Tropomyosin-mediated Regulation of Cytoplasmic Myosins

The ability of the actin-based cytoskeleton to rapidly reorganize is critical for maintaining cell organization and viability. The plethora of activities in which actin polymers participate require different biophysical properties, which can vary significantly between the different events that often occur simultaneously at separate cellular locations. In order to modify the biophysical properties of an actin polymer for a particular function, the cell contains diverse actin-binding proteins that modulate the growth, regulation and molecular interactions of actin-based structures according to functional requirements. In metazoan and yeast cells, tropomyosin is a key regulator of actin-based structures. Cells have the capacity to produce multiple tropomyosin isoforms, each capable of specifically associating as copolymers with actin at distinct cellular locations to fine-tune the functional properties of discrete actin structures. Here, we present a unifying theory in which tropomyosin isoforms critically define the surface landscape of copolymers with cytoplasmic ?- or ?-actin. Decoration of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins. Conversely, changes in the nucleotide state of actin and posttranslational modifications affect the composition, morphology, subcellular localization and allosteric coupling of the associated actin-based superstructures

Identification and characterization of an oncogenically modified actin associated protein

The actin cytoskeleton is crucial for a variety of cellular events such as cell locomotion, phagocytosis, cytokinesis and cell surface receptor movement. The control mechanisms for these actin based cellular events are provided by a large number of actin-associated proteins which, acting in concert, regulate the polymerization status, interactions and geometry of actin. I have characterized a new actin associated 21 kDa polypeptide doublet, protein C4. In normal mesenchymal cells, protein C4 is associated with and uniformly distributed on actin stress fibres, while in cells and tissues that lack stress fibres, C41 is the only protein C4 isoform present. Protein C4 is present in all cells and tissues so far examined apart from neurones, erythrocytes and skeletal muscle. Protein C4 is evolutionarily conserved as it has been found in yeast. A number of actin associated proteins are down regulated in transformed cells in parallel with the reorganization of the actin cytoskeleton that often accompanies transformation. I have shown that the higher Mr protein C4 isoform, transgelin, is absent in oncogenically transformed cells where actin stress fibres are reduced in number or absent, while in contrast, the lower Mr protein C4 isoform, C41, is always present. Expression of transgelin can also be blocked by culturing normal, non-transformed mesenchymal cells in suspension. Re-expression of transgelin occurs 24 hours after these cells are returned to normal adherent culture conditions, but can be blocked by either actinomycin D or cycloheximide, suggesting that the expression of transgelin is regulated at the transcriptional level. I have purified transgelin and shown that it binds directly to actin filaments at a ratio of 1:6 actin monomers, with a binding constant (Ka) of 7.5 x 105M-1 and that it induces actin filament gelation within 2 minutes, without affecting actin polymerization. Although both actin binding and gelation activities of transgelin are controlled by ionic strength, and may be mediated by positively charged amino acid residues, the molecule remains as a monomer irrespective of ionic conditions. Electron microscopy reveals that transgelin converts actin filaments from a loose random distribution into tightly tangled aggregates. An 'add-back' permeabilization system shows that transgelin specifically rebinds to actin filaments in cells from which it has previously been removed by detergent extraction

Comparative Proteomics: Assessing the Variation in Molecular Physiology Within the Adductor Muscle Between \u3ci\u3eMytilus galloprovincialis\u3c/i\u3e and \u3ci\u3eMytilus trossulus\u3c/i\u3e in Response to Acute Heat Stress

Increases in seawater temperatures have imposed physiological constraints which are partially thought to contribute to recently observed shifts in biogeographic distribution among closely related intertidal ectotherms. For instance, Mytilus galloprovincialis an introduced warm-adapted species from the Mediterranean, has displaced the native cold-adapted congener, M. trossulus, over large latitudinal expanses off the California coast. Several comparative physiological studies have revealed interspecific differences in thermal tolerance, including variation in aerobic metabolism and gape behavior, which suggest the invasive congener is better adapted to acclimate to increasing seawater conditions as predicted due to climate change. However, current analyses seek to discover the cellular process which contribute to thermal plasticity at the level of the whole organism in response to temperature stress. Since proteins represent the primary molecular machinery capable of responding to thermal stress, we quantified the proteomic response of the adductor muscles (AM) of M. galloprovincialis and M. trossulus to acute heat stress. After acclimation to 13°C, we exposed mussels to 24°C, 28°C and 32 °C (at a heating rate of 6C/h), kept mussels at the temperature for 1 h and then added a 24-h recovery period. Posterior adductor muscle samples were then excised and utilized for proteomic analysis. We were able to detect 273 protein spots within M. galloprovincialis and 286 protein spots within M. trossulus. Roughly 33% of these protein spots exhibited significant changes in abundance in response to heat stress within M. trossulus as compared to only 19% in M. galloprovincialis. In both data sets, most proteins changing abundance are part of the cytoskeleton or proteins controlling actin thin filament dynamics and stress fiber formation. Specifically, M. galloprovincialis increased the abundance of proteins involved in thin filament stabilization and cytoskeletal maintenance. In contrast, M. trossulus increased proteins involved in thin filament destabilization and filament turnover. In addition, only M. trossulus increased proteins involved in the cellular stress response at the highest temperature, suggesting its AM proteome is more thermolabile. In return, our results suggest that cytoskeletal architecture is more thermally stable in M. galloprovincialis. The differences in the proteomic responses suggest that M. galloprovincialis is capable of protecting itself from heat stress through valve closure at a higher temperature due to the increase in actin stabilizing proteins

Calponin And Cytoskeleton Dynamics In Macrophage Functions And The Pathogenesis Of Atherosclerosis

Arterial atherosclerosis is an inflammatory disease. Macrophages play a major role in the pathogenesis and progression of atherosclerotic lesions. Modulation of macrophage function is a therapeutic target for the treatment of atherosclerosis. Calponin is an actin-filament-associated regulatory protein that inhibits the activity of myosin-ATPase and dynamics of the actin cytoskeleton. Encoded by the Cnn2 gene, calponin isoform 2 is expressed at significant levels in macrophages. Deletion of calponin 2 increases macrophage migration and phagocytosis. In the present study, we investigated the effect of deletion of calponin 2 in macrophages on the pathogenesis and development of atherosclerosis. The results showed that macrophages isolated from Cnn2 knockout mice ingested the same level of acetylated low-density lipoprotein (LDL) as that of wild type (WT) macrophages but the resulting foam cells had significantly less impaired velocity of migration. Systemic or myeloid cell-specific Cnn2 knockouts effectively attenuated the development of arterial atherosclerosis lesions with less macrophage infiltration in apolipoprotein E knockout mice. Consistently, calponin 2-null macrophages produced less pro-inflammatory cytokines than that of WT macrophages, and the up-regulation of pro-inflammatory cytokines in foam cells was also attenuated by the deletion of calponin 2. Calponin 2-null macrophages and foam cells have significantly weakened cell adhesion, indicating a role of cytoskeleton regulation in macrophage functions and inflammatory responses, and a novel therapeutic target for the treatment of arterial atherosclerosis

Association of Smooth Muscle Myosin and its Carboxyl Isoforms with Actin Isoforms in Aorta Smooth Muscle

The contraction mechanism of smooth muscle is not fully understood. The primary interaction that leads to the formation of tension, the myosin-actin crossbridge, has been studied extensively. However, even this aspect of the contraction has proven not to be as simple as it might seem. There are several isoforms of smooth muscle myosin and actin, and the differences in the activities of these isoforms and their interactions during the contractile process are largely unknown. The studies to be discussed are directed at the determination of the interaction of these isoforms during the contraction of rat aortic smooth muscle. Chapter II describes the association of smooth muscle myosin with two of the actin isoforms found in smooth muscle, α-actin and β-actin, using a novel method of fluorescence resonance energy transfer (FRET) to examine this association in both the A7r5 cell model and in intact tissue. We show that the contractile apparatus undergoes significant remodeling during contraction and that the interaction of myosin with α-actin and β-actin is different at the various time points of contraction. In Chapter III, we describe more detailed experiments examining the two different myosin tail isoforms, SM1 and SM2. The results of these studies confirm our findings of remodeling of the cytoskeleton and the contractile apparatus during contraction and show that α-actin and β-actin interact differently with these myosin isoforms. The results provide the first direct evidence of contractile remodeling in smooth muscle and suggest that complex changes in actin-myosin interaction may be important in the contraction of this muscle type

Actin Dynamics in Muscle Cells

In every cell, actin is a key component involved in migration, cytokinesis, endocytosis and generation of contraction. In non-muscle cells, actin filaments are very dynamic and regulated by an array of proteins that interact with actin filaments and/or monomeric actin. Interestingly, in non-muscle cells the barbed ends of the filaments are the predominant assembly place, whereas in muscle cells actin dynamics was reported to predominate at the pointed ends of thin filaments. The actin-based thin filament pointed (slow growing) ends extend towards the middle of the sarcomere's M-line where they interact with the thick filaments to generate contraction. The actin filaments in muscle cells are organized into a nearly crystalline array and are believed to be significantly less dynamic than the ones in other cell types. However, the exact mechanisms of the sarcomere assembly and turnover are largely unknown. Interestingly, although sarcomeric actin structures are believed to be relatively non-dynamic, many proteins promoting actin dynamics are expressed also in muscle cells (e.g ADF/cofilin, cyclase-associated protein and twinfilin). Thus, it is possible that the muscle-specific isoforms of these proteins promote actin dynamics differently from their non-muscle counterparts, or that actin filaments in muscle cells are more dynamic than previously thought. To study protein dynamics in live muscle cells, I used primary cell cultures of rat cardiomyocytes. My studies revealed that a subset of actin filaments in cardiomyocyte sarcomeres displays rapid turnover. Importantly, I discovered that the turnover of actin filaments depends on contractility of the cardiomyocytes and that the contractility-induced actin dynamics plays an important role in sarcomere maturation. Together with previous studies those findings suggest that sarcomeres undergo two types of actin dynamics: (1) contractility-dependent turnover of whole filaments and (2) regulatory pointed end monomer exchange to maintain correct thin filament length. Studies involving an actin polymerization inhibitor suggest that the dynamic actin filament pool identified here is composed of filaments that do not contribute to contractility. Additionally, I provided evidence that ADF/cofilins, together with myosin-induced contractility, are required to disassemble non-productive filaments in developing cardiomyocytes. In addition, during these studies we learned that isoforms of actin monomer binding protein twinfilin, Twf-1 and Twf-2a localise to myofibrils in cardiomyocytes and may thus contribute to actin dynamics in myofibrils. Finally, in collaboration with Roberto Dominguez s laboratory we characterized a new actin nucleator in muscle cells - leiomodin (Lmod). Lmod localises towards actin filament pointed ends and its depletion by siRNA leads to severe sarcomere abnormalities in cardiomyocytes. The actin filament nucleation activity of Lmod is enhanced by interactions with tropomyosin. We also revealed that Lmod expression correlates with the maturation of myofibrils, and that it associates with sarcomeres only at relatively late stages of myofibrillogenesis. Thus, Lmod is unlikely to play an important role in myofibril formation, but rather might be involved in the second step of the filament arrangement and/or maintenance through its ability to promote tropomyosin-induced actin filament nucleation occurring at the filament pointed ends. The results of these studies provide valuable new information about the molecular mechanisms underlying muscle sarcomere assembly and turnover. These data offer important clues to understanding certain physiological and pathological behaviours of muscle cells. Better understanding of the processes occurring in muscles might help to find strategies for determining, diagnosis, prognosis and therapy in heart and skeletal muscles diseases.Human heart cells - cardiomoycytes - are constantly contracting cells; about three billion times during an average lifespan - to pump in average 7000 litres of blood per day. To ensure its proper function the multiprotein cytoskeletal complexes (myofibrils) have to be assembled and maintained correctly. The myofibrils consist of set of filaments organized in a paracrystal structure - sarcomere - to ensure maximal contractile force. Sarcomeres are composed by actin and myosin filaments. Their proper interaction is regulated and accompany by vary type of supporting proteins. The actin filaments in muscle cells are organized into a nearly crystalline array and are believed to be significantly less dynamic than the ones in other cell types (eg. skin cells). However, the exact mechanisms of the sarcomere assembly and turnover are largely unknown. This study revealed that two types of actin dynamics exist in muscle cells. First, actin filaments undergo slow dynamics at their ends to maintain the correct length of the filaments. Secondly, entire actin filaments are replaced by new ones during sarcomere assembly and maintenance, and this phenomenon is dependent on the contractility of the muscle cells. Finally, we characterized a new actin-binding protein, Lmod, which nucleates the formation of new actin filaments in muscle cells. These data provide valuable new information about the molecular mechanisms underlying muscle sarcomere assembly and turnover. These data offer important clues to understanding certain physiological and pathological behaviours of muscle cells. Better understanding of the processes occurring in muscles might help to find strategies for determining, diagnosis, prognosis and therapy in heart and skeletal muscles diseases

The molecular phenotype of human cardiac myosin associated with hypertrophic obstructive cardiomyopathy

AIM: The aim of the study was to compare the functional and structural properties of the motor protein, myosin, and isolated myocyte contractility in heart muscle excised from hypertrophic cardiomyopathy patients by surgical myectomy with explanted failing heart and non-failing donor heart muscle. METHODS: Myosin was isolated and studied using an in vitro motility assay. The distribution of myosin light chain-1 isoforms was measured by two-dimensional electrophoresis. Myosin light chain-2 phosphorylation was measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using Pro-Q Diamond phosphoprotein stain. RESULTS: The fraction of actin filaments moving when powered by myectomy myosin was 21% less than with donor myosin (P = 0.006), whereas the sliding speed was not different (0.310 +/- 0.034 for myectomy myosin vs. 0.305 +/- 0.019 microm/s for donor myosin in six paired experiments). Failing heart myosin showed 18% reduced motility. One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M). In myectomy myosin, the level of atrial light chain-1 relative to ventricular light chain-1 was 20 +/- 5% compared with 11 +/- 5% in donor heart myosin and the level of myosin light chain-2 phosphorylation was decreased by 30-45%. Isolated cardiomyocytes showed reduced contraction amplitude (1.61 +/- 0.25 vs. 3.58 +/- 0.40%) and reduced relaxation rates compared with donor myocytes (TT(50%) = 0.32 +/- 0.09 vs. 0.17 +/- 0.02 s). CONCLUSION: Contractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation. The presence of a myosin heavy chain mutation causing hypertrophic cardiomyopathy can be predicted from a simple functional assay

Regulation of mechanics and dynamics of actin filaments and networks by actin-binding proteins

Actin is a highly ubiquitous and evolutionarily conserved protein capable of polymerizing and forming filamentous polymers which play a central role in cell mechanics and motility. Here, we study the in vitro regulation of actin mechanics and dynamics by calponin and caldesmon, two actin binding proteins believed to be involved in regulating cytoskeletal mechanics and structure through mechanisms not currently well understood. Chapters 1 and 2 introduce the reader to actin and its roles in the cell, as well as to the methods and theoretical foundations used in this work. In Chapter 3, we use total internal reflection and confocal fluorescence microscopy to investigate the polymerization dynamics of actin in the presence of a caldesmon C terminal fragment, H32K. We show that H32K stabilizes a nascent structural state of actin without altering the polymerization dynamics of the filament. We also show that H32K stabilized nascent actin has increased affinity for the actin branching protein complex Arp2/3 involved in driving membrane protrusions during cell motility, and propose the nascent state of actin as a possible transient differentiator targeting certain actin binding proteins to actin in vivo. This is to our knowledge the first reported direct functional effect of nascent actin. In Chapter 4, we use fluorescence microscopy to quantify actin bending mechanics in the presence of the binding protein calponin and show that calponin reduces the persistence length of actin. We compare our results to the literature and compare the mechanical change to electron microscopy reconstructions, which suggest that calponin affects actin intermonomer contacts through interactions with actin subdomain 2. In Chapter 5, we expand on the results from Chapter 4 using bulk rheology and show that calponin increases the tensile strength of reconstituted actin networks, similar to the effect seen in whole cells and tissues. We discuss these data within an affine network model and show that the results can be entirely explained in terms of the reduced actin persistence length. We use this to propose a novel physical mechanism for calponin function in vivo. This work elucidates the physical mechanisms of calponin and caldesmon function and their role in regulating the cellular cytoskeleton.2031-01-01T00:00:00

Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms

Cardiac hypertrophy is an adaptive response that normalizes wall stress and compensates for increased workload. It is accompanied by distinct qualitative and quantitative changes in the expression of protein isoforms concerning contractility, intracellular Ca2+-homeostasis and metabolism. Changes in the myosin subunit isoform expression improves contractility by an increase in force generation at a given Ca2+-concentration (increased Ca2+-sensitivity) and by improving the economy of the chemo-mechanical transduction process per amount of utilised ATP (increased duty ratio). In the human atrium this is achieved by partial replacement of the endogenous fast myosin by the ventricular slow-type heavy and light chains. In the hypertrophic human ventricle the slow-type β-myosin heavy chains remain unchanged, but the ectopic expression of the atrial myosin essential light chain (ALC1) partially replaces the endogenous ventricular isoform (VLC1). The ventricular contractile apparatus with myosin containing ALC1 is characterised by faster cross-bridge kinetics, a higher Ca2+-sensitivity of force generation and an increased duty ratio. The mechanism for cross-bridge modulation relies on the extended Ala-Pro-rich N-terminus of the essential light chains of which the first eleven residues interact with the C-terminus of actin. A change in charge in this region between ALC1 and VLC1 explains their functional difference. The intracellular Ca2+-handling may be impaired in heart failure, resulting in either higher or lower cytosolic Ca2+-levels. Thus the state of the cardiomyocyte determines whether this hypertrophic adaptation remains beneficial or becomes detrimental during failure. Also discussed are the effects on contractility of long-term changes in isoform expression of other sarcomeric proteins. Positive and negative modulation of contractility by short-term phosphorylation reactions at multiple sites in the myosin regulatory light chain, troponin-I, troponin-T, α-tropomyosin and myosin binding protein-C are considered in detai

Cellular mechanisms in sympatho‐modulation of the heart

Cardiovascular function relies on complex servo-controlled regulation mechanisms that involve both fast-acting feedback responses and long-lasting adaptations affecting the gene expression. The adrenergic system, with its specific receptor subtypes and intracellular signalling cascades provides the major regulatory system, while the parasympathetic system plays a minor role. At the molecular level, Ca2+ acts as the general signal trigger for the majority of cell activities including contraction, metabolism and growth. During recent years, important new results have emerged allowing an integrated view of how the multifarious Ca2+-signalling mechanisms transmit adrenergic impulses to intracellular target sites. These insights into cellular and molecular mechanisms are pivotal in improving pharmacological control of the sympathetic responses to surgical trauma and perioperative stress. They are examined in detail in this review, with particular emphasis being given to the differences in intracellular signalling between cardiomyocytes and vascular smooth muscle cell