Myosin II Regulatory Light Chain. Overall, these studies provide new insight into how PGs regulate both actin bundle formation and cellular contraction, properties that are not only essential for development, but are misregulated in diseases.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

scholarly journals Release of myosin II from the membrane-cytoskeleton of Dictyostelium discoideum mediated by heavy-chain phosphorylation at the foci within the cortical actin network

1992 ◽  
Vol 117 (6) ◽  
pp. 1231-1239 ◽  
Author(s):  
S Yumura ◽  
T Kitanishi-Yumura
Keyword(s):  
Heavy Chain ◽  
Myosin Ii ◽  
Cortical Region ◽  
Actin Network ◽  
Cortical Actin ◽  
Filamentous Form ◽  
Heavy Chains ◽  
Membrane Cytoskeleton ◽  
Myosin Filaments ◽  
Gamma S

Membrane-cytoskeletons were prepared from Dictyostelium amebas, and networks of actin and myosin II filaments were visualized on the exposed cytoplasmic surfaces of the cell membranes by fluorescence staining (Yumura, S., and T. Kitanishi-Yumura. 1990. Cell Struct. Funct. 15:355-364). Addition of ATP caused contraction of the cytoskeleton with aggregation of part of actin into several foci within the network, but most of myosin II was released via the foci. However, in the presence of 10 mM MgCl2, which stabilized myosin II filaments, myosin II remained at the foci. Ultrastructural examination revealed that, after contraction, only traces of monomeric myosin II remained at the foci. By contrast, myosin II filaments remained in the foci in the presence of 10 mM MgCl2. These observations suggest that myosin II was released not in a filamentous form but in a monomeric form. Using [gamma 32P]ATP, we found that the heavy chains of myosin II released from membrane-cytoskeletons were phosphorylated, and this phosphorylation resulted in disassembly of myosin filaments. Using ITP (a substrate for myosin II ATPase) and/or ATP gamma S (a substrate for myosin II heavy-chain kinase [MHCK]), we demonstrated that phosphorylation of myosin heavy chains occurred at the foci within the actin network, a result that suggests that MHCK was localized at the foci. These results together indicate that, during contraction, the heavy chains of myosin II that have moved toward the foci within the actin network are phosphorylated by a specific MHCK, with the resultant disassembly of filaments which are finally released from membrane-cytoskeletons. This series of reactions could represent the mechanism for the relocation of myosin II from the cortical region to the endoplasm.

scholarly journals Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein

1992 ◽  
Vol 117 (5) ◽  
pp. 1067-1076 ◽  
Author(s):  
JF Amatruda ◽  
JA Cooper
Keyword(s):  
Sequence Similarity ◽  
Critical Concentration ◽  
Beta Subunits ◽  
Cortical Actin ◽  
Gene Encoding ◽  
Capping Protein ◽  
Bud Emergence ◽  
End Capping ◽  
Actin Cables

Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro. We purified capping protein from Saccharomyces cervisiae. One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J. F., J. F. Cannon, K. Tatchell, C. Hug, and J. A. Cooper. 1990. Nature (Lond.) 344:352-354). Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed-end capping activity. Like chicken capping protein, yeast capping protein is inhibited by PIP2. By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos. In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis.

scholarly journals UNC-45A Weakens and Breaks MT Lattice Independent of its Effect on Non-Muscle Myosin II

2020 ◽  
Author(s):  
Juri Habicht ◽  
Ashley Mooneyham ◽  
Asumi Hoshino ◽  
Mihir Shetty ◽  
Xiaonan Zhang ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Myosin Ii ◽  
Human Diseases ◽  
Therapeutic Implications ◽  
Associated Functions ◽  
Muscle Myosin ◽  
First Time ◽  
In Cells

AbstractIn invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only and UNC-45A, expressed in all cells and implicated in regulating both Non-Muscle Myosin II (NMII)- and microtubule (MT)-associated functions. Here we show for the first time that: a) in vitro UNC-45A binds to the MT lattice and weakens its integrity leading to MT bending, breakage and depolymerization, b) in cells, UNC-45A overexpression causes loss of MT mass and increase in MT breakages, c) both in vitro and in cells, UNC-45A destabilizes MTs independent of its NMII C-terminal binding domain and destabilization occurs even in presence of the NMII inhibitor blebbistatin. These findings are consistent with a not mutually exclusive but rather dual role of UNC-45A in regulating NMII activity and MT stability.Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability our findings have profound implications in both, the biology of MTs as well as the biology of human diseases and possible therapeutic implications for their treatment.

scholarly journals Changes in the expression and functional activities of Myosin II isoforms in human hyperplastic prostate

2021 ◽  
Author(s):  
Weixiang He ◽  
Xiao Wang ◽  
Daxing Zhan ◽  
Mingzhou Li ◽  
Qian Wang ◽  
...  
Keyword(s):  
Myosin Ii ◽  
Human Prostate ◽  
Common Disease ◽  
Organ Bath ◽  
Prostate Cell ◽  
Functional Activities ◽  
Prostate Cells ◽  
Induced Apoptosis ◽  
Muscle Myosin

Benign prostatic hyperplasia (BPH) is a common disease among aging males with the etiology remaining unclear. We recently found myosin II was abundantly expressed in rat and cultured human prostate cells with permissive roles in the dynamic and static components. This study aimed to explore the expression and functional activities of myosin II isoforms including smooth muscle myosin II (SMM II) and non-muscle myosin (NMM II) in the hyperplastic prostate. Human prostate cell lines and tissues from normal human and BPH patients were used. H&E, Masson’s trichrome, immunohistochemical staining, in vitro organ bath, RT-PCR and Western-blotting were performed. We further created cell models with NMM II isoforms silenced and proliferation, cycle, and apoptosis of prostate cells were determined by CCK-8 assay and flow cytometry. Hyperplastic prostate SM expressed more SM1 and LC17b isoforms compared to their alternatively spliced counterparts, favoring a slower more tonic-type contraction and greater force generation. For BPH group, blebbistatin (BLEB, a selective myosin II inhibitor), exhibited a stronger effect on relaxing phenylephrine (PE) pre-contracted prostate strips and inhibiting PE induced contraction. Additionally, NMMHC-A and NMMHC-B were upregulated in hyperplastic prostate with no change in NMMHC-C. Knockdown of NMMHC-A or NMMHC-B inhibited prostate cell proliferation and induced apoptosis, with no changes in cell cycle. Our novel data demonstrates that expression and functional activities of myosin II isoforms are altered in human hyperplastic prostate, suggesting a new pathological mechanism for BPH. Thus, the myosin II system may provide potential new therapeutic targets for BPH/lower urinary tract symptoms (LUTS).

scholarly journals In vivo optochemical control of cell contractility at single cell resolution by Ca2+ induced myosin activation

2018 ◽  
Author(s):  
Deqing Kong ◽  
Zhiyi Lv ◽  
Matthias Häring ◽  
Fred Wolf ◽  
Joerg Grosshans
Keyword(s):  
Single Cell ◽  
Temporal Dynamics ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Target Cells ◽  
Tissue Morphogenesis ◽  
Cross Sectional ◽  
Cell Contractility ◽  
Muscle Myosin

The spatial and temporal dynamics of cell contractility plays a key role in tissue morphogenesis, wound healing and cancer invasion. Here we report a simple, single cell resolution, optochemical method to induce minute-scale cell contractions in vivo during morphogenesis. We employed the photolabile Ca2+ chelator o-nitrophenyl EGTA to induce bursts of intracellular free Ca2+ by laser photolysis. Ca2+ bursts appear within seconds and are restricted to individual target cells. Cell contraction reliably followed within a minute, to about half of the cross-sectional area. Increased Ca2+ levels and contraction were reversible and the target cells further participated in tissue morphogenesis. Depending on Rho kinase (Rok) activity but not RhoGEF2, cell contractions are paralleled with non-muscle myosin-II accumulation in the apico-medial cortex, indicating that Ca2+ bursts trigger non-muscle myosin II activation. Our approach can be easily adapted to many experimental systems and species, as no specific genetic elements are required and a widely used reagent is employed.

P-cresol, a uremic retention solute, alters the endothelial barrier function in vitro

2004 ◽  
Vol 92 (07) ◽  
pp. 140-150 ◽  
Author(s):  
Laetitia Dou ◽  
Francine Anfosso ◽  
Florence Sabatier ◽  
Valérie Moal ◽  
Griet Glorieux ◽  
...  
Keyword(s):  
Barrier Function ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Endothelial Permeability ◽  
Endothelial Barrier ◽  
Strong Increase ◽  
Endothelial Barrier Function ◽  
Cortical Actin ◽  
Vitro Effect

SummaryPatients with chronic renal failure (CRF) exhibit endothelial dysfunction, which may involve uremic retention solutes that accumulate in blood and tissues. In this study, we investigated the in vitro effect of the uremic retention solute p-cresol on the barrier function of endothelial cells (HUVEC). P-cresol was tested at concentrations found in CRF patients, and since p-cresol is protein-bound, experiments were performed with and without physiological concentration of human albumin (4 g/dl).With albumin, we showed that p-cresol caused a strong increase in endothelial permeability after a 24-hour exposure. Concomitant with this increase in endothelial permeability, p-cresol induced a reorganization of the actin cytoskeleton and an alteration of adherens junctions. These molecular events were demonstrated by the decreased staining of cortical actin, associated with the formation of stress fibers across the cell, and by the decreased staining of junctional VE-cadherin. This decrease in junctional VE-cadherin staining was not associated with a reduction of membrane expression. Without albumin, the effects of p-cresol were more pronounced. The specific Rho kinase inhibitor, Y-27632, inhibited the effects of p-cresol, indicating that p-cresol mediates the increase in endothelial permeability in a Rho kinase-dependent way. In conclusion, these results show that p-cresol causes a severe dysfunction of endothelial barrier function in vitro and suggest this uremic retention solute may participate in the endothelium dysfunction observed in CRF patients.

scholarly journals Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis.

1992 ◽  
Vol 118 (3) ◽  
pp. 595-605 ◽  
Author(s):  
L L Satterwhite ◽  
M J Lohka ◽  
K L Wilson ◽  
T Y Scherson ◽  
L J Cisek ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Chromosome Segregation ◽  
Light Chain ◽  
Kinase Activity ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Nonmuscle Myosin ◽  
Xenopus Egg ◽  
Muscle Myosin

To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis.

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