Academic literature on the topic 'Myosin mechanics'

This thesis reports the realization and first application of a synthetic nanomachine, able to reproduce in vitro the performance emerging from the array arrangement of myosin II motors in the sarcomere of the striated muscle. The nanomachine consists of an ensemble of less than ten myosin dimers from fast skeletal muscle disposed on a functionalized support carried by a piezoelectric nanopositioner and brought to interact with an actin filament attached with the correct polarity via gelsolin to a bead (Bead Tailed Actin, BTA) trapped into the focus of a Dual Laser Optical Tweezers (DLOT). In solution with [ATP] = 2 mM the nanomachine is able to produce steady force and shortening, delivering a maximum power of 5 aW. The nanomachine performances are interpreted with a kinetic model based on mechanics and energetics of fast skeletal muscle. In this way it is possible to define the minimal conditions that allow an actomyosin system in vitro to produce force and power with the efficiency of the striated muscle, in the absence of the confusing contribution of the other sarcomeric proteins. In turn, since the system is assembled one piece at a time, it allows different degrees of reconstitution of the sarcomeric assembly. Therefore it will be possible to characterize the function of native and engineered contractile, regulatory and accessory proteins. For future investigations on the Ca2+-dependent thin filament activation, the preparation of BTA has been implemented using a Ca2+-independent gelsolin fragment and the procedure for thin filament reconstitution has been established during my visit to the Institute for Biophysical Chemistry, MHH, Germany.

Almost all animal cells maintain a thin layer of actin filaments and associated proteins underneath the cell membrane. The actomyosin cortex is subject to internal stress patterns which result from the spatiotemporally regulated activity of non-muscle myosin II motors in the actin network. We study how these active stresses drive changes in cell shape and flows within the cortical layer, and how these cytoskeletal deformations and flows govern processes such as cell migration, cell division and organelle transport. Following a continuum mechanics approach, we develop theoretical descriptions for three different cellular processes, to obtain - in collaboration with experimental groups - a detailed and quantitative understanding of the underlying cytoskeletal mechanics. We investigate the forces and cortex flows involved in adhesion-independent cell migration in confinement. Many types of cell migration rely on the extension of protrusions at the leading edge, where the cells attach to the substrate with specific focal adhesions, and pull themselves forward, exerting stresses in the kPa range. In confined environments however, cells exhibit migration modes which are independent of specific adhesions. Combining hydrodynamic theory, microfluidics and quantitative imaging of motile, non-adherent carcinosarcoma cells, we analyze the mechanical behavior of cells during adhesion-independent migration. We find that the accumulation of active myosin motors in the rear part of these cells results in a retrograde cortical flow as well as the contraction of the cell body in the rear and expansion in the front, and we describe how both processes contribute to the translocation of the cells, depending on the geometric and mechanical parameters of the system. Importantly, we find that the involved propulsive forces are several orders of magnitude lower than during adhesive motility while the achieved migration velocities are similar. Moreover, the distribution of forces on the substrate during non-adhesive migration is fundamentally different, giving rise to a positive force dipole. In contrast to adhesive migration modes, non-adhesive cells move by exerting pushing forces at the rear, acting to expand rather than contract their substrate as they move. These differences may strongly affect hydrodynamic and/or deformational interactions between collectively migrating cells. In addition to the work outlined above, we study contractile ring formation in the actin cytoskeleton before and during cell division. While in disordered actin networks, myosin motor activity gives rise to isotropic stresses, the alignment of actin filaments in the cortex during cell division introduces a preferred direction for motor-filament interactions, resulting in anisotropies in the cortical stress. Actin filaments align in myosin-dependent shear flows, resulting in possible feedback between motor activity, cortical flows and actin organization. We investigate how the mechanical interplay of these different cortical properties gives rise to the formation of a cleavage furrow during cell division, describing the level of actin filament alignment at different points on the cortex with a nematic order parameter, in analogy to liquid crystal physics. We show that cortical anisotropies arising from shear-flow induced alignment patterns are sufficient to drive the ingression of cellular furrows, even in the absence of localized biochemical myosin up-regulation. This mechanism explains the characteristic appearance of pseudocleavage furrows in polarizing cells. Finally, we study the characteristic nuclear movements in pseudostratified epithelia during development. These tissues consist of highly proliferative, tightly packed and elongated cells, with nuclei actively travelling to the apical side of the epithelium before each cell division. We explore how cytoskeletal properties act together with the mechanics of the surrounding tissue to control the shape of single cells embedded in the epithelium, and investigate potential mechanisms underlying the observed nuclear movements. These findings form a theoretical basis for a more detailed characterization of processes in pseudostratified epithelia. Taken together, we present a continuum mechanics description of the actomyosin cell cortex, and successfully apply it to several different cell biological processes. Combining our theory with experimental work from collaborating groups, we provide new insights into different aspects of cell mechanics.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by the absence of dystrophin in the muscle cell membranes, rendering them susceptible to mechanical damage. DMD leads to respiratory or cardiac muscle failure and death. We hypothesized that alterations in contractile protein function contribute to DMD muscle weakness. We measured muscle strip stress, myosin heavy chain (MHC) isoform composition, velocity (ʋmax) of actin propulsion by myosin, and relative myosin force in control and mdx mouse (C57Bl/10) diaphragms. Stress was statistically smaller for mdx (0.23kg/cm2±0.11; mean±SE) than control (0.69kg/cm2±0.01) whereas the MHC isoform composition was not statistically different (type I: p=0.423, type IIa/IIx: p=0.804, type IIb: p=0.401). υmax of mdx myosin (1.24µm/s±0.07) was not statistically different from control (1.37µm/s±0.12; p=0.353). Relative myosin force was not statistically different between control and mdx myosin (p=0.932). Thus, alterations in myosin molecular function do not contribute to the weakness of the mdx mouse diaphragm.<br>La dystrophie musculaire de Duchenne (DMD) est une maladie génétique caracterisée par un manque de dystrophine dans la membrane des cellules musculaires, les rendant susceptibles au dommage mécanique. La mort survient suite à l'insuffisance des muscles cardiaques ou respiratoires. Notre hypothèse est que des altérations au niveau des protéines contractiles jouent un rôle dans la faiblesse musculaire de la DMD. Dans cette étude, nous avons mesuré le stress généré par des faisceaux musculaires, la composition des isoformes de la chaîne lourde de myosine (MHC), la vélocité (υmax) de propulsion de l'actine par la myosine, et la force relative de la myosine de diaphragme de souris control et mdx (C57Bl/10). Nous avons observé que le stress est statistiquement plus petit pour la souris mdx (0.23±0.11; moyenne±SE) que pour le control (0.69±0.01), mais que la composition de MHC n'est pas statistiquement différente (type I: p=0.423, type IIa/IIx: p=0.804, type IIb: p=0.401). υmax de la myosine mdx (1.24µm/s±0.07) n'est pas statistiquement différente du control (1.37µm/s±0.12; p=0.353). La force relative n'est pas statistiquement différente entre la myosine control et mdx (p=0.932). Donc des altérations de la fonction moléculaire de la myosine ne contribuent pas à la faiblesse du diaphragme de la souris mdx.

Two smooth muscle (SM) myosin heavy chain isoforms, generated by alternative mRNA splicing, differ by the presence (SM-B) or absence (SM-A) of a 7 amino acid insert in the motor domain. The rate of actin filament propulsion (nu max) of SM-B, as measured in the in vitro motility assay, is 2-fold greater than that of SM-A. I investigated the expression and function of these isoforms in healthy SM and in asthma. First, I determined the sequence of the SM-B isoform in human SM and quantified its expression at the mRNA and protein levels in several human organs. The SM-B isoform was mostly expressed in rapidly contracting phasic SM. I then purified myosin from multiple rat organs and found a rank correlation between SM-B content and numax.<br>I then quantified the expression of SM-B and several other contractile protein genes in endobronchial biopsies from normal and asthmatic subjects. SM-B, myosin light chain kinase (MLCK), which is responsible for myosin activation, and transgelin, a ubiquitously expressed actin binding protein but whose function is unknown, were overexpressed in the asthmatic biopsies. The increased SM-B expression and myosin activation, due to the increased MLCK expression, both contribute to the increased rate of shortening of the asthmatic airway SM. In addition, I showed that beyond its enzymatic effects, MLCK mechanically enhances numax. The binding of SM22 to actin, however, did not alter numax.<br>Finally, I addressed the mechanisms behind the unique capacity of SM to maintain force at low energy cost, namely the latch-state. This property is mostly observed in SM-A containing, tonic muscle. Using a laser trap, I measured the binding force of unphosphorylated (non-active) SM-A and SM-B myosin isoforms and found that they can both attach to actin and maintain force. I also measured numax at different MgADP concentrations and found that SM-A has a greater affinity for MgADP. Because MgADP must be released before myosin can detach from actin, these results suggest that the SMA isoform remains attached longer to actin, allowing it to get into the latch-state. These findings explain the greater propensity of tonic muscle to get into the latch-state.

Thesis (M.S.)--University of California, San Diego, 2009.<br>Title from first page of PDF file (viewed May 4, 2009). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 43-48).

Tendon transfer surgery is used to improve the hand function of patients with nerve injuries, spinal cord lesions, cerebral palsy (CP), stroke, or muscle injuries. The tendon of a muscle, usually with function opposite that of the lost muscle function, is transferred to the tendon of the deficient muscle. The aim is to balance the wrist and fingers to achieve better hand function. The position, function, and length at which the donor muscle is sutured is essential for the outcome for the procedure. In these studies the significance of the transferred muscle’s morphology, length and apillarization was investigated using both animal and human models. Immunohistochemical, biochemical, and laser diffraction techniques were used to examine muscle structure. In animal studies (rabbit), the effects of immobilization and of tendon transfers at different muscle lengths were analyzed. Immobilization of highly stretched muscles resulted in fibrosis and aberrant regeneration. A greater pull on the tendon while suturing a tendon transfer resulted in larger sarcomere lengths as measured in vivo. On examination of the number of sarcomeres per muscle fiber and the sarcomere lengths after 3 weeks of immobilization and healing time, we found a cut-off point up to which the sarcomerogenesis was optimal. Transfer at too long sarcomere lengths inhibited adaptation of the muscle to its new length, probably resulting in diminished function. In human studies we defined the sarcomere lengths of a normal human flexor carpi ulnaris muscle through the range of motion, and then again after a routinely performed tendon transfer to the finger extensor. A calculated model illustrated that after a transfer the largest force was predicted to occur with the wrist in extension. Morphological studies of spastic biceps brachii muscle showed, compared with control muscle, smaller fiber areas and higher variability in fiber size. Similar changes were also found in the more spastic wrist flexors comparing with wrist extensors in children with CP. In flexors, more type 2B fibers were found. These observations could all be due to the decreased use in the spastic limb, but might also represent a specific effect of the spasticity. In children and adults with spasticity very small fibers containing developmental myosin were present in all specimens, while none were found in controls. These fibers probably represent newly formed fibers originating from activated satellite cells. Impaired supraspinal control of active motion as well as of spinal reflexes, both typical of upper motor syndrome, could result in minor eccentric injuries of the muscle, causing activation of satellite cells. Spastic biceps muscles had fewer capillaries per cross-sectional area compared to age-matched controls, and also a smaller number of capillaries around each fiber. Nevertheless, the number of capillaries related to the specific fiber area was normal, and hence the spastic fibers are sufficiently supplied with capillaries. This study shows that the length of the muscle during tendon transfer is crucial for optimization of force output. Laser diffraction can be used for accurate measurement of sarcomere length during tendon transfer surgery. Wrist flexor muscles have more morphological alterations typical of spasticity compared to extensors.

Cette thèse interdisciplinaire a été dédiée à la caractérisation des propriétés mécaniques de myoblastes (murins et humains) et de myotubes (murins) à l'aide de la microscopie à force atomique (AFM). En modifiant ou en inhibant la dynamique du cytosquelette (CSK) d’actine de ces cellules, nous avons pu montrer que ces propriétés mécaniques variaient. L’enregistrement de courbes de force indentation nous a permis de montrer que la présence de cellules adhérentes introduisait sur les leviers d’AFM un amortissement visqueux supplémentaire à celui d’une paroi solide, et que cet amortissement visqueux dépendait de sa vitesse d’approche et que celui-ci restait non négligeable pour les plus faibles vitesses (1μm/s). Nous avons observé que les propriétés mécaniques des précurseurs de muscles devenaient non linéaires (comportement plastiques) pour des grandes déformations (&gt;1μm) et qu’elles dépendaient de l’état, du type de cellule et de leur environnement. En combinant des expériences d’AFM, des modèles visco-élastiques et des méthodes d'analyse multi-échelle basées sur la transformation en ondelettes, nous avons illustré la variabilité des réponses mécaniques de ces cellules (de visco-élastiques à visco-plastiques). À l'aide de courbes de force-indentation, de l’imagerie morpho-structurale (DIC, microscopie à fluorescence) et de traitements pharmacologiques, nous avons éclairé le rôle essentiel des processus actifs (dépendants de l’ATP) dans la mécanique de myoblastes, en discutant tout particulièrement ceux des moteurs moléculaires (myosine II) couplés aux filaments d’actine. En particulier, nous avons montré que les fibres de stress du cytosquelette d’actine situées autour du noyau pouvaient présenter des évènements de remodelage soudains (ruptures) et que ces ruptures étaient une mesure indirecte de l’aptitude de ces cellules à tendre leur CSK. Nous avons enfin montré qu’il était possible de généraliser cette approche à des cas cliniques humains, en l’occurrence des myoblastes primaires de porteurs sains et de patients atteints de dystrophie musculaire de Duchenne, ouvrant la voie à des études plus larges sur d’autres types cellulaires et pathologies<br>This interdisciplinary thesis was dedicated to the atomic force microscopy (AFM) characterization of the mechanical properties of myoblasts (murine and human) and myotubes (murine). We reported that the mechanical properties of these cells were modified when their actin cytoskeleton (CSK) dynamics was inhibited or altered. Recording single AFM force indentation curves, we showed that adherent layers of myoblasts and myotubes introduced on the AFM cantilever an extra hydrodynamic drag as compared to a solid wall. This phenomenon was dependent on the cantilever scan speed and not negligible even at low scan velocities (1μm/s). We observed that the mechanical properties of the muscle precursor cells became non-linear (plastic behaviour) for large local deformations (&gt;1μm) and that they varied depending on the state, type and environment of the cells. Combining AFM experiments, viscoelastic modeling and multi-scale analyzing methods based on the wavelet transform, we illustrated the variability of the mechanical responses of these cells (from viscoelastic to viscoplastic). Through AFM force indentation curves analysis, morpho-structural imaging (DIC, fluorescence microscopy) and pharmacological treatments, we enlightened the important role of active (ATP-dependent) processes in myoblast mechanics, focusing especially on those related to the molecular motors (myosin II) coupled to the actin filaments. In particular, we showed that the perinuclear actin stress fibers could exhibit some abrupt remodelling events (ruptures), which are characteristic of the ability of these cells to tense their CSK. Finally, we showed that this approach can be generalized to some human clinical cases, namely primary human myoblasts from healthy donors and patients affected by Duchenne muscular dystrophy, paving the way for broader studies on different cell types and diseases