Cellular Contractility Research Articles

Synaptopodin couples epithelial contractility to α-actinin-4-dependent junction maturation.

The epithelial junction experiences mechanical force exerted by endogenous actomyosin activities and from interactions with neighboring cells. We hypothesize that tension generated at cell-cell adhesive contacts contributes to the maturation and assembly of the junctional complex. To test our hypothesis, we used a hydraulic apparatus that can apply mechanical force to intercellular junction in a confluent monolayer of cells. We found that mechanical force induces α-actinin-4 and actin accumulation at the cell junction in a time- and tension-dependent manner during junction development. Intercellular tension also induces α-actinin-4-dependent recruitment of vinculin to the cell junction. In addition, we have identified a tension-sensitive upstream regulator of α-actinin-4 as synaptopodin. Synaptopodin forms a complex containing α-actinin-4 and β-catenin and interacts with myosin II, indicating that it can physically link adhesion molecules to the cellular contractile apparatus. Synaptopodin depletion prevents junctional accumulation of α-actinin-4, vinculin, and actin. Knockdown of synaptopodin and α-actinin-4 decreases the strength of cell-cell adhesion, reduces the monolayer permeability barrier, and compromises cellular contractility. Our findings underscore the complexity of junction development and implicate a control process via tension-induced sequential incorporation of junctional components.

Active Biochemical Regulation of Cell Volume and a Simple Model of Cell Tension Response

Active contractile forces exerted by eukaryotic cells play significant roles during embryonic development, tissue formation, and cell motility. At the molecular level, small GTPases in signaling pathways can regulate active cell contraction. Here, starting with mechanical force balance at the cell cortex, and the recent discovery that tension-sensitive membrane channels can catalyze the conversion of the inactive form of Rho to the active form, we show mathematically that this active regulation of cellular contractility together with osmotic regulation can robustly control the cell size and membrane tension against external mechanical or osmotic shocks. We find that the magnitude of active contraction depends on the rate of mechanical pulling, but the cell tension can recover. The model also predicts that the cell exerts stronger contractile forces against a stiffer external environment, and therefore exhibits features of mechanosensation. These results suggest that a simple system for maintaining homeostatic values of cell volume and membrane tension could explain cell tension response and mechanosensation in different environments.

Review of cellular mechanotransduction on micropost substrates.

As physical entities, living cells can sense and respond to various stimulations within and outside the body through cellular mechanotransduction. Any deviation in cellular mechanotransduction will not only undermine the orchestrated regulation of mechanical responses, but also lead to the breakdown of their physiological function. Therefore, a quantitative study of cellular mechanotransduction needs to be conducted both in experiments and in computational simulations to investigate the underlying mechanisms of cellular mechanotransduction. In this review, we present an overview of the current knowledge and significant progress in cellular mechanotransduction via micropost substrates. In the aspect of experimental studies, we summarize significant experimental progress and place an emphasis on the coupled relationship among cellular spreading, focal adhesion and contractility as well as the influence of substrate properties on force-involved cellular behaviors. In the other aspect of computational investigations, we outline a coupled framework including the biochemically motivated stress fiber model and thermodynamically motivated adhesion model and present their predicted biomechanical responses and then compare predicted simulation results with experimental observations to further explore the mechanisms of cellular mechanotransduction. At last, we discuss the future perspectives both in experimental technologies and in computational models, as well as facing challenges in the area of cellular mechanotransduction.

Abstract 313: Examining the role of ABCA1 cholesterol transporter in ovarian cancer spheroids

Abstract Epithelial ovarian cancer (EOC) is a devastating disease which accounts for a large proportion of gynaecological cancer-related deaths. Poor survival is largely due to late diagnosis of the disease which typically presents with peritoneal dissemination. In order to achieve reduced tumor burden and improved survival, it is imperative to identify new prognostic markers and potential therapeutic targets. Recent work by our group identified high ATP- Binding Cassette A1 (ABCA1) transporter expression as being associated with poor outcome in EOC. ABCA1 has been widely studied as a cholesterol transporter, however there is little research investigating its potential roles in cancer biology. The aim of this work was to ascertain whether ABCA1 may promote aggressive characteristics in EOC. siRNA-mediated suppression of ABCA1 in EOC cell lines led to reduced colony forming ability, growth and migration by comparison with controls. By culturing EOC cell lines (Skov3, 27/87 and HEY) in a 3-dimensional spheroid model, we have shown that ABCA1 suppression significantly reduced overall spheroid volume (p = ≤0.0003, ANOVA with Dunnett's multiple comparison test). In Skov3 and 27/87 spheroids, reduced volume was associated with cell death; however in HEY spheroids, ABCA1 suppression significantly reduced cell numbers in the absence of detectable cell death (p = ≤0.0002, ANOVA with Dunnett's multiple comparison test). Detailed characterisation of spheroid architecture revealed an increased volume-to-cell ratio in those depleted of ABCA1, indicating a lower degree of compaction compared to controls. It was further observed that whilst HEY spheroids derived from control siRNA-transfected cells develop a prominent necrotic core this feature was dramatically impaired by ABCA1 suppression. Compaction and core necrosis are both well-documented processes in spheroid biology that are indicative of cellular contractility and a hypoxic tumour environment, both key characteristics of more aggressive, chemo-resistant and recurrent tumors. Collectively these data suggest that ABCA1 may be involved in regulating multiple processes associated with aggressive and invasive characteristics of EOC, highlighting ABCA1 as a potential therapeutic target. Citation Format: Rebekka Williams, Amanda Russell, Angelika Bongers, Sharon Sagnella, Christopher Fife, Wendy Jessup, Anna DeFazio, Georgia Chenevix-Trench, Michelle Haber, Murray Norris, Michelle Henderson. Examining the role of ABCA1 cholesterol transporter in ovarian cancer spheroids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 313. doi:10.1158/1538-7445.AM2015-313

Abstract 339: DWORF: a Novel Cardiac Micropeptide That Enhances SERCA Activity and Cardiomyocyte Contractility

Background: Our lab has discovered a novel conserved micropeptide of 34 amino acids encoded by a muscle-specific RNA that was previously annotated as a putative long non-coding RNA (lncRNA). This micropeptide, which we named DWORF (DWarf Open Reading Frame), shares structural similarity with the known SERCA modulators phospholamban (PLN), sarcolipin (SLN) and myoregulin (MLN). Objective: To define the functional role of DWORF in the heart and elucidate its mechanism of action. Methods and Results: Immunofluorescence staining and imaging in isolated adult mouse cardiac myocytes indicates that DWORF specifically localizes to sarcoplasmic reticulum (SR) membranes where it co-localizes with SERCA. Co-IP experiments performed in COS or HEK cells co-transfected with DWORF and SERCA show that DWORF forms a stable complex with various SERCA isoforms. Further biochemical analysis indicates that DWORF binds to the same site on SERCA as PLN, and overexpression of DWORF is capable of competing with PLN for SERCA binding. Co-expression of GFP-tagged DWORF and PLN in the presence of SERCA followed by SERCA pull-down and GFP Western indicates that PLN and DWORF have similar affinities for SERCA binding. Transgenic mice with cardiac specific over-expression of DWORF show enhanced cellular contractility as evidenced by increased peak Ca2+ transient amplitude and faster cytosolic Ca2+ decay rates (reduced Tau values). Furthermore, Ca2+-dependent Ca2+-uptake assays performed in homogenates from DWORF transgenic and littermate control hearts show a statistically significant leftward shift of the Ca2+-dependence curve for SERCA indicating a higher affinity of SERCA for Ca2+. Intriguingly, DWORF expression is silenced in the heart in response to pathological calcineurin signaling, implicating this micropeptide in the cellular pathway for calcineurin action. Conclusions: We have identified a novel muscle-specific micropeptide, DWORF, which is expressed in the heart at high levels and has the capacity to bind SERCA and modulate its function to increase contractility in transgenic mice. Our findings provide exciting data in support of DWORF as a previously unrecognized endogenous SERCA activator that plays a role in cardiac contractility.

TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility.

Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility.

E-cadherin junctions as active mechanical integrators in tissue dynamics.

During epithelial morphogenesis, E-cadherin adhesive junctions play an important part in mechanically coupling the contractile cortices of cells together, thereby distributing the stresses that drive cell rearrangements at both local and tissue levels. Here we discuss the concept that cellular contractility and E-cadherin-based adhesion are functionally integrated by biomechanical feedback pathways that operate on molecular, cellular and tissue scales.

Enhancer of zeste homolog-2 (EZH2) methyltransferase regulates transgelin/smooth muscle-22α expression in endothelial cells in response to interleukin-1β and transforming growth factor-β2

Smooth muscle-22α (SM22α), encoded by transgelin (TAGLN), is expressed in mesenchymal lineage cells, including myofibroblasts and smooth muscle cells. It is an F-actin binding protein that regulates the organization of actin cytoskeleton, cellular contractility and motility. SM22α is crucial for the maintenance of smooth muscle cell phenotype and its function. SM22α is also expressed in the processes of mesenchymal transition of epithelial (EMT) or endothelial cells (EndMT). The expression of TAGLN/SM22α is induced by transforming growth factor-β (TGFβ) signaling and enhanced by concomitant interleukin-1β (IL-1β) signaling. We investigated the epigenetic regulation of TAGLN expression by enhancer of zeste homolog-2 (EZH2), the methyltransferase of Polycomb, in the context of TGFβ and IL-1β signaling in endothelial cells. We demonstrate that the expression of EZH2 in endothelial cells was regulated by the inflammatory cytokine IL-1β. A decrease in both expression and activity of EZH2 led to an increase in TAGLN expression. Inhibition of EZH2 augmented TGFβ2-induced SM22α expression. The decrease of EZH2 levels in endothelial cells co-stimulated with IL-1β and TGFβ2 correlated with decreased H3K27me3 levels at the TAGLN proximal promoter. Moreover, the SM22α expression increased. Taken together, this suggests that EZH2 regulates the chromatin structure at the TAGLN promoter through tri-methylation of H3K27. EZH2 therefore acts as an epigenetic integrator of IL-1β and TGFβ2 signaling, providing an example of how cellular signaling can be resolved at the level of epigenetic regulation. Since IL-1β and TGFβ2 represent the pro-inflammatory and pro-fibrotic conditions during vascular fibroproliferative disease, we surmise that EZH2, as the molecule that integrates their signaling, could also be a promising target for development of future therapy.

Blood and immune cell engineering: Cytoskeletal contractility and nuclear rheology impact cell lineage and localization: Biophysical regulation of hematopoietic differentiation and trafficking.

Clinical success with human hematopoietic stem cell (HSC) transplantation establishes a paradigm for regenerative therapies with other types of stem cells. However, it remains generally challenging to therapeutically treat tissues after engineering of stem cells in vitro. Recent studies suggest that stem and progenitor cells sense physical features of their niches. Here, we review biophysical contributions to lineage decisions, maturation, and trafficking of blood and immune cells. Polarized cellular contractility and nuclear rheology are separately shown to be functional markers of a hematopoietic hierarchy that predict the ability of a lineage to traffic in and out of the bone marrow niche. These biophysical determinants are regulated by a set of structural molecules, including cytoplasmic myosin-II and nuclear lamins, which themselves are modulated by a diverse range of transcriptional and post-translational mechanisms. Small molecules that target these mechanobiological circuits, along with novel bioengineering methods, could prove broadly useful in programming blood and immune cells for therapies ranging from blood transfusions to immune attack of tumors.

Polychlorinated biphenyl quinone induces endothelial barrier dysregulation by setting the cross talk between VE-cadherin, focal adhesion, and MAPK signaling.

Environmental hazardous material polychlorinated biphenyl (PCB) exposure is associated with vascular endothelial dysfunction, which may increase the risk of cardiovascular diseases and cancer metastasis. Our previous studies illustrated the cytotoxic, antiproliferative, and genotoxic effects of a synthetic, quinone-type, highly reactive metabolite of PCB, 2,3,5-trichloro-6-phenyl-[1,4]benzoquinone (PCB29-pQ). Here, we used it as the model compound to investigate its effects on vascular endothelial integrity and permeability. We demonstrated that noncytotoxic doses of PCB29-pQ induced vascular endothelial (VE)-cadherin junction disassembly by increasing the phosphorylation of VE-cadherin at Y658. We also found that focal adhesion assembly was required for PCB29-pQ-induced junction breakdown. Focal adhesion site-associated actin stress fibers may serve as holding points for cytoskeletal tension to regulate the cellular contractility. PCB29-pQ exposure promoted the association of actin stress fibers with paxillin-containing focal adhesion sites and enlarged the size/number of focal adhesions. In addition, PCB29-pQ treatment induced phosphorylation of paxillin at Y118. By using pharmacological inhibition, we further demonstrated that p38 activation was necessary for paxillin phosphorylation, whereas extracellular signal-regulated kinases-1/2 activation regulated VE-cadherin phosphorylation. In conclusion, these results indicated that PCB29-pQ stimulates endothelial hyperpermeability by mediating VE-cadherin disassembly, junction breakdown, and focal adhesion formation. Intervention strategies targeting focal adhesion and MAPK signaling could be used as therapeutic approaches for preventing adverse cardiovascular health effects induced by environmental toxicants such as PCBs.

Mechanical Stress and Network Structure Drive Protein Dynamics during Cytokinesis

Cell-shape changes associated with processes like cytokinesis and motility proceed on several-second timescales but are derived from molecular events, including protein-protein interactions, filament assembly, and force generation by molecular motors, all of which occur much faster [1-4]. Therefore, defining the dynamics of such molecular machinery is critical for understanding cell-shape regulation. In addition to signaling pathways, mechanical stresses also direct cytoskeletal protein accumulation [5-7]. A myosin-II-based mechanosensory system controls cellular contractility and shape during cytokinesis and under applied stress [6, 8]. In Dictyostelium, this system tunes myosin II accumulation by feedback through the actin network, particularly through the crosslinker cortexillin I. Cortexillin-binding IQGAPs are major regulators of this system. Here, we defined the short timescale dynamics of key cytoskeletal proteins during cytokinesis and under mechanical stress, using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy, to examine the dynamic interplay between these proteins. Equatorially enriched proteins including cortexillin I, IQGAP2, and myosin II recovered much more slowly than actin and polar crosslinkers. The mobility of equatorial proteins was greatly reduced at the furrow compared to the interphase cortex, suggesting their stabilization during cytokinesis. This mobility shift did not arise from a single biochemical event, but rather from a global inhibition of protein dynamics by mechanical-stress-associated changes in the cytoskeletal structure. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to cytokinesis fidelity.

Mechanics of epithelial closure over non-adherent environments.

The closure of gaps within epithelia is crucial to maintain its integrity during biological processes such as wound healing and gastrulation. Depending on the distribution of extracellular matrix, gap closure occurs through assembly of multicellular actin-based contractile cables or protrusive activity of border cells into the gap. Here we show that the supracellular actomyosin contractility of cells near the gap edge exerts sufficient tension on the surrounding tissue to promote closure of non-adherent gaps. Using traction force microscopy, we observe that cell-generated forces on the substrate at the gap edge first point away from the centre of the gap and then increase in the radial direction pointing into the gap as closure proceeds. Combining with numerical simulations, we show that the increase in force relies less on localized purse-string contractility and more on large-scale remodelling of the suspended tissue around the gap. Our results provide a framework for understanding the assembly and the mechanics of cellular contractility at the tissue level.

Activation of the IGF1 pathway mediates changes in cellular contractility and motility in single-suture craniosynostosis.

Insulin growth factor 1 (IGF1) is a major anabolic signal that is essential during skeletal development, cellular adhesion and migration. Recent transcriptomic studies have shown that there is an upregulation in IGF1 expression in calvarial osteoblasts derived from patients with single-suture craniosynostosis (SSC). Upregulation of the IGF1 signaling pathway is known to induce increased expression of a set of osteogenic markers that previously have been shown to be correlated with contractility and migration. Although the IGF1 signaling pathway has been implicated in SSC, a correlation between IGF1, contractility and migration has not yet been investigated. Here, we examined the effect of IGF1 activation in inducing cellular contractility and migration in SSC osteoblasts using micropost arrays and time-lapse microscopy. We observed that the contractile forces and migration speeds of SSC osteoblasts correlated with IGF1 expression. Moreover, both contractility and migration of SSC osteoblasts were directly affected by the interaction of IGF1 with IGF1 receptor (IGF1R). Our results suggest that IGF1 activity can provide valuable insight for phenotype-genotype correlation in SSC osteoblasts and might provide a target for therapeutic intervention.

Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks.

ABSTRACTMutations in the cytoskeletal linker protein plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a plectin-organized vimentin scaffold keeps actomyosin contractility ‘in-check’ and maintains AJ homeostasis.

The Dynamic Interplay between Cleavage Furrow Proteins in Cellular Mechanoresponsiveness

Cell shape changes associated with processes like cytokinesis and motility proceed on several second time-scales. However, they are derived from molecular events occurring much faster, including protein-protein interactions, filament assembly, and force generation. How these fast dynamics define cellular outcomes remain unknown. While accumulation of cytoskeletal elements is often portrayed as being driven by signaling pathways, mechanical stresses also direct proteins. A myosin II-based mechanosensory system controls cellular contractility and shape during cytokinesis and under applied stress. In Dictyostelium, this system tunes myosin II accumulation under mechanical stress by feedback through the actin network, particularly through the crosslinker cortexillin I. Cortexillin-binding IQGAP proteins are major regulators of this system. We examined the dynamic interplay between these key cytoskeletal proteins using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy, defining the short time-scale dynamics of these players during cytokinesis and under mechanical stress. Actin and its polar cortex-enriched crosslinkers showed sub-second recovery, while equatorially enriched proteins including cortexillin I, IQGAP2, and myosin II recovered in 1-5 seconds. Mobility of these equatorial proteins was greatly reduced at the furrow, compared to their interphase dynamics. This mobility shift did not arise from a single biochemical event, but rather from global inhibition of protein dynamics by mechanical stress-associated changes in cytoskeletal structure. We further expanded our genetic and biochemical understanding of this mechanosensory system using a proteomics approach to identify relevant protein-protein interactions. We identified that, in addition to binding to each other, both cortexillin I and IQGAP2 also interact with myosin II under conditions that prevent myosin II-F-actin binding. This validates the high crosstalk occurring between various mechanosensitive elements. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to the fidelity of cytokinesis.

Transient Membrane Localization of SPV-1 Drives Cyclical Actomyosin Contractions in the C. elegans Spermatheca

Actomyosin contractility is the major cellular force driving changes in cell and tissue shape. A principal regulator of contractility is the small GTPase RhoA. External mechanical forces have been shown to impact RhoA activity and cellular contractility. However, the mechanotransduction pathway from external forces to actomyosin contractility is poorly understood. Here, we show that actomyosin contractility in the C. elegans spermatheca is under control of RHO-1/RhoA, which, in turn, is regulated by the F-BAR and RhoGAP protein SPV-1. In the relaxed spermatheca, SPV-1 localizes through its F-BAR domain to the apical membrane, where it inhibits RHO-1/RhoA activity through its RhoGAP domain. Oocyte entry forces the spermatheca cells to stretch, and subsequently SPV-1 detaches from the membrane, permitting RHO-1 activity to increase. The increase in RHO-1 activity facilitates spermatheca contraction and expulsion of the newly fertilized embryo into the uterus, leading to relaxation of the spermatheca, SPV-1 membrane localization, and initiation of a new cycle. Our results demonstrate how transient membrane localization of a novel F-BAR domain, likely via specific binding to curved membranes, coupled to a RhoGAP domain, can provide feedback between a mechanical signal (membrane stretching) and actomyosin contractility. We anticipate this to be a widely utilized feedback mechanism used to balance actomyosin forces in the face of externally applied forces, as well as intrinsic processes involving cell deformation, from single-cell migration to tissue morphogenesis.

NH2-terminal truncations of cardiac troponin I and cardiac troponin T produce distinct effects on contractility and calcium homeostasis in adult cardiomyocytes.

Cardiac troponin I (TnI) has an NH2-terminal extension that is an adult heart-specific regulatory structure. Restrictive proteolytic truncation of the NH2-terminal extension of cardiac TnI occurs in normal hearts and is upregulated in cardiac adaptation to hemodynamic stress or β-adrenergic deficiency. NH2-terminal truncated cardiac TnI (cTnI-ND) alters the conformation of the core structure of cardiac TnI similarly to that produced by PKA phosphorylation of Ser(23/24) in the NH2-terminal extension. At organ level, cTnI-ND enhances ventricular diastolic function. The NH2-terminal region of cardiac troponin T (TnT) is another regulatory structure that can be selectively cleaved via restrictive proteolysis. Structural variations in the NH2-terminal region of TnT also alter the molecular conformation and function. Transgenic mouse hearts expressing NH2-terminal truncated cardiac TnT (cTnT-ND) showed slower contractile velocity to prolong ventricular rapid-ejection time, resulting in higher stroke volume. Our present study compared the effects of cTnI-ND and cTnT-ND in cardiomyocytes isolated from transgenic mice on cellular morphology, contractility, and calcium kinetics. Resting cTnI-ND, but not cTnT-ND, cardiomyocytes had shorter length than wild-type cells with no change in sarcomere length. cTnI-ND, but not cTnT-ND, cardiomyocytes produced higher contractile amplitude and faster shortening and relengthening velocities in the absence of external load than wild-type controls. Although the baseline and peak levels of cytosolic Ca(2+) were not changed, Ca(2+) resequestration was faster in both cTnI-ND and cTnT-ND cardiomyocytes than in wild-type control. The distinct effects of cTnI-ND and cTnT-ND demonstrate their roles in selectively modulating diastolic or systolic functions of the heart.

Systems Perspective on Mechanobiology: Producing the Right Proteins in the Right Place at the Right Time

Systems Perspective on Mechanobiology: Producing the Right Proteins in the Right Place at the Right Time

Abstract 20311: MYBPC3 Mutations Causative for Hypertrophic Cardiomyopathy Result in Locus-Dependent Alterations in Cellular Localization and Contractility

Introduction: Mutations in MYBPC3 resulting in truncated proteins are the most common genetic cause of hypertrophic cardiomyopathy (HCM). Whether lack of sufficient MYBPC3 protein is the primary disease mechanism is controversial. We hypothesize instead that truncated MYBPC3 proteins exert dominant-negative effects on sarcomere structure and function. Methods and Results: MYBPC3 mutations resulting in short (Ile154LeuFs*5, 17 kD) or long (Asp1076Valfs*6 and Trp1098*, each ~120 kD) truncated proteins were studied. For each truncated protein, in silico analysis (I-TASSER) predicted marked deviation from the predicted wild-type (WT) structure. Immunofluorescence of adenoviral-expressed proteins in neonatal rat ventricular cardiomyocytes revealed that WT MYBPC3 was correctly localized at sarcomere A-bands. Near-complete mislocalization (predominantly nuclear) was observed for Ile154LeuFs*5 MYBPC3. Both Asp1076Valfs*6 and Trp1098*MYBPC3 mutant proteins localized imprecisely within the sarcomere, but also were diffusely present in the cytosol. Western blot of myofilament preparations confirmed no sarcomere incorporation of Ile154LeuFs*5, and reduced incorporation of Asp1076Valfs*6 and Trp1098* truncated MYBPC3 (Asp1076Valfs*6 28.1+0.4% and Trp1098* 20.3+0.2% vs. WT 72.4+0.2%, p<0.001). Ile154LeuFs*5 MYBPC3 was exclusively present in the non-myofilament fraction, while Asp1076Valfs*6 and Trp1098* MYBPC3 were 3-fold enriched in this fraction (p=0.01). Analysis of sarcomere shortening (Ionoptix) in adenoviral-infected adult rat ventricular cardiomyocytes indicated reduced peak shortening for Trp1098* (5.30+0.92% vs. WT 8.35+0.49%, p<0.05) and time to peak contraction (43+2 ms vs. WT 67+3 ms, p<0.05). In contrast, no significant effect of Ile154LeuFs*5 MYBPC3 on cellular contractility was observed. Conclusions: Truncating MYBPC3 mutations are predicted to markedly alter protein structure. Longer truncated proteins partially incorporate into the sarcomere lattice and alter contractile function while shorter truncated proteins accumulate in other cellular compartments. These results suggest distinct mutation-specific negative effects of truncating MYBPC3 mutations on myocyte function.

The actin targeting compound Chondramide inhibits breast cancer metastasis via reduction of cellular contractility.

BackgroundA major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent.Methods In vivo, the effect of Chondramide on metastasis was tested employing a 4T1-Luc BALB/c mouse model. In vitro, Chondramide was tested using the highly invasive cancer cell line MDA-MB-231 in Boyden-chamber assays, fluorescent stainings, Western blot and Pull down assays. Finally, the contractility of MDA-MB-231 cells was monitored in 3D environment and analyzed via PIV analysis.Results In vivo, Chondramide treatment inhibits metastasis to the lung and the migration and invasion of MDA-MB-231 cells is reduced by Chondramide in vitro. On the signaling level, RhoA activity is decreased by Chondramide accompanied by reduced MLC-2 and the stretch induced guanine nucleotide exchange factor Vav2 activation. At same conditions, EGF-receptor autophosphorylation, Akt and Erk as well as Rac1 are not affected. Finally, Chondramide treatment disrupted the actin cytoskeleton and decreased the ability of cells for contraction.ConclusionsChondramide inhibits cellular contractility and thus represents a potential inhibitor of tumor cell invasion.