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- Dynamin contributes to cytokinesis by stabilizing actin filaments in the contractile ring. [JOURNAL ARTICLE]
- Genes Cells 2013 May 17.
Dynamin has been proposed to play an important role in cytokinesis, although the nature of its contribution has remained unclear. Dictyostelium discoideum has five dynamin-like proteins: DymA, DymB, DlpA, DlpB and DlpC. Cells mutant for dymA, dlpA or dlpB presented defects in cytokinesis that resulted in multinucleation when the cells were cultured in suspension. However, the cells could divide normally when attached to the substratum; this latter process depends on traction-mediated cytokinesis B. A dynamin GTPase inhibitor also blocked cytokinesis in suspension, suggesting an important role for dynamin in cytokinesis A, which requires a contractile ring powered by myosin II. Myosin II did not properly localize to the cleavage furrow in dynamin mutant cells, and the furrow shape was distorted. DymA and DlpA were associated with actin filaments at the furrow. Fluorescence recovery after photobleaching and a DNase I binding assay showed that actin filaments in the contractile ring were significantly fragmented in mutant cells. Dynamin is therefore involved in the stabilization of actin filaments in the furrow, which, in turn, maintain proper myosin II organization. We conclude that the lack of these dynamins disrupts proper actomyosin organization and thereby disables cytokinesis A.
- Ca2+-Pumping Impairment during Repetitive Fatiguing Contractions in Single Myofibers: Role of Cross-Bridge Cycling. [JOURNAL ARTICLE]
- Am J Physiol Regul Integr Comp Physiol 2013 May 15.
The energy cost of contractions in skeletal muscle involves activation of both actomyosin and sarcoplasmic reticulum (SR) Ca(2+)-pump (SERCA) ATPases, which together determine the overall ATP demand. During repetitive contractions leading to fatigue, the relaxation rate and Ca(2+)-pumping become slowed, possibly due to intracellular metabolite accumulation. The role of the energy cost of cross-bridge cycling during contractile activity on Ca(2+)-pumping properties has not been investigated. Therefore, we inhibited cross-bridge cycling by incubating isolated Xenopus single fibers with N-benzyl-p-toluene sulfonamide (BTS) to study the mechanisms by which SR Ca(2+)-pumping is impaired during fatiguing contractions. Fibers were stimulated in the absence (control) and presence of BTS and cytosolic calcium ([Ca(2+)]c) transients or intracellular pH (pHi) changes were measured. BTS treatment allowed normal [Ca(2+)]c transients during stimulation without cross-bridge activation. At the time-point that tension was reduced to 50% in the control condition, the fall in the peak [Ca(2+)]c and the increase in basal [Ca(2+)]c did not occur with BTS incubation. The progressive slower Ca(2+)-pumping rate, and the fall in pHi, during repetitive contractions were reduced during BTS conditions. However, when mitochondrial ATP supply was blocked during contractions with BTS present (BTS + cyanide), there was no further slowing in SR Ca(2+)-pumping during contractions compared to the BTS-alone condition. Furthermore, the fall in pHi was significantly less during the BTS + cyanide condition than in the control conditions. These results demonstrate that factors related to the energetic cost of cross-bridge cycling, possibly the accumulation of metabolites, inhibit the Ca(2+)-pumping rate during fatiguing contractions.
- ALKBH4-dependent demethylation of actin regulates actomyosin dynamics. [Journal Article]
- Nat Commun 2013.:1832.
Regulation of actomyosin dynamics by post-transcriptional modifications in cytoplasmic actin is still poorly understood. Here we demonstrate that dioxygenase ALKBH4-mediated demethylation of a monomethylated site in actin (K84me1) regulates actin-myosin interaction and actomyosin-dependent processes such as cytokinesis and cell migration. ALKBH4-deficient cells display elevated K84me1 levels. Non-muscle myosin II only interacts with unmethylated actin and its proper recruitment to and interaction with actin depend on ALKBH4. ALKBH4 co-localizes with the actomyosin-based contractile ring and midbody via association with methylated actin. ALKBH4-mediated regulation of actomyosin dynamics is completely dependent on its catalytic activity. Disorganization of cleavage furrow components and multinucleation associated with ALKBH4 deficiency can all be restored by reconstitution with wild-type but not catalytically inactive ALKBH4. Similar to actin and myosin knock-out mice, homozygous Alkbh4 mutant mice display early embryonic lethality. These findings imply that ALKBH4-dependent actin demethylation regulates actomyosin function by promoting actin-non-muscle myosin II interaction.
- Ultrafast molecular motor driven nanoseparation and biosensing. [JOURNAL ARTICLE]
- Biosens Bioelectron 2013 Apr 6.:145-152.
Portable biosensor systems would benefit from reduced dependency on external power supplies as well as from further miniaturization and increased detection rate. Systems built around self-propelled biological molecular motors and cytoskeletal filaments hold significant promise in these regards as they are built from nanoscale components that enable nanoseparation independent of fluidic pumping. Previously reported microtubule-kinesin based devices are slow, however, compared to several existing biosensor systems. Here we demonstrate that this speed limitation can be overcome by using the faster actomyosin motor system. Moreover, due to lower flexural rigidity of the actin filaments, smaller features can be achieved compared to microtubule-based systems, enabling further miniaturization. Using a device designed through optimization by Monte Carlo simulations, we demonstrate extensive myosin driven enrichment of actin filaments on a detector area of less than 10μm(2), with a concentration half-time of approximately 40s. We also show accumulation of model analyte (streptavidin at nanomolar concentration in nanoliter effective volume) detecting increased fluorescence intensity within seconds after initiation of motor-driven transportation from capture regions. We discuss further optimizations of the system and incorporation into a complete biosensing workflow.
- Nanotopography Modulates Mechanotransduction of Stem Cell and Induces Differentiation Through Focal Adhesion Kinase. [JOURNAL ARTICLE]
- ACS Nano 2013 May 13.
Regulated biophysical cues, such as nanotopography, have been shown to be integral for tissue regeneration and embryogenesis in the stem cell niche. Tissue homeostasis involves the interaction of multipotent cells with nano-scaled topographical features in their ECM to regulate aspects of cell behaviour. Synthetic nanostructures can drive specific cell differentiation but the sensing mechanism of nanocues remains poorly understood. Here we report that nanotopography-induced human mesenchymal stem cells (hMSC) differentiation through cell mechanotransduction is modulated by the integrin-activated focal adhesion kinase (FAK). On nano-gratings with 250nm line width on polydimethylsiloxane, hMSCs developed aligned stress fibers and showed an upregulation of neurogenic and myogenic differentiation markers. The observed cellular focal adhesions within these cells were also significantly smaller and more elongated on the nano-gratings compared to micro-gratings or unpatterned control. In addition, our mechanistic study confirmed that the regulation was dependent on actomyosin contractility, suggesting a direct force dependent mechanism. The topography induced differentiation could be observed on different ECM compositions but the response is independent of direct ECM-induced hMSC differentiation pathway. Essentially, FAK phosphorylation was required for topography-induced hMSC differentiation while FAK overexpression overruled the topographical cues in cell lineage bias. The results obtained indicated a direct effect of FAK activity on topography-induced gene expression but less dependent on cell shape. Mechanically, the findings may explain how hMSCs can sense and transduce nanotopographical signals through focal adhesions and actomyosin cytoskeleton contractility to induce differential gene expression.
- Forces measured with micro-fabricated cantilevers during actomyosin interactions produced by filaments containing different myosin isoforms and loop 1 structures. [Journal Article, Research Support, Non-U.S. Gov't]
- Biochim Biophys Acta 2013 Mar; 1830(3):2710-9.
There is evidence that the actin-activated ATP kinetics and the mechanical work produced by muscle myosin molecules are regulated by two surface loops, located near the ATP binding pocket (loop 1), and in a region that interfaces with actin (loop 2). These loops regulate force and velocity of contraction, and have been investigated mostly in single molecules. There is a lack of information of the work produced by myosin molecules ordered in filaments and working cooperatively, which is the actual muscle environment.We use micro-fabricated cantilevers to measure forces produced by myosin filaments isolated from mollusk muscles, skeletal muscles, and smooth muscles containing variations in the structure of loop 1 (tonic and phasic myosins). We complemented the experiments with in-vitro assays to measure the velocity of actin motility.Smooth muscle myosin filaments produced more force than skeletal and mollusk myosin filaments when normalized per filament overlap. Skeletal muscle myosin propelled actin filaments in a higher sliding velocity than smooth muscle myosin. The values for force and velocity were consistent with previous studies using myosin molecules, and suggest a close correlation with the myosin isoform and structure of surface loop 1.The technique using micro-fabricated cantilevers to measure force of filaments allows for the investigation of the relation between myosin structure and contractility, allowing experiments to be conducted with an array of different myosin isoforms. Using the technique we observed that the work produced by myosin molecules is regulated by amino-acid sequences aligned in specific loops.
- A viscous two-phase model for contractile actomyosin bundles. [JOURNAL ARTICLE]
- J Math Biol 2013 May 14.
A mathematical model in one dimension for a non-sarcomeric actomyosin bundle featuring anti-parallel flows of anti-parallel F-actin is introduced. The model is able to relate these flows to the effect of cross-linking and bundling proteins, to the forces due to myosin-II filaments and to external forces at the extreme tips of the bundle. The modeling is based on a coarse graining approach starting with a microscopic model which includes the description of chemical bonds as elastic springs and the force contribution of myosin filaments. In a second step we consider the asymptotic regime where the filament lengths are small compared to the overall bundle length and restrict to the lowest order contributions. There it becomes apparent that myosin filaments generate forces which are partly compensated by drag forces due to cross-linking proteins. The remaining local contractile forces are then propagated to the tips of the bundle by the viscosity effect of bundling proteins in the filament gel. The model is able to explain how a disordered bundle of comparatively short actin filaments interspersed with myosin filaments can effectively contract the two tips of the actomyosin bundle. It gives a quantitative description of these forces and of the anti-parallel flows of the two phases of anti-parallel F-actin. An asymptotic version of the model with infinite viscosity can be solved explicitly and yields an upper bound to the contractile force of the bundle.
- Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein-C slow. [JOURNAL ARTICLE]
- FASEB J 2013 May 8.
Myosin binding protein C (MyBP-C) is expressed in striated muscles, where it plays key roles in the modulation of actomyosin cross-bridges. Slow MyBP-C (sMyBP-C) consists of multiple variants sharing common domains but also containing unique segments within the NH2 and COOH termini. Two missense mutations in the NH2 terminus (W236R) and COOH terminus (Y856H) of sMyBP-C have been causally linked to the development of distal arthrogryposis-1 (DA-1), a severe skeletal muscle disorder. Using a combination of in vitro binding and motility assays, we show that the COOH terminus mediates binding of sMyBP-C to thick filaments, while the NH2 terminus modulates the formation of actomyosin cross-bridges in a variant-specific manner. Consistent with this, a recombinant NH2-terminal peptide that excludes residues 34-59 reduces the sliding velocity of actin filaments past myosin heads from 9.0 ± 1.3 to 5.7 ± 1.0 μm/s at 0.1 μM, while a recombinant peptide that excludes residues 21-59 fails to do so. Notably, the actomyosin regulatory properties of sMyBP-C are completely abolished by the presence of the DA-1 mutations. In summary, our studies are the first to show that the NH2 and COOH termini of sMyBP-C have distinct functions, which are regulated by differential splicing, and are compromized by the presence of missense point mutations linked to muscle disease.-Ackermann, M. A., Patel, P. D., Valenti, J., Takagi, Y., Homsher, E., Sellers, J. R., Kontrogiannni-Konstantopoulos, A. Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein-C slow.
- E-cadherin supports steady-state Rho signaling at the epithelial zonula adherens. [JOURNAL ARTICLE]
- Differentiation 2013 May 2.
In simple polarized epithelial cells, the Rho GTPase commonly localizes to E-cadherin-based cell-cell junctions, such as the zonula adherens (ZA), where it regulates the actomyosin cytoskeleton to support junctional integrity and tension. An important question is how E-cadherin contributes to Rho signaling, notably whether junctional Rho may depend on cadherin adhesion. We sought to investigate this by assessing Rho localization and activity in epithelial monolayers depleted of E-cadherin by RNAi. We report that E-cadherin depletion reduced both Rho and Rho-GTP at the ZA, an effect that was rescued by expressing a RNAi-resistant full-length E-cadherin transgene. This impact on Rho signaling was accompanied by reduced junctional localization of the Rho GEF ECT2 and the centralspindlin complex that recruits ECT2. Further, the Rho signaling pathway contributes to the selective stabilization of E-cadherin molecules in the apical zone of the cells compared with E-cadherin at the lateral surface, thereby creating a more defined and restricted pool of E-cadherin that forms the ZA. Thus, E-cadherin and Rho signaling cooperate to ensure proper ZA architecture and function.
- Magnetic capture from blood rescues molecular motor function in diagnostic nanodevices. [JOURNAL ARTICLE]
- J Nanobiotechnology 2013 May 3; 11(1):14.