Actin Domains Research Articles

Centrosome-organized plasma membrane infoldings linked to growth of a cortical actin domain.

Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.

HPV disrupt the cytoskeleton in oral squamous cell carcinomas from non-oropharyngeal sites via the E-cadherin/Mena/SMA pathway

In this paper, we aimed to evaluate the mechanism of actin cytoskeleton disruption, in oral squamous cell carcinoma (OSCC). A total of 43 patients with surgically resected OSCCs located in non-oropharyngeal regions were randomly selected. The expression of E-cadherin, β-catenin, smooth muscle actin (SMA), Mena, maspin, V-set and immunoglobulin domain containing 1 (VSIG1), β human chorionic gonadotropin (βhCG), and angiotensin-converting enzyme (ACE) was assessed via immunohistochemistry (IHC) and evaluated in association with the prevalence of high-risk human papillomavirus (HPV). Mena positivity (n = 30; 69.77%) was more frequent in poorly differentiated OSCC of the tongue and lips with high-risk HPV viral DNA and a lymph node ratio (LNR) ≤ 2.5. Loss of E-cadherin was more prevalent among poorly differentiated stage pT4N1 tumors with an LNR ≤ 2.5 and perineural invasion. These cases were classified as SMA-high tumors. Independent negative prognostic factors included high Mena expression, loss of E-cadherin, high SMA expression, and the presence of high-risk HPV. No VSIG1 positivity was observed. In conclusion, in non-oropharyngeal OSCC, cytoskeleton activity might be driven by the Mena/E-cadherin/SMA axis, reflecting active epithelial-mesenchymal interaction. High Mena intensity is an indicator of poorly differentiated carcinomas with high-risk HPV and unfavorable prognosis.

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

The role of myosin loop-4 in localizing tropomyosin on actin during muscle activation and relaxation.

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, namely myosin loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct. In the mutant, the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366 to 372. Cryo-EM analysis of fully-decorated actin-tropomyosin filaments with either wild-type or mutant S1, yielded 3.4- to 3.6-Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum site, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the relatively high potential energy associated with wild-type tropomyosin positioning favors an effective transition to B- and C-states following release of myosin from thin filaments during relaxation.

LincRNA_XR209691.3 could promote Bombyx mori nucleopolyhedrovirus replication by interacting with BmHSP70.

Long non-coding RNAs (lncRNAs), a class of transcripts exceeding 200 nucleotides and lacking protein coding potential, have been proven to play important roles in viral infection and host immunity. Bombyx mori nucleopolyhedrovirus (BmNPV) is an important pathogen, which causes the silkworm disease and leads to a huge challenge to the sericultural industry. At present, research on the roles of insect lncRNAs in host-virus interaction are relatively few. In this study, we explored the function of lincRNA_XR209691.3 that was significantly up-regulated in the silkworm fat body upon BmNPV infection. Firstly, the subcellular localization experiment confirmed that lincRNA_XR209691.3 was present in both the nucleus and cytoplasm. Enhancing the expression of lincRNA_XR209691.3 in BmN cells could promote the proliferation of BmNPV, while inhibition of lincRNA_XR209691.3 by RNA interference suppresses the proliferation of BmNPV. Combining RNA pull-down and mass spectrometry, we identified the host and BmNPV proteins that could interact with lincRNA_XR209691.3. Next, by using truncation experiment and RNA immunoprecipitation (RIP) assay, it was found that lincRNA_XR209691.3 could bind to the Actin domain of BmHSP70. Subsequently, overexpression of lncRNA_XR209691.3 in BmN cells promoted the expression of BmHSP70, while knockdown of BmHsp70 suppressed the replication of BmNPV. Based on the above results, it is speculated that lincRNA_XR209691.3 could promote the proliferation of BmNPV through interaction with BmHSP70, possibly by improving the stability of BmHSP70 and thereby enhancing the expression of BmHSP70. Our results shed light on the lncRNA function in insect-pathogen interactions and provide a new clue to elucidate the molecular mechanism of BmNPV infection.

Myosin loop-4 is critical for optimal tropomyosin repositioning on actin during muscle activation and relaxation.

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, specifically loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct, where the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366-372. Cryo-EM analysis of fully decorated actin-tropomyosin filaments with wild-type and mutant S1, yielded 3.4-3.6 Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other when filaments were decorated with either mutant or wild-type S1. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of filaments containing the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the high potential energy associated with tropomyosin positioning in wild-type filaments favors an effective transition to B- and C-states following release of myosin from the thin filaments during relaxation.

Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: Toward improved gene therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full- length dystrophin. Thus increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin-binding domain1 (ABD1). We engineered a novel fluorescence resonance energy transfer (FRET) biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C- terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, high-throughput screening (HTS) fluorescence lifetime assay, identifying several compounds from a FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.

Studies of functional properties of espin 1: Its interaction to actin filaments.

Actin is a multifunctional biomolecule that forms not only basic structural bodies such as filopodia and lamellipodia, but also large microvilli-like organelles like stereocilia. Actin consists of four sub-domains (S1, S2, S3, and S4), and the "target-binding groove" formed between S1 and S3 is the major binding site for various actin binding proteins. Actin filament dynamics are regulated by numerous actin binding proteins with different mechanisms of actin binding, assembly, and disassembly such as actin severing, branching, and bundling. Ectoplasmic specialization protein 1 (espin 1) is an actin binding and bundling protein that is specifically implicated in the elongation and stabilization of stereocilia as a binding partner with myosin III. However, little is known about the molecular structure, actin bundling, and stabilizing mechanism of espin 1; hence, we investigated the interaction between actin and espin 1 through structural data. In this study, we first purified human espin 1 in an E. coli system following a new detergent-free approach and then demonstrated the 2D structure of full-length espin 1 using transmission electron microscopy along with Nickel nitrilotriacetic acid nanogold labeling and 2D averaging using SPIDER. Furthermore, we also determined the espin 1 binding domain of actin using a co-sedimentation assay along with gelsolin and myosin S1. These findings are not only beneficial for understanding the actin binding and bundling mechanism of espin 1, but also shed light on its elongation, stabilization, and tip-localization mechanisms with myosin III. This study thus provides a basis for understanding the molecular structure of espin 1 and can contribute to various hearing-related diseases, such as hearing loss and vestibular dysfunction.

Is the small heat shock protein HSPB7 (cvHsp) a genuine actin-binding protein?

It is postulated that the small heat shock proteins directly interact with actin, affect formation and stabilize actin filaments. To verify this suggestion, we have analyzed interaction of recombinant human small heat shock protein HspB7 with skeletal muscle actin. In blot overlay HspB7 binds both G- and F-actin. The sites of interaction are located in the C-terminal large core domain of actin. In the course of ultracentrifugation F-actin and F-actin/tropomyosin complexes were pelleted and trapped HspB7. However, HspB7 pelleting was nonspecific and saturation was not achieved even at very high HspB7 concentration. HspB7 was unable to retard or prevent heat-induced F-actin aggregation. Native gel electrophoresis and chemical crosslinking failed to detect interaction of G-actin with HspB7, although both these methods clearly demonstrated formation of complexes formed by G-actin with DNAse I and cofilin-2. It is concluded that HspB7 is not a genuine actin-binding protein and its effect on actin filaments seems to be determined by interaction of HspB7 with minor regulatory proteins of actin filaments.

Nucleation causes an actin network to fragment into multiple high-density domains

Actin networks rely on nucleation mechanisms to generate new filaments because spontaneous nucleation is kinetically disfavored. Branching nucleation of actin filaments by actin-related protein (Arp2/3), in particular, is critical for actin self-organization. In this study, we use the simulation platform for active matter MEDYAN to generate 2000 s long stochastic trajectories of actin networks, under varying Arp2/3 concentrations, in reaction volumes of biologically meaningful size (>20 μm3). We find that the dynamics of Arp2/3 increase the abundance of short filaments and increases network treadmilling rate. By analyzing the density fields of F-actin, we find that at low Arp2/3 concentrations, F-actin is organized into a single connected and contractile domain, while at elevated Arp2/3 levels (10 nM and above), such high-density actin domains fragment into smaller domains spanning a wide range of volumes. These fragmented domains are extremely dynamic, continuously merging and splitting, owing to the high treadmilling rate of the underlying actin network. Treating the domain dynamics as a drift-diffusion process, we find that the fragmented state is stochastically favored, and the network state slowly drifts toward the fragmented state with considerable diffusion (variability) in the number of domains. We suggest that tuning the Arp2/3 concentration enables cells to transition from a globally coherent cytoskeleton, whose response involves the entire cytoplasmic network, to a fragmented cytoskeleton, where domains can respond independently to locally varying signals.

Myofibroblast transdifferentiation is associated with changes in cellular and extracellular vesicle miRNA abundance

Transforming growth factor-beta 1 (TGF-β1), a pro-fibrotic tumour-derived factor promotes fibroblast differentiation in the tumour microenvironment and is thought to contribute to the development of pro-tumourigenic cancer-associated fibroblasts (CAFs) by promoting myofibroblast differentiation. miRNA dysregulation has been demonstrated in myofibroblast transdifferentiation and CAF activation, however, their expression varies among cell types and with the method of fibroblast induction. Here, the expression profile of miRNA in human primary oral fibroblasts treated with TGF-β1, to derive a myofibroblastic, CAF-like phenotype, was determined compared to untreated fibroblasts. Myofibroblast transdifferentiation was determined by the expression of alpha-smooth muscle actin (α-SMA) and fibronectin-1 extra domain A (FN-EDA1) using quantitative real-time PCR (qRT-PCR) and western blot. The formation of stress fibres was assessed by fluorescence microscopy, and associated changes in contractility were assessed using collagen contraction assays. Extracellular vesicles (EVs) were purified by using size exclusion chromatography and ultracentrifugation and their size and concentration were determined by nanoparticle tracking analysis. miRNA expression profiling in oral fibroblasts treated with TGF-β1 and their extracellular vesicles was carried out using tiling low-density array cards. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform functional and pathway enrichment analysis of target genes. In this study, TGF-β1 induced a myofibroblastic phenotype in normal oral fibroblasts as assessed by expression of molecular markers, the formation of stress fibres and increased contractility. TaqMan Low-Density Array (TLDA) analysis demonstrated that miR-503 and miR-708 were significantly upregulated, while miR-1276 was significantly downregulated in TGF-β1-treated oral fibroblasts (henceforth termed experimentally-derived CAF, eCAF). The gene functional enrichment analysis showed that the candidate miRNAs have the potential to modulate various pathways; including the Ras associated protein 1 (Rap1), PI3K-Akt, and tumour necrosis factor (TNF) signalling pathways. In addition, altered levels of several miRNAs were detected in eCAF EV, including miR-142 and miR-222. No differences in size or abundance of EV were detected between eCAF and normal oral fibroblast (NOF). Little overlap was observed between changes in cellular and EV miRNA profiles, suggesting the possibility of selective loading of EV miRNA. The study reveals miRNA expression signature could be involved in myofibroblast transdifferentiation and the miRNA cargo of their EV, providing novel insight into the involvement of miRNA in CAF development and function.

Primate-specific POTE-actin gene could play a role in human folliculogenesis by controlling the proliferation of granulosa cells

Patients with primary ovarian insufficiency (POI) often have a high prevalence of autoimmune disorders. To identify antigenic molecules associated with ovarian autoimmunity, we performed immunoprecipitation (IP) screening using serum from patients with POI and the established human granulosa cell line (HGrC1). POTE ankyrin domain family member E (POTEE) and POTE ankyrin domain family member F (POTEF), proteins specific to primates, were identified as candidate antigens. Using immunohistochemistry (IHC) with human ovarian tissue, POTEE or POTEF was weakly seen in the granulosa cells (GCs) of primordial follicles and primary follicles, and strongly in large antral follicles and luteal cells. Interestingly, no signals were detected in growing GCs in secondary, preantral, and small antral follicles. Thus, to explore the function of POTEE and POTEF in human folliculogenesis, we established HGrC1 cell lines with drug-inducible expression of POTEF. Expression of POTEF significantly suppressed cell proliferation in HGrC1 cells. Furthermore, chaperonin containing TCP-1 complex (CCT) components, which affect folding proteins required for cell proliferation, was bound to the actin domain of POTEF protein. Although CCT is normally localized only around the Golgi apparatus, TCP-1α, a component of CCT, co-migrated closer to the cell membrane when POTEF expression was induced. These data suggest that the interaction between POTEF and CCT components impairs the usual function of CCT during cell growth. In addition, over-accumulation of POTEF in HGrC1 cells leads to autophagic failure. It was recently reported that knockout of an autophagic gene in mice leads to a phenotype similar to human POI. These results suggested that a proper amount of POTEF is required for the maintenance of GCs in follicle pools, whereas POTEF overaccumulation might be involved in follicle atresia and the development of POI. We also showed the possibility that POTEF could be an antigen involved in ovarian autoimmunity.

Molecular Mechanisms of the Deregulation of Muscle Contraction Induced by the R90P Mutation in Tpm3.12 and the Weakening of This Effect by BDM and W7.

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca2+-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin’s ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca2+. These changes in the behavior of tropomyosin and the troponin–tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca2+-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca2+-dependent movement and the ability of the troponin–tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.

Probing the Role of Actin in Membrane Organization

The actin cortex has long been hypothesized to organize membrane components, especially ordered membrane domains thought to play important functional roles at the plasma membrane. Recent developments in super-resolution fluorescence localization microscopy have enabled imaging of the actin cortex in chemically fixed cells through the use of fluorophores that spatially sample filaments at densities required to trace actin meshworks. This poster describes our ongoing efforts to quantify how actin impacts the domain structure of cell plasma membranes, utilizing these actin binding probes in conjunction with well characterized peptide markers of membrane domains. We aim to employ super-resolution imaging to validate past studies, which have characterized meshwork compartment sizes in several cell types based on diffusion data. The majority of our studies are conducted in B cells, where past work indicates that actin structure plays important roles in both antigen mediated B cell receptor signaling and in the tonic signaling that occurs in the absence of ligands. Through this approach, we explore the extent to which these signaling functions are impacted by coupling between actin and membrane domains.

Generation of transgene-free induced pluripotent stem cells from cardiac fibroblasts of goat embryos.

Induced pluripotent stem cells (iPSCs) hold a great potential for therapeutic regenerative medicine. The aim of this study was to generate induced pluripotent stem cells from goat embryonic cardiac tissue derived fibroblasts. The isolated cardiac fibroblasts from the cardiac tissue of goat embryos were positive for alfa smooth muscle actin, vimentin and discoidin domain receptor2. From these cells, we generated transgene free iPSCs using piggyBac transposons / transposase using five transcription factors (Oct4, Sox2, Klf, Myc and Lin 28). The generated iPSCs were SSEA1, SSEA4 and Oct4 positive. They were cultured on neofeeders using 20% Serum replacement - IMDM with bFGF. They could form cystic and compact embryoid bodies that showed differentiated ectodermal and mesodermal like cells when cultured using 20% FBS-IMDM without bFGF. The iPSCs, generated in the frame of this approach were produced without the use of integrating virus and the reprogramming transgenes were removed at the end of the process. Though there were limitations in the approach used, a substantial sign of reprogramming was obtained.

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.

Looking for Targets to Restore the Contractile Function in Congenital Myopathy Caused by Gln147Pro Tropomyosin.

We have used the technique of polarized microfluorimetry to obtain new insight into the pathogenesis of skeletal muscle disease caused by the Gln147Pro substitution in β-tropomyosin (Tpm2.2). The spatial rearrangements of actin, myosin and tropomyosin in the single muscle fiber containing reconstituted thin filaments were studied during simulation of several stages of ATP hydrolysis cycle. The angular orientation of the fluorescence probes bound to tropomyosin was found to be changed by the substitution and was characteristic for a shift of tropomyosin strands closer to the inner actin domains. It was observed both in the absence and in the presence of troponin, Ca2+ and myosin heads at all simulated stages of the ATPase cycle. The mutant showed higher flexibility. Moreover, the Gln147Pro substitution disrupted the myosin-induced displacement of tropomyosin over actin. The irregular positioning of the mutant tropomyosin caused premature activation of actin monomers and a tendency to increase the number of myosin cross-bridges in a state of strong binding with actin at low Ca2+.

Characterization of primary adult mouse cardiac fibroblast cultures.

The role of cardiac fibroblasts (CFs) in disease states has been a focus of cardiovascular research over the past decade. Here, we briefly describe methods for isolation and characterization of CFs from adult mouse ventricles. Primary cultures were stained using antibodies for several marker proteins such as α-smooth muscle actin (αSMA), vimentin, and discoidin domain receptor 2 (DDR2) to confirm the identity of CFs or cardiac myofibroblasts (CMFs). Most cells in primary cultures consisted of CFs, with very low frequencies of endothelial cells, cardiomyocytes, and smooth muscle cells. We compared marker expression between cultures that were not passaged (P0) or passaged for few times (P1-3). When compared with P1-3 cultures, P0 cultures consistently displayed a lower percentage of cells positive for αSMA and DDR2, whereas vimentin expression was significantly higher in P0 cultures compared with P1-3 cultures. P0 cells were also smaller in area than P1-3 cells. Further, P1-3 mouse CFs were found to express both β1 and β2 adrenergic receptors (ARs) and β1ARs were more readily detected on the cell surface compared with β2ARs. In summary, mouse CF cultures underwent phenotype conversion into CMFs after passaging, consistent with what is seen with CF cultures from other species.

Molecular Mechanisms of Muscle Weakness Associated with E173A Mutation in Tpm3.12. Troponin Ca2+ Sensitivity Inhibitor W7 Can Reduce the Damaging Effect of This Mutation.

Substitution of Ala for Glu residue in position 173 of γ-tropomyosin (Tpm3.12) is associated with muscle weakness. Here we observe that this mutation increases myofilament Ca2+-sensitivity and inhibits in vitro actin-activated ATPase activity of myosin subfragment-1 at high Ca2+. In order to determine the critical conformational changes in myosin, actin and tropomyosin caused by the mutation, we used the technique of polarized fluorimetry. It was found that this mutation changes the spatial arrangement of actin monomers and myosin heads, and the position of the mutant tropomyosin on the thin filaments in muscle fibres at various mimicked stages of the ATPase cycle. At low Ca2+ the E173A mutant tropomyosin shifts towards the inner domains of actin at all stages of the cycle, and this is accompanied by an increase in the number of switched-on actin monomers and myosin heads strongly bound to F-actin even at relaxation. Contrarily, at high Ca2+ the amount of the strongly bound myosin heads slightly decreases. These changes in the balance of the strongly bound myosin heads in the ATPase cycle may underlie the occurrence of muscle weakness. W7, an inhibitor of troponin Ca2+-sensitivity, restores the increase in the number of myosin heads strongly bound to F-actin at high Ca2+ and stops their strong binding at relaxation, suggesting the possibility of using Ca2+-desensitizers to reduce the damaging effect of the E173A mutation on muscle fibre contractility.

In silico analysis of PFN1 related to amyotrophic lateral sclerosis.

Profilin 1 (PFN1) protein plays key roles in neuronal growth and differentiation, membrane trafficking, and regulation of the actin cytoskeleton. Four natural variants of PFN1 were described as related to ALS, the most common adult-onset motor neuron disorder. However, the pathological mechanism of PFN1 in ALS is not yet completely understood. The goal of this work is to thoroughly analyze the effects of the ALS-related mutations on PFN1 structure and function using computational simulations. Here, PhD-SNP, PMUT, PolyPhen-2, SIFT, SNAP, SNPS&GO, SAAP, nsSNPAnalyzer, SNPeffect4.0 and I-Mutant2.0 were used to predict the functional and stability effects of PFN1 mutations. ConSurf was used for the evolutionary conservation analysis, and GROMACS was used to perform the MD simulations. The mutations C71G, M114T, and G118V, but not E117G, were predicted as deleterious by most of the functional prediction algorithms that were used. The stability prediction indicated that the ALS-related mutations could destabilize PFN1. The ConSurf analysis indicated that the mutation C71G, M114T, E117G, and G118V occur in highly conserved positions. The MD results indicated that the studied mutations could affect the PFN1 flexibility at the actin and PLP-binding domains, and consequently, their intermolecular interactions. It may be therefore related to the functional impairment of PFN1 upon C71G, M114T, E117G and G118V mutations, and their involvement in ALS development. We also developed a database, SNPMOL (http://www.snpmol.org/), containing the results presented on this paper for biologists and clinicians to exploit PFN1 and its natural variants.