Differential fluorescence nanoparticle tracking analysis for enumeration of the extracellular vesicle content in mixed particulate solutions.

BACKGROUND: A major concern for the extracellular vesicle (EV) field is the current lack of accurate methods for EV quantification. Total protein measurement fails to reliably quantify EVs from serum-containing conditioned media and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate between membrane-bounded EVs, lipids and protein aggregates. However, EVs can be fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. Fluorescence-based NTA (F-NTA) is thus emerging as a method for counting and phenotyping of EVs. We have validated a differential NTA/F-NTA method using specific antibodies against surface markers in analogy to flow cytometric analyses. METHODS: EVs from umbilical cord mesenchymal stromal cells (UC-MSCs) were isolated by a combined tangential flow filtration and ultracentrifugation protocol. EV preparations from 2 × 107 cells were stained with AlexaFluor 488-conjugated specific antibodies or corresponding isotype controls. Amount and size of particles in normal scattering light mode (N mode) versus fluorescence mode (F mode, laser wavelength 488 nm) was measured using ZetaView Nanoparticle Tracking Analyzer (Particle Metrix). Cryo electron microscopy (EM) was used to verify the presence of membrane bilayer surrounded nanoparticles. RESULTS: All UC-MSC-EV preparations were found positive for typical EV marker proteins and negative for MHC class I. Novel and improved devices that include more sensitive cameras for detection in the fluorescent mode further increase the detection limit. CONCLUSION: Differential NTA/F-NTA facilitates determination of the percentage of EV marker protein-positive nanoparticles within a mixed particulate solution. The set of markers can be extended to other MSC-EV positive and negative surface proteins in order to establish F-NTA-based profiling as a supporting method for the quantification of EVs.

Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions.

Actin polymerization is accompanied by the formation of protein complexes that link extracellular signals to sites of actin assembly such as membrane ruffles and focal adhesions. One candidate recently implicated in these processes is the LIM domain protein zyxin, which can bind both Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the actin filament cross-linking protein alpha-actinin. To characterize the localization and dynamics of zyxin in detail, we generated both monoclonal antibodies and a green fluorescent protein (GFP)-fusion construct. The antibodies colocalized with ectopically expressed GFP-VASP at focal adhesions and along stress fibers, but failed to label lamellipodial and filopodial tips, which also recruit Ena/VASP proteins. Likewise, neither microinjected, fluorescently labeled zyxin antibodies nor ectopically expressed GFP-zyxin were recruited to these latter sites in live cells, whereas both probes incorporated into focal adhesions and stress fibers. Comparing the dynamics of zyxin with that of the focal adhesion protein vinculin revealed that both proteins incorporated simultaneously into newly formed adhesions. However, during spontaneous or induced focal adhesion disassembly, zyxin delocalization preceded that of either vinculin or paxillin. Together, these data identify zyxin as an early target for signals leading to adhesion disassembly, but exclude its role in recruiting Ena/VASP proteins to the tips of lamellipodia and filopodia.

GEF at work: Vav in protruding filopodia.

The Dbl family proto-oncogene vav is a nucleotide exchange factor for Rho family GTPases and is involved in triggering cytoskeletal changes contributing to the alterations of cell shape and motility, as well as in the induction of gene expression. In vitro and in vivo Vav is regulated by multiple tyrosine phosphorylation and binding to phosphatidylinositol phosphates. Although recruitment of Vav to the plasma membrane appears important for the activation of Vav function, there is little information on the precise subcellular localization of Vav in living cells. Employing live video fluorescence and immunoelectron microscopy, we show that GFP-tagged full-length Vav, and several mutants in which the N-terminal regulatory calponin homology (CH) domain has been deleted, specifically localize to the tips of filopodia. This localization was congruent with a high content of tyrosine phosphorylation in these regions. Consistent with earlier observations, mutants lacking the C-terminal SH domain region were unable to translocate to the filopodia tips. The enrichment in filopodial tips persisted despite their lateral movement but was dependent on forward growth. Upon retraction, the signal was rapidly lost, indicating that Vav undergoes a specific and transient translocation in response to actin-based, protrusive events in filopodia.

UNC-87 is an actin-bundling protein.

The Caenorhabditis elegans unc-87 gene product is essential for the maintenance of the nematode body wall muscle where it is found colocalized with actin in the I band. The molecular domain structure of the protein reveals similarity to the C-terminal repeat region of the smooth muscle actin-binding protein calponin. In this study we investigated the in vitro function of UNC-87 using both the full-length recombinant molecule and several truncated mutants. According to analytical ultracentrifugation UNC-87 occurs as a monomer in solution. UNC-87 cosedimented with both smooth and skeletal muscle F-actin, but not with monomeric G-actin, and exhibited potent actin filament bundling activity. Actin binding was independent of the presence of tropomyosin and the actin cross-linking proteins filamin and alpha-actinin. Consistent with its actin bundling activity in vitro, UNC-87 tagged with green fluorescent protein associated with and promoted the formation of actin stress fiber bundles in living cells. These data identify UNC-87 as an actin-bundling protein and highlight the calponin-like repeats as a novel actin-binding module.

Live dynamics of GFP-calponin: isoform-specific modulation of the actin cytoskeleton and autoregulation by C-terminal sequences.

The calponin family of F-actin-, tropomyosin- and calmodulin-binding proteins currently comprises three genetic variants. Their functional roles implicated from in vitro studies include the regulation of actomyosin interactions in smooth muscle cells (h1 calponin), cytoskeletal organisation in non-muscle cells (h2 calponin) and the control of neurite outgrowth (acidic calponin). We have now investigated the effects of calponin (CaP) isoforms and their C-terminal deletion mutants on the actin cytoskeleton by time lapse video microscopy of GFP fusion proteins in living smooth muscle cells and fibroblasts. It is shown that h1 CaP associates with the actin stress fibers in the more central part of the cell, whereas h2 CaP localizes to the ends of stress fibres and in the motile lamellipodial protrusions of spreading cells. Cells expressing h2 CaP spread more efficiently than those expressing h1 CaP and expression of GFP h1 CaP resulted in reduced cell motility in wound healing experiments. Notably, expression of GFP h1 CaP, but not GFP h2 CaP, conferred increased resistance of the actin cytoskeleton to the actin polymerization antagonists cytochalasin B and latrunculin B, as well as to the protein kinase inhibitors H7-dihydrochloride and rho-kinase inhibitor Y-27632. These data point towards a dual role of CaP in the stabilization and regulation of the actin cytoskeleton in vivo. Deletion studies further identify an autoregulatory role for the unique C-terminal tail sequences in the respective CaP isoforms.

Preliminary crystallographic study of turkey gizzard vinculin.

Vinculin is a 117 kDa microfilament-associated protein located at the cytoplasmic aspects of focal contacts and cell-cell adherens type junctions. In both sites, vinculin participates in the formation of a submembrane 'plaque' structure which is responsible for the attachment of actin filaments to the plasma membrane. Vinculin consists of 1066 amino acids, which form a large 90 kDa globular head domain and a rod-like 29 kDa tail domain. The two domains are separated by several stretches of proline residues where the major proteolytic cleavage sites are located. The experimental procedure for isolation and purification of vinculin from smooth muscle has been developed and crystals of native vinculin suitable for X-ray analysis have been obtained. The homogeneity of the vinculin solution was analyzed prior to crystallization using dynamic light scattering. Crystals of vinculin have been obtained in buffer containing 2 mg ml(-1) protein, 0.9 M ammonium sulfate, 0.1 M MES pH 6.5 using both the hanging-drop and sitting-drop vapour-diffusion methods. The crystals have the form of rhombic plates and grow to maximal dimensions of 0.3 x 0.3 x 0.05 mm in two weeks. Preliminary X-ray data show that the crystals diffract to 3.5 A resolution at the X11 beamline of DESY and belong to the monoclinic space group P2(1). Crystal unit-cell parameters are estimated to be a = 57, b = 351, c = 70 A, alpha = 90, beta = 113, gamma = 90 degrees.

Enforced polarisation and locomotion of fibroblasts lacking microtubules.

The polarisation and locomotion of fibroblasts requires an intact microtubule cytoskeleton [1]. This has been attributed to an influence of microtubule-mediated signals on actin cytoskeleton dynamics, either through the generation of active Rac to promote protrusion of lamellipodia [2], or through the modulation of substrate adhesion via microtubule targeting events [3] [4]. We show here that the polarizing role of microtubules can be mimicked by externally imposing an asymmetric gradient of contractility by local application of the contractility inhibitor ML-7. Apolar fibroblasts lacking microtubules could be induced to polarize and to move by application of ML-7 by micropipette to one side of the cell and then to the trailing vertices that developed. The release and retraction of trailing adhesions could be correlated with a relaxation of traction on the substrate and a differential shortening of stress-fibre bundles, with their distal tips relaxed. Although retraction and protrusion in these conditions resembled control cell locomotion, the normal turnover of adhesion sites that form behind the protruding cell front was blocked. These findings show that microtubules are dispensable for fibroblast protrusion, but are required for the turnover of substrate adhesions that normally occurs during cell locomotion. We conclude that regional contractility is modulated by the interfacing of microtubule-linked events with focal adhesions and that microtubules determine cell polarity via this route.

Mapping the zinc ligands of S100A2 by site-directed mutagenesis.

S100 family proteins are characterized by short individual N and C termini and a conserved central part, harboring two Ca(2+)-binding EF-hands, one of them highly conserved among EF-hand family proteins and the other characteristic for S100 proteins. In addition to Ca(2+), several members of the S100 protein family, including S100A2, bind Zn(2+). Two regions in the amino acid sequences of S100 proteins, namely the helices of the N-terminal EF-hand motif and the very C-terminal loop are believed to be involved in Zn(2+)-binding due to the presence of histidine and/or cysteine residues. Human S100A2 contains four cysteine residues, each of them located at positions that may be important for Zn(2+) binding. We have now constructed and purified 10 cysteine-deficient mutants of human S100A2 by site-directed mutagenesis and investigated the contribution of the individual cysteine residues to Zn(2+) binding. Here we show that Cys(1(3)) (the number in parentheses indicating the position in the sequence of S100A2) is the crucial determinant for Zn(2+) binding in association with conformational changes as determined by internal tyrosine fluorescence. Solid phase Zn(2+) binding assays also revealed that the C-terminal residues Cys(3(87)) and Cys(4(94)) mediated a second type of Zn(2+) binding, not associated with detectable conformational changes in the molecule. Cys(2(22)), by contrast, which is located within the first EF hand motif affected neither Ca(2+) nor Zn(2+) binding, and a Cys "null" mutant was entirely incapable of ligating Zn(2+). These results provide new information about the mechanism and the site(s) of zinc binding in S100A2.

Ca(2+)-dependent association of S100A6 (Calcyclin) with the plasma membrane and the nuclear envelope.

Expression of S100A6 (Calcyclin), a member of the S100 family and of Zn(2+)-binding proteins is elevated in a number of malignant tumors. In vitro the protein associates with several actin-binding proteins and annexins in a Ca(2+)-dependent manner. We have now studied the subcellular localization of S100A6 using a new, specific monoclonal antibody. Immunofluorescence microscopy of unfixed, ultrathin, frozen sections demonstrated a dual localization of S100A6 at the nuclear envelope and the plasma membrane of porcine smooth muscle only in the presence of Ca(2+). The same localization was found by immunofluorescence and immunogold electron microscopy as well as by confocal laser scanning microscopy with cultured, fixed, human CaKi-2 and porcine ST interphase cells. Upon cell division, however, S100A6 was found exclusively in the cytoplasm. Cell fractionation studies showed that S100A6 was present in the microsomal fraction in the presence of Ca(2+) and was released from this fraction by the addition of EGTA/EDTA but not by Triton X-100. The data demonstrate that S100A6 is localized both at the plasma membrane and the nuclear envelope in vivo and suggest a Ca(2+)-dependent interaction with annexins or other components of the nuclear envelope.

N-terminally truncated Vav induces the formation of depolymerization-resistant actin filaments in NIH 3T3 cells.

The Dbl family proto-oncogene vav is a guanine nucleotide exchange factor (GEF) for Rho family GTPases. Deletion of the N-terminus of Vav, harboring the single calponin homology (CH) domain, activates Vav's transforming potential, suggesting an important role of the CH domain in influencing Vav function. Since calponin binds actin, it has been suggested that the CH domain may mediate association with the actin cytoskeleton. In this study we have analyzed the subcellular localization and investigated the putative actin association of the Vav protein using enhanced green fluorescent protein (EGFP) fusion constructs. Our data show that both EGFP-tagged full length Vav and the CH domain-depleted EGFPvav 143-845 construct localize throughout the cytoplasm but fail to colocalize with F-actin. However, the latter construct of Vav was more strongly retained in the Triton-insoluble cytoskeleton fraction than full length Vav. Whereas removal of the CH domain had no apparent influence on the subcellular localization of Vav, deletion of the SH domains caused nuclear localization, indicating that Vav contains a functional nuclear localization signal. Expression of N-terminally truncated Vav constructs caused depolarization of fibroblasts and triggered the bundling of actin stress fibers into parallel arrays in NIH 3T3 cells. Notably, the parallel actin bundles showed prolonged resistance to the actin polymerization antagonists cytochalasin B and latrunculin B. These data point towards a regulatory role for the CH domain in Vav and suggest an actin cross-linking or bundling protein as a downstream effector molecule of vav-mediated signalling pathways.

CH domains revisited.

A sequence motif of about 100 amino acids, termed the 'calponin homology domain' has been suggested to confer actin binding to a variety of cytoskeletal and signalling molecules. Here we analyse and compare the sequences of all calponin homology domain-containing proteins identified to date. We propose that single calponin homology domains do not confer actin-binding per se and that the actin-binding motifs of cross-linking proteins, which comprise two disparate calponin homology domains, represent a unique protein module.

The single CH domain of calponin is neither sufficient nor necessary for F-actin binding.

Calponins have been implicated in the regulation of actomyosin interactions in smooth muscle cells, cytoskeletal organisation in nonmuscle cells, and the control of neurite outgrowth. Domains homologous to the amino-terminal region of calponin have been identified in a variety of actin cross-linking proteins and signal transduction molecules, and by inference these 'calponin homology (CH) domains' have been assumed to participate in actin binding. We here report on the actin binding activities of the subdomains of the calponin molecule. All three mammalian isoforms of calponin (basic h1, neutral h2 and acidic) possess a single CH domain at their amino terminus as well as three tandem repeats proximal to the carboxyl terminus. Calponin h2 differs, however, from h1 in lacking a consensus actin-binding motif in the region 142-163, between the CH domain and the tandem repeats, which in h1 calponin can be chemically cross-linked to actin. Despite the absence of this consensus actin-binding motif, recombinant full-length h2 calponin co-sediments in vitro with F-actin, suggesting the presence of another binding site in the molecule. It could be shown that this binding site resides in the C-terminal tandem repeats and not in the CH domain. Thus, constructs of h2 calponin bearing partial or complete deletions of the triple repeated sequences failed to co-localise with actin stress fibres despite the presence of a CH domain. Deletion of the acidic carboxyl terminus, beyond the repeats, increased actin binding, suggesting that the carboxy-terminal tail may modulate actin association. Results obtained from transient transfections of amino- and carboxy-terminal truncations in h1 calponin were consistent with the established location of the actin binding motif outside and carboxy-terminal to the CH domain, and confirm that the presence of a single CH domain alone is neither sufficient nor necessary to mediate actin binding. Instead, the carboxy-terminal tandem repeats of h1 and h2 calponin are shown to harbour a second, independent actin binding motif.

The cytoskeleton of the vertebrate smooth muscle cell.

Smooth muscle cells possess a structural lattice composed of two primary parts: the 'cytoskeleton' that pervades the cytoplasm and the 'membrane skeleton' that provides anchorage for the cytoskeleton and contractile apparatus at the cell surface. The cytoskeleton contains two major components: first, a complement of actin filaments that links the cytoplasmic dense bodies at equispaced intervals in longitudinal fibrils; and second, a network of desmin intermediate filaments that co-distributes with the cytoskeletal actin. The actin filaments of the contractile apparatus are presumed to interface with the cytoskeleton at the cytoplasmic dense bodies and with the longitudinal rib-like arrays of dense plaques of the membrane skeleton that couple to the extracellular matrix. The present report focuses attention on the functional role of intermediate filaments and on the molecular domain structure of the protein calponin, which is found both in the cytoskeleton and the contractile apparatus. New information about the role of intermediate filaments in smooth muscle has come from studies of transgenic mice in which desmin expression has been ablated. These have shown that while desmin is dispensable for normal development and viability its absence has significant consequences for the mechanical properties of muscle tissue. Thus, the visceral smooth muscles develop only 40% of the normal contractile force and the maximal shortening velocity is reduced by 25-40%. Intermediate filaments therefore play an active role in force transmission and do not contribute solely to cell shape maintenance, as has hitherto been presumed. Recent studies on calponin have revealed a second actin binding domain at the C-terminus of the molecule and have also pinpointed an N-terminal domain that shares homology with a growing family of actin binding and signalling molecules. How these newly identified features of calponin relate to its function in vivo remains to be established.

Localization of smoothelin in avian smooth muscle and identification of a vascular-specific isoform.

Smoothelin is a smooth muscle-specific protein of minor abundance first identified via a monoclonal antibody obtained using an avian gizzard extract as antigen. Dual labelling of ultrathin sections with antibodies to smoothelin together with antibodies to other smooth muscle proteins showed that smoothelin was co-distributed with filamin and desmin in the cytoskeleton domain of the smooth muscle cell. From the finding that smoothelin, unlike desmin, was readily extracted by Triton X-100 as well as under conditions that solubilized myosin, beta-actin and filamin, we conclude that smoothelin is most likely associated with the actin cytoskeleton. Western blot analysis of gizzard smooth muscle tissue revealed an immunoreactive protein band with an apparent molecular weight of 59 kDa that separated into 3-4 isolated variants, while avian vascular muscle showed a polypeptide band of 95 kDa. These results point to the presence of specific isoforms in visceral and vascular smooth muscles. The 59 kDa isoform was shown to be distinct from the 60 kDa filamin-binding protein, described by Maekawa and Sakai (FEBS Lett. 221, 68-72, 1987). As compared to other smooth muscle markers, such as calponin and SM22, smoothelin appeared very late during differentiation in the chick gizzard, on about the 18th embryonic day.

Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins.

Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S]methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.

Forced expression of tropomyosin 2 or 3 in v-Ki-ras-transformed fibroblasts results in distinct phenotypic effects.

Transformation of cells in tissue culture results in a variety of cellular changes including alterations in cell growth, adhesiveness, motility, morphology, and organization of the cytoskeleton. Morphological and cytoskeletal changes are perhaps the most readily apparent features of transformed cells. Although a number of studies have documented a decrease in the expression of specific tropomyosin (TM) isoforms in transformed cells, it remains to be determined if the suppression of TM synthesis is essential in the establishment and maintenance of the transformed pheno-type. To address the roles of different TM isoforms in transformed cells we have examined the effects of expressing specific TM isoforms in transformed cells using a Kirsten virus-transformed cell line (ATCC NRK1569) as a model system. In contrast to normal fibroblasts, the NRK 1569 cells contain reduced levels of TM-1 and undetectable levels of TM-2 and TM-3. These cells have a rounded morphology and are devoid of stress fibers. Employing expression plasmids for TM-2 and TM-3, stable cell lines were established from the NRK 1569 cells that express these isoforms individually. We demonstrate that expression of TM-2 or TM-3 leads to increased cell spreading accompanied by the formation of identifiable microfilament bundles, as well as significant restoration of well-defined vinculin-containing focal adhesion plaques, although expression of each isoform exhibited distinct properties. In addition, cells expressing TM-2, but not TM-3, exhibited contact-inhibited cell growth and a requirement for serum.

Expressing functional domains of mouse calponin: involvement of the region around alanine 145 in the actomyosin ATPase inhibitory activity of calponin.

Previously, we attributed the binding of F-actin to the 38-residue stretch of gizzard calponin encompassing the sequence A145-Y182 and postulated the hexapeptide motif VKYAEK, representing residues 142-147, as a putative actin-binding site [Mezgueldi, M., Fattoum, A., Derancourt, J. & Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951]. Herein, the nature of the ATPase inhibitory amino acids of calponin and their relative position within the actin binding domain was investigated by expressing the following fragments of mouse calponin with or without substitution or deletion of the hexapeptide V142-K147: amino acids 1-228 (CaP1-228), 45-228 (CaP45-228), 131-228 (CaP131-228), and CaP1-228 with substitution of A145 with S (CaP1-228A145S) or deletion of V142-K147 (CaP1-228de1142-147). All the recombinant fragments displayed most of the biochemical properties of the smooth muscle purified calponin including (a) expected electrophoretic mobility, (b) heat stability, (c) binding to actin, tropomyosin and calmodulin, and (d) zero-length cross-linking to actin switched by calmodulin in a calcium-dependent fashion. However, while the wild-type recombinant fragments inhibit the acto-S-1 ATPase activity to the same extent as do the parent calponin, modulation of the hexapeptide by either substitution or deletion strongly affect the inhibitory activity with only slightly decreasing actin binding capacity. The data indicate that the stretch VKYAEK is crucial for ATPase inhibition by calponin but represents only part of the actin-binding domain. These results are discussed in terms of multiple contact sites between actin and calponin.

Specificity of dimer formation in tropomyosins: influence of alternatively spliced exons on homodimer and heterodimer assembly.

Tropomyosins consist of nearly 100% alpha-helix and assemble into parallel and in-register coiled-coil dimers. In vitro it has been established that nonmuscle as well as native muscle tropomyosins can form homodimers. However, a mixture of muscle alpha and beta tropomyosin subunits results in the formation of the thermodynamically more stable alpha/beta heterodimer. Although the assembly preference of the muscle tropomyosin heterodimer can be understood thermodynamically, the presence of multiple tropomyosin isoforms expressed in nonmuscle cells points toward a more complex principle for determining dimer formation. We have investigated the dimerization of rat tropomyosins in living cells by the use of epitope tagging with a 16-aa sequence of the influenza hemagglutinin. Employing transfection and immunoprecipitation techniques, we have analyzed the dimers formed by muscle and nonmuscle tropomyosins in rat fibroblasts. We demonstrate that the information for homo- versus heterodimerization is contained within the tropomyosin molecule itself and that the information for the selectivity is conferred by the alternatively spliced exons. These results have important implications for models of the regulation of cytoskeletal dynamics.

Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres.

Calponin (4.1-5.9 microM, pig stomach) inhibited maximal shortening velocity (Vmax) by 20-25% with only minor influence on force in skinned smooth muscle from guinea-pig taenia coli activated at different Ca2+ levels and with thiophosphorylation. Similar results were obtained with a fragment of the N-terminal 1-228 amino acids engineered using a mouse cDNA construct (5.4 microM). Both the native calponin and the fragment inhibited actin filament sliding in a graded manner in an in vitro motility assay. We conclude that calponin influences the kinetics of the actin-myosin interaction in the organised smooth muscle contractile system and that engineered fragments of calponin can be used to probe its action in muscle fibres. The effects can be due to an introduction of an internal load during filament sliding, possibly by decreasing the detachment rates and increasing the cross-bridge time spent in the attached state.