Disassembly Of Stress Fibers Research Articles

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P(2). Unexpectedly, we observed that at low doses (10-25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P(2) production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P(2) synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62-71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P(2) fell by only 28-46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P(2) at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool.

Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament

Intracellular and extracellular mechanical forces affect the structure and dynamics of the actin cytoskeleton. However, the underlying molecular and biophysical mechanisms, including how mechanical forces are sensed, are largely unknown. Actin-depolymerizing factor/cofilin proteins are actin-modulating proteins that are ubiquitously distributed in eukaryotes, and they are the most likely candidate as proteins to drive stress fiber disassembly in response to changes in tension in the fiber. In this study, we propose a novel hypothesis that tension in an actin filament prevents the filament from being severed by cofilin. To test this, we placed single actin filaments under tension using optical tweezers. When a fiber was tensed, it was severed after the application of cofilin with a significantly larger delay in comparison with control filaments suspended in solution. The binding rate of cofilin to an actin bundle decreased when the bundle was tensed. These results suggest that tension in an actin filament reduces the cofilin binding, resulting in a decrease in its effective severing activity.

The Arf Family GTPase Arl4A Complexes with ELMO Proteins to Promote Actin Cytoskeleton Remodeling and Reveals a Versatile Ras-binding Domain in the ELMO Proteins Family

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is indispensable during processes such as cell migration and myoblast fusion. The biological activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that autoinhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate the ELMO-DOCK180 complex appears to be the recruitment of this complex to the membrane via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel ELMO-interacting proteins to further define the molecular events capable of controlling ELMO recruitment to the membrane. To do so, we performed two independent interaction screens: one specifically interrogated an active GTPase library while the other probed a brain cDNA library. Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. Biochemically, Arl4A is constitutively GTP-loaded, and our binding assays confirm that both wild-type and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In addition, we report that membrane targeting of ELMO via Arl4A promotes cytoskeletal reorganization including membrane ruffling and stress fiber disassembly via an ELMO-DOCK1800-Rac signaling pathway. We conclude that ELMO is capable of interacting with GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile RBD. Furthermore, via binding of an Arf family GTPase, the ELMO-DOCK180 is uniquely positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton.

Altered osteogenic commitment of human mesenchymal stem cells by ERM protein-dependent modulation of cellular biomechanics

Cellular mechanics is known to play an important role in many cellular functions including adhesion, migration, proliferation, and differentiation. Human mesenchymal stem cells (hMSCs) demonstrate unique mechanical properties distinct from fully differentiated cells. This observation suggests that the stem cell mechanics may be modulated to regulate the hMSCs' lineage commitment. Specifically, ERM (ezrin, radixin, moesin) proteins are known to mediate the membrane–cytoskeleton adhesion, cell elasticity, actin cytoskeleton organization, and therefore could serve as potential targets for modulation of the cellular mechanics. Combining silencing RNA, atomic force microscopy, and laser optical tweezers, the role of the ERM proteins involved in the regulation of stem cell biomechanics and osteogenic differentiation was quantitatively determined. Transient ERM knockdown by RNAi causes disassembly of actin stress fibers and focal adhesions, a decrease in the cell stiffness, and membrane separation from the cytoskeleton. The silencing RNA treatment not only induced mechanical changes in stem cells but impaired biochemically-directed osteogenic differentiation. The intact actin cytoskeleton and focal adhesions of hMSCs appear critical for the osteogenic induction. Thus, ERM knockdown modulates the dynamics of cell mechanical changes during hMSC differentiation and regulates the expression of tissue specific molecular markers. These findings are of particular interest for modulation of the cellular biomechanics to control hMSCs' activities and fate in tissue engineering, regenerative medicine, and other stem cell-based therapeutic applications.

Nanoengineering life: from cell to tissue

Nanoengineering, which brings several traditional disciplines including physics, engineering, biology and medicine together, is an emerging tool for manipulating major constituents of living organisms such as cells and tissues. Generally, the scopes of nanoengineering techniques include

Non-muscle myosin II induces disassembly of actin stress fibres independently of myosin light chain dephosphorylation

Dynamic remodelling of actin stress fibres (SFs) allows non-muscle cells to adapt to applied forces such as uniaxial cell shortening. However, the mechanism underlying rapid and selective disassembly of SFs oriented in the direction of shortening remains to be elucidated. Here, we investigated how myosin crossbridge cycling induced by MgATP is associated with SF disassembly. Moderate concentrations of MgATP, or [MgATP], induced SF contraction. Meanwhile, at [MgATP] slightly higher than the physiological level, periodic actin patterns emerged along the length of SFs and dispersed within seconds. The actin fragments were diverse in length, but comparable to those in characteristic sarcomeric units of SFs. These results suggest that MgATP-bound non-muscle myosin II dissociates from the individual actin filaments that constitute the sarcomeric units, resulting in unbundling-induced disassembly rather than end-to-end actin depolymerization. This rapid SF disassembly occurred independent of dephosphorylation of myosin light chain. In terms of effects on actin-myosin interactions, a rise in [MgATP] is functionally equivalent to a temporal decrease in the total number of actin-myosin crossbridges. Actin-myosin crossbridges are known to be reduced by an assisting load on myosin. Thus, the present study suggests that reducing the number of actin-myosin crossbridges promotes rapid and orientation-dependent disassembly of SFs after cell shortening.

Semaphorin 5A and plexin-B3 regulate human glioma cell motility and morphology through Rac1 and the actin cytoskeleton

Semaphorins are implicated in glioma progression, although little is known about the underlying mechanisms. We have reported plexin-B3 expression in human gliomas, which upon stimulation by Sema5A causes significant inhibition of cell migration and invasion. The concomitant inactivation of Rac1 is of mechanistic importance because forced expression of constitutively active Rac1 abolishes these inhibitory effects. Furthermore, Sema5A induces prominent cell collapse and ramification of processes reminiscent of astrocytic morphology, which temporally associate with extensive disassembly of actin stress fibers and disruption of focal adhesions, followed by accumulation of actin patches in protrusions. Mechanistically, Sema5A induces transient protein kinase C (PKC) phosphorylation of fascin-1, which can reduce its actin-binding/bundling activities and temporally parallels its translocation from cell body to extending processes. PKC inhibition or fascin-1 knockdown is sufficient to abrogate Sema5A-induced morphological differentiation, whereas the process is hastened by forced expression of fascin-1. Intriguingly, Sema5A induces re-expression of glial fibrillary acidic protein (GFAP), which when silenced restricts differentiation of glioma cells to bipolar instead of multipolar morphology. Therefore, we hypothesize complementary functions of fascin-1 and GFAP in the early and late phases of Sema5A-induced astrocytic differentiation of gliomas, respectively. In summary, Sema5A and plexin-B3 impede motility but promote differentiation of human gliomas. These effects are plausibly compromised in high-grade human astrocytomas in which Sema5A expression is markedly reduced, hence leading to infiltrative and anaplastic characteristics. This is evident by increased invasiveness of glioma cells when endogenous Sema5A is silenced. Therefore, Sema5A and plexin-B3 represent potential novel targets in counteracting glioma progression.

Multiple Roles for Myosin II in Tensional Homeostasis Under Mechanical Loading

Cyclic stretching of adherent cells regulates cell morphology, signal transduction and cell function. It is well established that actin stress fibers are mechano-sensitive structural elements that reorganize in response to applied stress and strain. In this review, we discuss studies revealing the roles of myosin II in stress fiber remodeling including stress fiber assembly, disassembly, and tension maintenance. The results of these studies are interpreted with mathematical models that describe a mechanism by which stress fibers reorganize in response to cyclic stretch and predict how changes in stress fiber tension regulate signal transduction dependent on the spatial and temporal patterns of the applied strain.

SKAP2, a novel target of HSF4b, associates with NCK2/F-actin at membrane ruffles and regulates actin reorganization in lens cell

In addition to roles in stress response, heat shock factors (HSFs) play crucial roles in differentiation and development. Heat shock transcription factor 4 (HSF4) deficiency leads to defect in lens epithelial cell (LEC) differentiation and cataract formation. However, the mechanism remains obscure. Here, we identified Src kinase-associated phosphoprotein 2 (SKAP2) as a downstream target of HSF4b and it was highly expressed at the anterior tip of lens elongating fibre cells in vivo. The HSF4-deficient lenses showed reduced SKAP2 expression and defects in actin reorganization. The disassembly of stress fibres and formation of cortical actin fibres are critical for the initiation of LEC differentiation. SKAP2 localized at actin-rich ruffles in human LECs (SRA01/04 cells) and knockdown SKAP2 using RNA interference impaired the disassembly of cellular stress fibres in response to fibroblast growth factor (FGF)-b. Overexpression of SKAP2, but not the N-terminal deletion mutant of SKAP2, induced the actin remodelling. We further found that SKAP2 interacted with the SH2 domain of non-catalytic region of tyrosine kinase adaptor protein 2 (NCK2) via its N-terminus. The complex of SKAP2-NCK2-F-actin accumulated at the leading edge of the lamellipodium, where FGF receptors and focal adhesion were also recruited. These results revealed an essential role for HSF4-mediated SKAP2 expression in the regulation of actin reorganization during lens differentiation, likely through a mechanism that SKAP2 anchors the complex of NCK2/focal adhesion to FGF receptors at the lamellipodium in lens epithelial cells.

9J-02 アクチンストレスファイバが張力依存的に脱重合するメカニズムの考察(OS-11(3) 細胞・生体分子のバイオメカニクス(1))

9J-02 アクチンストレスファイバが張力依存的に脱重合するメカニズムの考察(OS-11(3) 細胞・生体分子のバイオメカニクス(1))

1K1648 II型非筋ミオシンによるストレスファイバの脱重合(細胞生物的課題1,第49回日本生物物理学会年会)

1K1648 II型非筋ミオシンによるストレスファイバの脱重合(細胞生物的課題1,第49回日本生物物理学会年会)

Cyclic stretch-induced stress fiber dynamics – Dependence on strain rate, Rho-kinase and MLCK

Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor Y27632 reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.

A microfluidic bioreactor with integrated transepithelial electrical resistance (TEER) measurement electrodes for evaluation of renal epithelial cells

We have developed a bilayer microfluidic system with integrated transepithelial electrical resistance (TEER) measurement electrodes to evaluate kidney epithelial cells under physiologically relevant fluid flow conditions. The bioreactor consists of apical and basolateral fluidic chambers connected via a transparent microporous membrane. The top chamber contains microfluidic channels to perfuse the apical surface of the cells. The bottom chamber acts as a reservoir for transport across the cell layer and provides support for the membrane. TEER electrodes were integrated into the device to monitor cell growth and evaluate cell-cell tight junction integrity. Immunofluorescence staining was performed within the microchannels for ZO-1 tight junction protein and acetylated α-tubulin (primary cilia) using human renal epithelial cells (HREC) and MDCK cells. HREC were stained for cytoskeletal F-actin and exhibited disassembly of cytosolic F-actin stress fibers when exposed to shear stress. TEER was monitored over time under normal culture conditions and after disruption of the tight junctions using low Ca(2+) medium. The transport rate of a fluorescently labeled tracer molecule (FITC-inulin) was measured before and after Ca(2+) switch and a decrease in TEER corresponded with a large increase in paracellular inulin transport. This bioreactor design provides an instrumented platform with physiologically meaningful flow conditions to study various epithelial cell transport processes.

PI3K-dependent host cell actin rearrangements are required for Cronobacter sakazakii invasion of human brain microvascular endothelial cells

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that can cause neonatal sepsis and meningitis. The mechanism involved in the pathogenesis of C. sakazakii meningitis remains largely unknown. Previous studies indicated that bacterial invasion of brain microvascular endothelial cells is required for penetration into the central nervous system. In this study, we found that C. sakazakii invasion of human brain microvascular endothelial cells (HBMEC) was significantly inhibited by cytochalasin D, a disrupting agent of actin microfilaments. Disassembly of actin stress fibers and cortical actin fibers was observed in HBMEC infected with C. sakazakii. C. sakazakii infection leads to increased Akt phosphorylation in HBMEC, which was blocked by treatment with PI3K inhibitors. Meanwhile, PI3K and Akt inhibitors significantly inhibited C. sakazakii invasion of HBMEC. Our further results illustrated that the C. sakazakii-induced Akt activation and C. sakazakii invasion were attenuated in HBMEC transfected with dominant-negative PI3K (Δp110). More importantly, the actin filaments rearrangements in HBMEC induced by C. sakazakii were effectively blocked by PI3K inhibitors treatment and transfection with Δp110. Taken together, our findings demonstrated that PI3K-mediated actin rearrangements are required for C. sakazakii invasion of HBMEC.

Desmuslin gene knockdown causes altered expression of phenotype markers and differentiation of saphenous vein smooth muscle cells

Phenotypic alterations of vascular smooth muscle cells (VSMCs) appear critical to the development of primary varicose veins. Previous study indicated desmuslin, an intermediate filament protein, was differentially expressed in smooth muscle cells (SMCs) isolated from varicose veins; thus, it was naturally hypothesized that altered desmuslin expression might in turn affect the functioning of VSMCs, leading to the phenotypic alterations and varicose vein development. In this study, expression of desmuslin in normal human saphenous vein SMCs was knocked down using small interfering RNA (siRNA), and control cells were treated with a scrambled siRNA sequence. The levels of several phenotypic markers including smooth muscle (SM) alpha-actin and smooth muscle myosin heavy chain (SM-MHC) were assessed. Collagen formation, matrix metalloproteinase expression (MMP-2), and cytoskeletal and morphological changes were also examined. SMCs treated with desmuslin siRNA exhibited significantly increased levels of collagen synthesis and MMP-2 expression and decreased expression levels of SM alpha-actin, SM-MHC, and smoothelin and exhibited disassembly of actin stress fibers when compared with the control cells. Changes in cell morphology and actin fiber networks in VSMCs treated with desmuslin siRNA were consistent with a lower degree of differentiation. These results indicated desmuslin expression is required for the maintenance of VSMC phenotype. Decreased desmuslin expression may affect differentiation of VSMCs and ultimately contribute to the development of varicose veins.

Vicrostatin - an anti-invasive multi-integrin targeting chimeric disintegrin with tumor anti-angiogenic and pro-apoptotic activities.

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.

A kinematic model coupling stress fiber dynamics with JNK activation in response to matrix stretching

The role of the actin cytoskeleton in regulating mechanotransduction in response to external forces is complex and incompletely understood. Here, we develop a mathematical model coupling the dynamic disassembly and reassembly of actin stress fibers and associated focal adhesions to the activation of c-jun N-terminal kinase (JNK) in cells attached to deformable matrices. The model is based on the assumptions that stress fibers are pre-extended to a preferred level under static conditions and that perturbations from this preferred level destabilize the stress fibers. The subsequent reassembly of fibers upregulates the rate of JNK activation as a result of the formation of new integrin bonds within the associated focal adhesions. Numerical solutions of the model equations predict that different patterns of matrix stretch result in distinct temporal patterns in JNK activation that compare well with published experimental results. In the case of cyclic uniaxial stretching, stretch-induced JNK activation slowly subsides as stress fibers gradually reorient perpendicular to the stretch direction. In contrast, JNK activation is chronically elevated in response to cyclic equibiaxial stretch. A step change in either uniaxial or equibiaxial stretch results in a short, transient upregulation in JNK that quickly returns to the basal level as overly stretched stress fibers disassemble and are replaced by fibers assembled at the preferred level of stretch. In summary, the model describes a mechanism by which the dynamic properties of the actin cytoskeleton allow cells to adapt to applied forces through turnover and reorganization to modulate intracellular signaling.

A Single Actin Filament Works as a Mechanosensor

Force plays a variety of roles in cell biology, including cell signal pathways for cell survival, growth, development, and migration. Cells may use molecular mechanisms other than mechanosensitive ion channels such as cytoskeletons to sense force, since intracellular and extracellular mechanical environments greatly affect the disassembly of stress fibers, suggesting that the tension is sensed by unknown, intracellular mechanisms and the stress fibers are disassembled. Here, we report that loss of tension in stress fibers induced disassembly of stress fibers in permeabilized semi-intact cells, which was dependent on mouse brain derived cytosolic factor(s) in the bath solution, and purified Cofilin/ADF, a major actin modulating protein ubiquitously distributed in eukaryotes, was sufficient for this stress fiber disassembly. We hypothesize that the tension decline is sensed by the actin filament, which allows cofilin to sever the filament. To test this, we have prepared a single actin filament tensed with optical tweezers and examined the tension dependent severing of the filament by cofilin. The time to sever the filament by cofilin was prolonged about 2 times in the presence of mechanical tension in the filament compared with that of a filament that was not tensed. These results indicate that tension prevents binding of cofilin to actin filaments, or prevents severing of actin filaments by cofilin. We directly observed binding of Alexa488-labeled-cofilin to actin filaments tethered on the surface of cover slip via NEM-myosin by TIRF microscopy. Scratching the “motion restricted” tethered actin filaments by a fine tip of glass capillaries created freely moving filament ends. The rate of binding of cofilin to the actin filaments near (<2 μm) the scratching was significantly increased. These results strongly support the hypothesis that the actin filament acts as a “tension-sensor” and changes susceptibility of cofilin to the filament.

A stretching device for imaging real-time molecular dynamics of live cells adhering to elastic membranes on inverted microscopes during the entire process of the stretch

We present a device for stretching cells adhering to elastic membranes in equiaxial or uniaxial mode, meanwhile allowing real-time imaging of molecular dynamics of live cells at high resolution on an inverted microscope during the entire process of the stretch. We obtained high-resolution images of stress fibers at each stage of the stretch, and found that stress fibers were shortened after one stretching cycle. We, for the first time, captured real-time images of the process of stress fiber disassembly during stretching. Several adjacent stress fibers appeared to reassemble into a single one after stretching. All these indicated that mechanical stretching played important roles in the rearrangement of actin filaments. This device will be especially useful in studies of the molecular dynamics in the process of mechanotransduction. The device is fabricated on a glass slide through a simple procedure and is adaptable to most ordinary laboratories.

RhoE Inhibits 4E-BP1 Phosphorylation and eIF4E Function Impairing Cap-dependent Translation

The Rho GTPase family member RhoE inhibits RhoA/ROCK signaling to promote actin stress fiber and focal adhesion disassembly. We have previously reported that RhoE also inhibits cell cycle progression and Ras-induced transformation, specifically preventing cyclin D1 translation. Here we investigate the molecular mechanisms underlying those observations. RhoE inhibits the phosphorylation of the translational repressor 4E-BP1 in response to extracellular stimuli. However, RhoE does not affect the activation of mTOR, the major kinase regulating 4E-BP1 phosphorylation, as indicated by the phosphorylation levels of the mTOR substrate S6K, the dynamics of mTOR/Raptor association, and the observation that RhoE, as opposed to rapamycin, does not impair cellular growth. Interestingly, RhoE prevents the release of the eukaryotic initiation factor eIF4E from 4E-BP1, inhibiting cap-dependent translation. Accordingly, RhoE also inhibits the expression and the transcriptional activity of the eIF4E target c-Myc. Consistent with its crucial role in cell proliferation, we show that eIF4E can rescue both cell cycle progression and Ras-induced transformation in RhoE-expressing cells, indicating that the inhibition of eIF4E function is critical to mediate the anti-proliferative effects of RhoE.