scholarly journals UNC-45a promotes myosin folding and stress fiber assembly

2017 ◽  
Vol 216 (12) ◽  
pp. 4053-4072 ◽  
Author(s):  
Jaakko I. Lehtimäki ◽  
Aidan M. Fenix ◽  
Tommi M. Kotila ◽  
Giuseppe Balistreri ◽  
Lassi Paavolainen ◽  
...  
Keyword(s):  
Large Fraction ◽  
Gradient Centrifugation ◽  
Proteasome Inhibition ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Dynamic Component ◽  
Fiber Assembly

Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.

Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

2002 ◽  
Vol 227 (6) ◽  
pp. 412-424 ◽  
Author(s):  
Imre L. Szabó ◽  
Rama Pai ◽  
Michael K. Jones ◽  
George R. Ehring ◽  
Hirofumi Kawanaka ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Stress Fiber Formation ◽  
Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

Role of Rho and myosin phosphorylation in actin stress fiber assembly in mesangial cells

1997 ◽  
Vol 273 (2) ◽  
pp. F283-F288 ◽  
Author(s):  
J. I. Kreisberg ◽  
N. Ghosh-Choudhury ◽  
R. A. Radnik ◽  
M. A. Schwartz
Keyword(s):  
Cell Shape ◽  
Mesangial Cells ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Glomerular Mesangial Cells ◽  
Fiber Assembly ◽  
Camp Elevation ◽  
Actin Stress Fibers

Treatment of renal glomerular mesangial cells with adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents induces actin stress fiber disassembly, myosin light chain (MLC) dephosphorylation, loss of adhesion to the substratum and cell shape change [J. I. Kreisberg and M. A. Venkatachalam. Am. J. Physiol. 251 (Cell Physiol. 20): C505-C511, 1986]. Thrombin and vasopressin block the effects of cAMP. Because these agents are known to promote stress fiber formation via the small GTP-binding protein Rho, we investigated the effect of an activated variant of Rho on the response to cAMP elevation. Microinjecting V14-Rho completely blocked the effect of cAMP elevation on cell shape and the actin cytoskeleton, whereas inactivating Rho with botulinum C3 exoenzyme induced stress fiber disruption and cell retraction that was indistinguishable from that caused by elevations in intracellular levels of cAMP. Disruption of actin stress fibers by cAMP has previously been ascribed to MLC dephosphorylation; however, both C3 and cytochalasin D also caused dephosphorylation of MLC, whereas blocking MLC dephosphorylation failed to block the cAMP-induced loss of actin stress fibers. We conclude that Rho can modulate the effects of cAMP elevation and suggest that MLC dephosphorylation may be a consequence of actin stress fiber disassembly.

scholarly journals Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

2015 ◽  
Vol 112 (32) ◽  
pp. E4390-E4399 ◽  
Author(s):  
Mathew Stracy ◽  
Christian Lesterlin ◽  
Federico Garza de Leon ◽  
Stephan Uphoff ◽  
Pawel Zawadzki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Spatial Organization ◽  
Search Time ◽  
Population Level ◽  
Bacterial Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Superresolution Microscopy

Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.

scholarly journals Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes.

1989 ◽  
Vol 108 (6) ◽  
pp. 2355-2367 ◽  
Author(s):  
Z X Lin ◽  
S Holtzer ◽  
T Schultheiss ◽  
J Murray ◽  
T Masaki ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Stress Fiber ◽  
Protein Isoforms ◽  
Stress Fibers ◽  
Intercalated Discs ◽  
Adhesion Plaques ◽  
Alpha Actin ◽  
Muscle Myosin

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.

scholarly journals Muscle specific stress fibers give rise to sarcomeres and are mechanistically distinct from stress fibers in non-muscle cells

2017 ◽  
Author(s):  
Aidan M. Feinx ◽  
Nilay Taneja ◽  
Abigail C. Neininger ◽  
Mike R. Visetsouk ◽  
Benjamin R. Nixon ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Muscle Cells ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Myosin Filament ◽  
Human Cardiomyocytes ◽  
Fiber Assembly ◽  
Filament Assembly ◽  
Sarcomere Assembly

AbstractThe sarcomere is the basic contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating thin (i.e., actin) and thick (i.e., myosin) filament assembly during sarcomere formation. Thus, we developed an assay using human cardiomyocytes to test de novo sarcomere assembly. Using this assay, we report a population of muscle-specific stress fibers are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from these muscle stress fibers. This process requires formin-mediated but not Arp2/3-mediated actin polymerization and nonmuscle myosin IIB but not non-muscle myosin IIA. Furthermore, we show a short species of β cardiac myosin II filaments grows to form ~1.5 long filaments that then “stitch” together to form the stack of filaments at the core of the sarcomere (i.e., A-band). Interestingly, these are different from mechanisms that have previously been reported during stress fiber assembly in non-muscle cells. Thus, we provide a new model of cardiac sarcomere assembly based on distinct mechanisms of stress fiber regulation between non-muscle and muscle cells.

scholarly journals Isolation and Contraction of the Stress Fiber

1998 ◽  
Vol 9 (7) ◽  
pp. 1919-1938 ◽  
Author(s):  
Kazuo Katoh ◽  
Yumiko Kano ◽  
Michitaka Masuda ◽  
Hirofumi Onishi ◽  
Keigi Fujiwara
Keyword(s):  
Light Chain ◽  
Kinase Inhibitor ◽  
Gradient Centrifugation ◽  
Maximum Velocity ◽  
Apical Part ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Hypodermic Needle ◽  
Culture Dish

Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-α-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion–associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle.

scholarly journals Tension-dependent stretching and folding of ZO-1 controls the localization of its interactors

2017 ◽  
Author(s):  
Domenica Spadaro ◽  
Shimin Le ◽  
Thierry Laroche ◽  
Isabelle Mean ◽  
Lionel Jond ◽  
...  
Keyword(s):  
Tight Junction ◽  
Molecular Mechanisms ◽  
Magnetic Tweezers ◽  
Dependent Manner ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Epithelial Homeostasis ◽  
Junction Protein ◽  
Tensile Forces

Tensile forces regulate epithelial homeostasis, but the molecular mechanisms behind this regulation are poorly understood. Using structured illumination microscopy and proximity ligation assays we show that the tight junction protein ZO-1 undergoes actomyosin tension-dependent stretching and folding in vivo. Magnetic tweezers experiments using purified ZO-1 indicate that pN-scale tensions (~2-4 pN) are sufficient to maintain the stretched conformation of ZO-1, while keeping its structured domains intact. Actomyosin tension and substrate stiffness regulate the localization and expression of the transcription factor DbpA and the tight junction membrane protein occludin in a ZO-1/ZO-2-dependent manner, resulting in modulation of gene expression, cell proliferation, barrier function and cyst morphogenesis. Interactions between the N-terminal (ZPSG) and C-terminal domains of ZO-1 prevent binding of DbpA to the ZPSG, and folding is antagonized by heterodimerization with ZO-2. We propose that tensile forces regulate epithelial homeostasis by activating ZO proteins through stretching, to modulate their protein interactions and downstream signaling.

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