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The carcinoembryonic antigen-related cell adhesion molecule CEACAM1 (CD66a) and the scaffolding protein filamin A have both been implicated in tumor cell migration. In the present study we identified filamin A as a novel binding partner for the CEACAM1-L cytoplasmic domain in a yeast two-hybrid screen. Direct binding was shown by surface plasmon resonance analysis and by affinity precipitation assays. The association was shown for human and rodent CEACAM1-L in endogenous CEACAM1-L expressing cells. To address functional aspects of the interaction, we used a well-established melanoma cell system. We found in different migration studies that the interaction of CEACAM1-L and filamin A drastically reduced migration and cell scattering, whereas each of these proteins when expressed alone, acted promigratory. CEACAM1-L binding to filamin A reduced the interaction of the latter with RalA, a member of the Ras-family of GTPases. Furthermore, co-expression of CEACAM1-L and filamin A led to a reduced focal adhesion turnover. Independent of the presence of filamin A, the expression of CEACAM1-L led to an increased phosphorylation of focal adhesions and to altered cytoskeletal rearrangements during monolayer wound healing assays. Together, our data demonstrate a novel mechanism for how CEACAM1-L regulates cell migration via its interaction with filamin A.

The surgical closure of orofacial clefts is considered to impair maxillary growth and dento-alveolar development. Wound contraction and subsequent scar tissue formation, during healing of these surgical wounds, contribute largely to these growth disturbances. The potential to minimize wound contraction and subsequent scarring by clinical interventions depends on the surgeon's knowledge of the events responsible for these phenomena. Fibroblasts initiate wound contraction, but proto-myofibroblasts and mature myofibroblasts are by far the most important cells in this process. Myofibroblasts are characterized by their cytoskeleton, which contains alpha-smooth-muscle actin. Additionally, their contractile apparatus contains bundles of actin microfilaments and associated contractile proteins, such as non-muscle myosin. This contractile apparatus is thought to be the major force-generating element involved in wound contraction. After closure of the wound, the myofibroblasts disappear by apoptosis, and a less cellular scar is formed. A reduction of contraction and scarring might be obtained by inhibition of myofibroblast differentiation, stimulation of their de-differentiation, stimulation of myofibroblast apoptosis, or impairment of myofibroblast function. In this review, we will discuss all of these possibilities, which ultimately may lead to a better outcome of cleft palate surgery.

Anillin is an actin-binding protein that can bind septins and is a component of the cytokinetic ring. We assessed the anillin expression in 7,579 human tissue samples and cell lines by DNA microarray analysis. Anillin is expressed ubiquitously but with variable levels of expression, being highest in the central nervous system. The median level of anillin mRNA expression was higher in tumors than normal tissues (median fold increase 2.58; 95% confidence intervals, 2.19-5.68, P < 0.0001) except in the central nervous system where anillin mRNA levels were lower in tumors. We developed a sensitive reverse transcription-PCR strategy to show that anillin mRNA is expressed in cell lines and in cDNA panels derived from fetal and adult tissues, thus validating the microarray data. We compared anillin with Ki67 mRNA expression and found a significant linear relationship between anillin and Ki67 mRNA expression (Spearmann r approximately 0.6, P < 0.0001). Anillin mRNA expression was analyzed during tumor progression in breast, ovarian, kidney, colorectal, hepatic, lung, endometrial, and pancreatic tumors and in all tissues there was progressive increase in anillin mRNA expression from normal to benign to malignant to metastatic disease. Finally, we used anti-anillin sera and found nuclear anillin immunoreactivity to be widespread in normal tissues, often not correlating with proliferative compartments. These data provide insight into the existence of nonproliferation-associated activities of anillin and roles in interphase nuclei. Thus, anillin is overexpressed in diverse common human tumors, but not simply as a consequence of being a proliferation marker. Anillin may have potential as a novel biomarker.

The regenerative capacity of skeletal muscle is due to the myogenic progenitor cell population that is resident in adult skeletal muscle. To enhance our understanding of this cell population, we examined the temporal-spatial expression pattern for filamin C during murine embryogenesis, adult muscle regeneration and in selected myopathic models of human disease. Using in situ hybridization, we observed filamin C to be restricted to mesodermal lineages including the developing heart and skeletal muscle during embryogenesis. Following cardiotoxin-induced muscle injury of adult skeletal muscle, filamin C expression was dynamically regulated in activated myogenic progenitor cells and newly regenerated myotubes. This expression pattern was further supported using RT-PCR analysis of filamin C expression in differentiating C2C12 myotubes. These results support the paradigm that the regulatory mechanisms of muscle regeneration largely recapitulate the fundamental events observed during muscle development and that filamin C may function in signal transduction or cellular migration of the myogenic progenitor cell population.

Calcium-sensing receptors (CaR) regulate cell proliferation, differentiation, and apoptosis through the MAPK pathway. MAPK pathway activation requires the cytoskeletal scaffold protein filamin A. Here we examine the interactions of CaR with filamin A in HEK-293 and M2 or A7 melanoma cells to determine how interactions with filamin A facilitate signaling. Filamin A interacts with CaR through two predicted beta-strands from residues 962 to 981; interactions between filamin A and CaR are greatly enhanced by exposure to 5 mM Ca2+. Truncations or deletions (from 972 to 997 or 962 to 981) of the CaR carboxyl terminus eliminate high affinity interactions with filamin A, but CaR-mediated MAPK pathway activation still occurs. CaR-mediated ERK phosphorylation can be localized to a predicted alpha-helix proximal to the membrane, which has been shown to be important for G protein-mediated signaling (residues 868-879). In M2 cells (-filamin A), CaR expression levels are very low; cotransfection of CaR with filamin A increases total cellular CaR and increases plasma membrane localization of CaR, facilitating CaR signaling to the MAPK pathway; similar results were obtained in HEK-293 cells. Interaction with filamin A increases cellular CaR by preventing CaR degradation, thereby facilitating CaR signaling. In addition, filamin A facilitates signaling to the MAPK pathway even by CaR truncations or deletion mutants that cannot engage in high affinity interactions with filamin A, suggesting the targeting of critical signaling proteins to CaR.

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet-mediated hemostasis and thrombosis. The cytoplasmic domain of its largest polypeptide subunit GpIbalpha possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIbalpha directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIbalpha biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wild-type or mutant GpIbalpha with GpIbbeta, GpIX with and without filamin A. We observed that surface GpIbalpha expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIbalpha, GpIbbeta, and GpIX (CHO-GpIbalpha/betaIX), lysates from CHO-GpIbalpha/betaIX + filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIbalpha, but lesser amounts of the approximately 15-kDa C-terminal peptide released when the extracellular domain of GpIbalpha is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIbalpha at C-terminal residue 557 or by a deletion between amino acids 560-570, the decreased synthesis of mature GpIbalpha is accompanied by decreased immature GpIbalpha and by an increased immunodetectable C-terminal peptide. The synthesis of mature GpIbalpha in CHO-GpIbalpha/betaIX cells is eliminated by brefeldin A (which inhibits transport out of the endoplasmic reticulum (ER)) and restored by lactacystin (which inhibits proteasomal degradation). These results suggest that GpIbalpha binds to filamin A within the ER and that filamin A binding directs post-ER trafficking of GpIbalpha to the cell surface.

The acrosome reaction (AR) is an exocytotic process of spermatozoa, and an absolute requirement for fertilization. During AR, actin polymerization is necessary in the equatorial and postacrosomal regions of guinea pig sperm for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane (PM) fusion nor the early steps of egg activation. To identify the mechanisms involved in this sperm actin polymerization, we searched for the protein members, known to be involved in a highly conserved model, that may apply to any cellular process in which de novo actin polymerization occurs from G protein activation. WASP, Arp 2/3, profilins I and II, and Cdc42, RhoA and RhoB GTPases were localized by indirect immunofluorescence (IIF) in guinea pig spermatozoa and their presence corroborated by Western blotting. WASP and profilin II were translocated to the postacrosomal region (Arp2/3 already were there) in long-term capacitated and acrosome-reacted spermatozoa, at the same time as actin polymerization occurred. These events were inhibited by GDP-beta-S and promoted by lysophosphatidic acid (LPA) and GTP-gamma-S, a small GTPase inhibitor and two activators, respectively. By immunoprecipitation, Cdc42-WASp association was identified in capacitated but not in noncapacitated gametes. Polymerized actin in the postacrosomal region is apparently anchored both to the postacrosomal perinuclear theca region and the overlying PM. Results suggest that GTPases are involved in sperm actin polymerization, in the postacrosomal region and the mechanism for polymerization might fit a previously proposed model (Mullins, 2000: Curr Opin Cell Biol 12:91-96)., ((c) 2005 Wiley-Liss, Inc.)

Objective: To define the clinical, radiologic, and genetic features of periventricular heterotopia (PH) with Ehlers-Danlos syndrome (EDS)., Methods: Exonic sequencing and single stranded conformational polymorphism (SSCP) analysis was performed on affected individuals. Linkage analysis using microsatellite markers on the X-chromosome was performed on a single pedigree. Western blotting evaluated for loss of filamin A (FLNA) protein and Southern blotting assessed for any potential chromosome rearrangement in this region., Results: The authors report two familial cases and nine additional sporadic cases of the EDS-variant form of PH, which is characterized by nodular brain heterotopia, joint hypermobility, and development of aortic dilatation in early adulthood. MRI typically demonstrated bilateral nodular PH, indistinguishable from PH due to FLNA mutations. Exonic sequencing or SSCP analyses of FLNA revealed a 2762 delG single base pair deletion in one affected female. Another affected female harbored a C116 single point mutation, resulting in an A39G change. A third affected female had a 4147 delG single base pair deletion. One pedigree with no detectable exonic mutation demonstrated positive linkage to the FLNA locus Xq28, an affected individual in this family also had no detectable FLNA protein, but no chromosomal rearrangement was detected., Conclusion: These results suggest that the Ehlers-Danlos variant of periventricular heterotopia (PH), in part, represents an overlapping syndrome with X-linked dominant PH due to filamin A mutations.

Cytokinesis in eukaryotes involves the regulated assembly and contraction of a ring comprising filamentous (F)-actin and myosin II. Assembly of the contractile ring occurs through the accumulation of cortical cues at the specified division plane, followed by recruitment of F-actin, myosin II and accessory proteins involved in generating the mature ring. Ring contraction is temporally regulated to occur only after chromosome segregation and, in yeast, it is controlled by a conserved signaling cascade that becomes active only after Cdk1-Cyclin-B inactivation. In this article (which is part of the Cytokinesis series), we discuss recent studies that have begun to clarify both the spatial and the temporal order of ring assembly and that have illuminated the signals that trigger ring contraction in yeast. These studies add to the growing knowledge of the processes that control eukaryotic cell division.

Filamins were discovered as the first family of non-muscle actin-binding protein. They are lage cytoplasmic proteins that cross-link cortical actin into a dynamic three-dimensional structure. Filamins have also been reported to interact with a large number of cellular proteins of great functional diversity, suggesting that they are unusually versatile signalling scaffolds. More recently, genetic mutations in filamin A and B have been reported to cause a wide range of human diseases, suggesting that different diseases highlight distinct filamin interactions.

In the developing neocortex, most excitatory neurons are supplied and arranged through radial migration. Because neurons show global morphological changes and complicated behavior during that migration, precise regulation of cell shape and polarity is essential for proper migration and correct neocortical formation; however, how cell shape and polarity are regulated in migrating neuron remains elusive. We show here that Filamin A, a well known actin-binding protein, determines the shape of neocortical neurons during radial migration in vivo. Dysfunction of Filamin A, caused by a mutant Filamin A expression, prevents cells from acquiring consistent polarity toward specific direction and decreases motility in the subventricular and intermediate zones. In contrast, Filamin A overexpression, achieved by a short interfering RNA for Filamin A-interacting protein that induces Filamin A degradation (FILIP), promotes the development and maintenance of a bipolar shape also in the subventricular and intermediate zones. These results suggest that the amount of Filamin A helps migrating neurons determine their mode of migration, multipolar or bipolar, before entering the cortical plate and that FILIP is responsible, at least in part, for Filamin A content. In addition, our results also give a possible clue to understanding the pathogenesis of human malformation periventricular heterotopia, which is caused by various "loss-of-function" mutations in the filamin A gene.

The actin cytoskeleton plays a central part in the dynamic organization of eukaryotic cell structure. Nucleation of actin filaments is a crucial step in the establishment of new cytoskeletal structures or modification of existing ones, providing abundant targets for regulatory processes. A substantial part of our understanding of actin nucleation derives from studies on yeast and metazoan cells. However, recent advances in structural and functional genome analysis in less traditional models, such as plants or Dictyostelium discoideum, provide an emerging picture of an evolutionarily conserved core of at least two actin nucleation mechanisms, one mediated by the Arp2/3 complex and the other one by the formin-based module. A considerable degree of conservation is found also in the systems controlling the balance between filamentous and globular actin (profilin, actin-depolymerizing factor/cofilin) and even in certain regulatory aspects, such as the involvement of Rho-related small GTPases. Identification of such conserved elements provides a prerequisite for the characterization of evolutionarily variable aspects of actin regulation which may be responsible for the rich morphological diversity of eukaryotic cells.

Much of our understanding of animal cell cytokinesis centers on the regulation of the equatorial acto-myosin contractile ring that drives the rapid ingression of a deep cleavage furrow. However, the central part of the mitotic spindle collapses to a dense structure that impedes the furrow and keeps the daughter cells connected via an intercellular bridge. Factors involved in the formation, maintenance, and resolution of this bridge are largely unknown. Using a library of 7,216 double-stranded RNAs (dsRNAs) representing the conserved genes of Drosophila, we performed an RNA interference (RNAi) screen for cytokinesis genes in Schneider's S2 cells. We identified both familiar and novel genes whose inactivation induced a multi-nucleate phenotype. Using live video microscopy, we show that three genes: anillin, citron-kinase (CG10522), and soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (alpha-SNAP), are essential for the terminal (post-furrowing) events of cytokinesis. anillin RNAi caused gradual disruption of the intercellular bridge after furrowing; citron-kinase RNAi destabilized the bridge at a later stage; alpha-SNAP RNAi caused sister cells to fuse many hours later and by a different mechanism. We have shown that the stability of the intercellular bridge is essential for successful cytokinesis and have defined genes contributing to this stability.

Resistance exercise can result in both potentiating and fatiguing responses. These responses can acutely affect performance, which may affect subsequent exercise sessions in the same day. The purpose of this investigation was to study the acute neuromuscular responses to two high intensity training sessions in the same day. Twelve recreationally trained males performed two training sessions, each involving ten sets of five repetitions in the speed squat exercise. For the initial session (HIT-1), the barbell load was constant at 70% one repetition maximum, whereas during the second session (HIT-2), barbell load decreased if movement velocity decreased. Neuromuscular performance testing consisted of unilateral isometric knee extensor actions performed prior to the training day (PRE) and following each testing session. Prior to the sessions, subjects provided a muscle biopsy for myosin heavy chain analysis. Peak force was impaired 16.9 (9.5)% (P approximately 0.00; d=1.62) following HIT-1 and 19.9 (18.4)% (P approximately 0.00; d=1.94) following HIT-2. Initial rate of force development was depressed from PRE following HIT-1 (P approximately 0.00; d=1.74) and HIT-2 (P approximately 0.00; d=2.18); however, this was dependent on muscle fiber composition. Significant correlations existed between the change score for initial rate of force development from HIT-1 to HIT-2 and myosin heavy chain I (r= -0.60; P=0.04) and IIa (r=0.69; P=0.01) expression. Impaired neuromuscular performance following HIT-1 may occur due to low frequency fatigue. For individuals with predominantly myosin heavy chain IIa, HIT-2 appeared to induce post-activation potentiation, resulting in restoration of the initial rate of force development.

Profilins are small actin-binding proteins that are essential in all organisms that have been examined to date. In vitro, profilins regulate the dynamics of actin polymerization, which is their key role in vivo during cell motility. However, there is growing evidence that, apart from actin binding, profilins function as hubs that control a complex network of molecular interactions. Profilins interact with a plethora of proteins and the importance of this aspect of their function is just beginning to be understood. In this article, I will summarize recent findings in mammalian cells and mice, and discuss the evidence of a role for profilins in cellular processes such as membrane trafficking, small-GTPase signaling and nuclear activities, in addition to neurological diseases and tumor formation.

Defective cell signalling during embryonic development is a well-recognized modus operandi of mutations in genes that lead to congenital malformations. This signalling occurs within and around a dynamic cellular cytoskeleton that is continuously under modulating influences during morphogenesis. Evidence is accumulating to suggest that filamin A, an actin-binding protein and the product of one of three paralogous filamin genes in humans, represents a key molecule that connects such signalling events to modulation of the cellular cytoskeletal architecture. This review summarizes the clinical consequences of mutations in the gene encoding filamin A, FLNA. The molecular pathology of this gene suggests remarkable functional pleiotropy, indicative of diverse roles in embryonic, fetal and postnatal development.

The changes occurring at the luminal surface of endothelial cells in diabetes and their relevance to endothelial dysfunction are poorly characterized in vivo. In this study, we developed an integrated strategy to discover cell surface proteins associated with diabetes and to test their role in endothelial dysfunction. First, a peptide phage display library was screened over the endothelial surface of the intact aorta or in retinal endothelial cells from diabetic and control rats. Then, we purified profilin-1 as a binding partner for one of the diabetic aorta-specific phage. Profilin was increased in the aortic endothelium of human diabetic individuals and streptozotocin-diabetic rats. Furthermore, overexpressing profilin in rat aortic endothelial cells triggered 3 indicators of endothelial dysfunction: increased apoptosis, elevated expression of ICAM-1, and decreased phosphorylation of the vasodilator-stimulated phosphoprotein, a marker for nitric oxide signaling. The changes in ICAM-1 and vasodilator-stimulated phosphoprotein were recapitulated in the diabetic aorta in vivo. LDL and oxysterols elevated profilin in cultured aortic endothelial cells. Interference with the de novo synthesis of profilin abrogated the LDL-mediated increase in ICAM-1 expression. Finally, profilin expression was markedly elevated in atherosclerotic plaques. These data indicate that profilin contributes to endothelial dysfunction in a pathway that is downstream of LDL.

Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1.

Prostate specific membrane antigen (PSMA), is a unique membrane bound glycoprotein, which is overexpressed manifold on prostate cancer as well as neovasculature of most of the solid tumors, but not in the vasculature of the normal tissues. This unique expression of PSMA makes it an important marker as well as a large extracellular target of imaging agents. PSMA can serve as target for delivery of therapeutic agents such as cytotoxins or radionuclides. PSMA has two unique enzymatic functions, folate hydrolase and NAALADase and found to be recycled like other membrane bound receptors through clathrin coated pits. The internalization property of PSMA leads one to consider the potential existence of a natural ligand for PSMA. In this review we have discussed the regulation of PSMA expression within the cells, and significance of its expression in prostate cancer and metastasis., (Copyright 2003 Wiley-Liss, Inc.)

Reconstituted actin filament networks have been used extensively to understand the mechanics of the actin cortex and decipher the role of actin cross-linking proteins in the maintenance and deformation of cell shape. However, studies of the mechanical role of the F-actin cross-linking protein filamin have led to seemingly contradictory conclusions, in part due to the use of ill-defined mechanical assays. Using quantitative rheological methods that avoid the pitfalls of previous studies, we systematically tested the complex mechanical response of reconstituted actin filament networks containing a wide range of filamin concentrations and compared the mechanical function of filamin with that of the cross-linking/bundling proteins alpha-actinin and fascin. At steady state and within a well defined linear regime of small non-destructive deformations, F-actin solutions behave as highly dynamic networks (actin polymers are still sufficiently mobile to relax the stress) below the cross-linking-to-bundling threshold filamin concentration, and they behave as covalently cross-linked gels above that threshold. Under large deformations, F-actin networks soften at low filamin concentrations and strain-harden at high filamin concentrations. Filamin cross-links F-actin into networks that are more resilient, stiffer, more solid-like, and less dynamic than alpha-actinin and fascin. These results resolve the controversy by showing that F-actin/filamin networks can adopt diametrically opposed rheological behaviors depending on the concentration in cross-linking proteins.

Evolutionarily conserved in eukaryotes, formin homology (FH) proteins, or formins, exert their effects on the actin and microtubule (MT) networks during meiosis, mitosis, the maintenance of cell polarity, vesicular trafficking, signaling to the nucleus and embryonic development. Once thought to be only molecular scaffolds that indirectly affected cellular functions through the binding of other proteins, recent in vitro studies have illustrated that they can function as actin nucleators in the formation of new filaments. The connection between formins and MTs is less well understood. In yeast, the MT effects appear to be dependent on the ability of formins to generate polarized actin cables whereas, in mammalian cells, formin signals that cause MT stabilization and polarization might be more direct. A subclass of formins, the Diaphanous-related formins (Drfs), can act as effectors for Rho small GTPases, yet it is not clear what GTPase binding contributes to formin function.

The Filamin family of actin binding proteins is required to maintain cell shape and promote cell locomotion. Using the Drosophila ovary, we provide a detailed description of Filamin-deficient cells during morphogenesis. Reduced expression of Filamin in follicle cells causes defects in the initial encapsulation of germline cysts and in the migration of border cells through the germline cyst. However, follicle cell morphogenesis is unaffected by point mutations that produce truncated Filamin proteins. In addition, mutant follicle cell movements can be partially rescued by a transgene encoding only the actin-binding domain and the first six filamin repeats. These data show that Filamin function in cell motility can be provided by a truncated Filamin protein that resembles Dictyostelium Actin Binding Protein-120.

It is generally accepted that the cartilaginous frame of the reptilian cochlea has only a passive supportive function. In this study, a ribbon of contractile tissue was revealed within the cartilaginous frame of the cochlea of the gecko Teratoscincus scincus. It consisted of tightly packed cells and received an extensive blood supply. The cytoplasm of the cells was filled with cytoskeletal filaments 5-7 nm thick as revealed by electron microscopy. Isolated tissue permeabilized with Triton X-100 or glycerol reversibly contracted in the presence of ATP. Noradrenaline caused slow relaxation of the freshly isolated tissue placed in artificial perilymph. We suggest that slow motility of the contractile tissue may adjust passive cochlear mechanics to sounds of high intensities., (Copyright 2003 Wiley-Liss, Inc.)

Expression of the actin-binding protein profilin was disrupted in the ciliate Tetrahymena thermophila by an antisense ribosome method. In cells with the antisense disruption no profilin protein was detected. Cultures of cells with the antisense disruption could be maintained, indicating that profilin was not essential for cytokinesis or vegetative growth. Disruption of the expression of profilin resulted in many cells that were large and abnormally shaped. Formation of multiple micronuclei, which divide mitotically, was observed in cells with a single macronucleus, indicating a defect in early cytokinesis. Some cells with the antisense disruption contained multiple macronuclei, which in Tetrahymena may indicate a function late in cytokinesis. The lack of profilin also affected cytokinesis in the cells that could divide. Normal-sized and normal-shaped cells with the antisense disruption took significantly longer to divide than control cell types. The profilin disruption revealed two new processes in which profilin functions. In cells lacking profilin, micronuclei were not positioned at their normal site on the surface of the macronucleus and phagocytosis was defective. The defect in phagocytosis appeared to be due to disruption of the formation of oral apparatuses (stomatogenesis) and a possible failure in the internalization of phagocytic vacuoles.

We have examined the effect of covalently crosslinked profilin-actin (PxA), which closely matches the biochemical properties of ordinary profilin-actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of PH133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin-actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin-actin is important for actin-based processes, and show that it has a key function in Listeria motility.

The object of this theme is to offer new perspectives on the effect of aging on signal-transduction pathways associated with agonist-induced contraction of smooth muscle cells from the colon. Smooth muscle cells from old rats (32 mo old) exhibit limited cell length distribution and diminished contractility. The observed reduced contractile response may be due to the effect of aging on signal-transduction pathways, especially an inhibition of the tyrosine kinase-Src kinase pathway, a reduced activation of the PKC pathway, and a reduced association of contractile proteins [heat shock protein 27 (HSP27)-tropomyosin, HSP27-actin, actin-myosin]. Levels of HSP27 phosphorylation are also reduced compared with adult rats.

Cells remodel extracellular matrix during tissue development and wound healing. Similar processes occur when cells compress and stiffen collagen gels. An important task for cell biologists, biophysicists, and tissue engineers is to guide these remodeling processes to produce tissue constructs that mimic the structure and mechanical properties of natural tissues. This requires an understanding of the mechanisms by which this remodeling occurs. Quantitative measurements of the contractile force developed by cells and the extent of compression and stiffening of the matrix describe the results of the remodeling processes. Not only do forces exerted by cells influence the structure of the matrix but also external forces exerted on the matrix can modulate the structure and orientation of the cells. The mechanisms of these processes remain largely unknown, but recent studies of the regulation of myosin-dependent contractile force and of cell protrusion driven by actin polymerization provide clues about the regulation of cellular functions during remodeling.

The myocardium consists of three basic categories of proteins. The myofibrillar proteins trasform the chemical energy of ATP to the mechanical work of the heart. The metabolic proteins located both in the cytosol and in the mitochondrial compartments provide energy for the cardiac contraction. The interstitial space between myocytes is occupied by the extracellular proteins (collagens, glycoproteins, glycosaminoglycans, elastins). By far the greater percentage of myofibrillar proteins (about 80%) is that concerned with contraction (actin and myosin), with about 10% concerned with its regulation (troponin, tropomyosin and tropomodulin) and another 10% concerned with maintenance of the structure of myofibril (C, M-, H-proteins, myomesin, nebulette, alpha-actinin, titin, CapZ protein). Most collagenous and non-collagenous proteins exist in many isoforms that originate from the same genom but are the product of alternative splicing of a primary RNA transcript.

The conversion of physical signals, such as contractile forces or external mechanical perturbations, into chemical signaling events is a fundamental cellular process that occurs at cell-extracellular matrix contacts, known as focal adhesions. At these sites, transmembrane integrin receptors are associated via their cytoplasmic domains with the actin cytoskeleton. This interaction with actin is mediated by a submembrane plaque, consisting of numerous cytoskeletal and signaling molecules. Application of intrinsic or external forces to these structures dramatically affects their assembly and triggers adhesion-mediated signaling. In this review, we discuss the structure-function relationships of focal adhesions and the possible mode of action of the putative mechanosensor associated with them. We also discuss the general phenomenon of mechanosensitivity, and the approaches used to measure local forces at adhesion sites, the cytoskeleton-mediated regulation of local contractility, and the nature of the signaling networks that both affect contractility and are affected by it.

Human heart failure, a complex disease with heterogeneous etiologies, remains one of the most life-threatening diseases known. Identification of "candidate genes" and molecular and biochemical mediators of cardiac hypertrophy and failure has been vigorously pursued to dissect the pathogenesis and signaling pathways of this disease. With the availability of murine cardiac-specific promoters, transgenesis and gene targeting technologies have revolutionized the field of cardiac research. During the past decade, a large number of genetically engineered mouse models with altered cardiac function have been generated. The ability to engineer precise mutations in the heart, coupled with the technological sophistication to quantitate the effects of these mutations on cardiac function at cellular, organ and intact animal levels, has provided novel insights into the molecular mechanisms of heart failure and led to the recognition of a wide array of previously unknown molecular sensors, initiators, transducers, and effectors for the development of cardiac hypertrophy and its transition to heart failure.

Protein interaction screens have revealed an interaction between the D2 dopamine receptor and the actin cross-linking protein filamin A. However, the physiological significance of this interaction has not been explained. To better understand the role of filamin A in D2 receptor-mediated signaling, we examined the effect of disrupting filamin A/D2 receptor interaction. Overexpression of a truncated form of filamin A (repeat units 18-19 containing the D2, but not the actin, binding domain) caused a marked reduction in both the number and half-life of cell surface D2 receptors. These results suggest that disruption of the linkage between D2 receptors and the actin cytoskeleton destabilizes plasma membrane-associated D2 receptors. Several missense mutations within repeat unit 19 of filamin A were identified that abrogate filamin A/D2 receptor interaction. Introduction of mutant and wild-type filamin A into filamin A-deficient M2 cells demonstrated that wild-type filamin A, but not the filamin A-binding mutants, was able to promote cell-surface expression of D2 receptors. Together, these studies provide evidence that filamin A/D2 receptor interaction is required for the proper targeting or stabilization of D2 dopamine receptors at the plasma membrane., (Copyright 2002 S. Karger AG, Basel)

Although endothelin-1-stimulated contractile force generation by stellate cells is believed to play an important role in hepatic pathophysiology, the molecular signals that mediate this process are incompletely understood. The aim of this study was to test the hypothesis that myosin mediates the contractile force generated by stellate cells in response to endothelin-1. Contractile force generation by primary and immortalized stellate cells was directly and quantitatively measured. Myosin phosphorylation and reorganization, and actin stress fiber formation were investigated in immortalized stellate cells. Endothelin-1 stimulated a rapid and robust generation of contractile force by primary and immortalized stellate cells with a similar dose dependence. Myosin phosphorylation, actin stress fiber assembly, and reorganization of myosin to stress fibers were induced by concentrations of endothelin-1 that also stimulated stellate cell contraction. BQ-123, a selective endothelin receptor antagonist, inhibited myosin phosphorylation and contractile force generation. Y-27632, which selectively inhibits rho-associated kinase, also blocked endothelin-1-stimulated myosin phosphorylation and contractile force generation with a similar dose dependence. These results suggest that endothelin-1-stimulated contractile force generation by stellate cells is mediated by myosin., (Copyright 2002 National Science Council, ROC and S. Karger AG, Basel)

Myosin, the motor protein in skeletal muscle, is composed of two subunits, myosin heavy chain and myosin light chain. All vertebrates express a family of myosin heavy chain and myosin light chain isoforms that together are primary determinants of force, velocity, and power in muscle fibers. Therefore, appropriate expression of myosin isoforms in skeletal muscle is critical to proper motor function. Myosin isoform expression is highly plastic and undergoes significant changes in response to muscular injury, muscle disuse, and disease. Therefore, myosin isoform function and plasticity are highly relevant to clinical orthopaedic research, musculoskeletal surgery, and sports medicine. Muscle from frogs offers a special opportunity to study the structural basis of contractile protein function because single intact fibers can be isolated that maintain excellent mechanical stability, allowing for high-resolution studies of contractile performance in intact cells. The current authors summarize recent studies defining the myosin isoforms in muscle from frogs and the relationship between myosin isoforms and mechanical performance of intact single muscle cells. Preliminary studies also are described that show the potential for simple plasmid-based in vivo gene transfer approaches as a model system to elucidate the structural basis of muscle protein function in intact cells.

Strength conditioning will result in an increase in muscle size and this increase in size is largely the result of increased contractile proteins. The mechanisms by which the mechanical events stimulate an increase in ribonucleic acid synthesis and subsequent protein synthesis are not well understood. Lifting weight requires that a muscle shorten as it produces force (concentric contraction). Lowering the weight, however, forces the muscle to lengthen as it produces force (eccentric contraction). These lengthening muscle contractions have been shown to produce ultrastructural damage (microscopic tears in contractile proteins muscle cells) that may stimulate increased muscle protein turnover. This muscle damage produces a cascade of metabolic events that is similar to an acute phase response and includes complement activation, mobilization of neutrophils, increased circulation of skeletal muscle interleukin-1, macrophage accumulation in muscle, and an increase in muscle protein synthesis and degradation. Although endurance exercise increases the oxidation of essential amino acids and increases the requirement for dietary protein, resistance exercise results in a decrease in nitrogen excretion, lowering dietary protein needs. This increased efficiency of protein use may be important for wasting diseases such as human immunodeficiency virus infection and cancer and particularly in elderly people suffering from sarcopenia. Research has indicated that increased dietary protein intake (as much as 1.6 g protein x kg x day ) may enhance the hypertrophic response to resistance exercise. It also has been shown that in very old men and women the use of a protein-calorie supplement was associated with greater strength and muscle mass gains than the use of placebo.

The cellular processes that lead to changes in shape (morphogenesis) and organ formation (organogenesis) are poorly understood. Local contraction of microfilaments can change cell shape and lead to changes of tissue shape. To clarify the role of contractile microfilaments in the foregut morphogenesis of chick embryos, 2- to 4-day-old embryos were exposed to cytochalasin D (CD), which is known to disrupt microfilaments. Untreated age-matched embryos were used as controls. Sections of treated embryos and controls were stained with phalloidin, which binds to actin, and examined with a fluorescence microscope. Microdissected specimens were examined using a scanning electron microscope (SEM). Immunofluorescent staining showed a bright signal belt toward the apical cell region of the foregut epithelium in controls. This signal was not evident in CD-exposed embryos. SEM micrographs of the controls showed the cranial foregut as a smooth, even, cylindrical structure in all stages studied. The lumen was narrow and perfectly straight, the ventral and dorsal walls were in close apposition. The foregut of CD-exposed specimens, however, showed a wide lumen and the walls were separated from each other. The structure seemed atonic and appeared conical, curved, or tilted. We observed a dense microfilament network toward the apical cell pole of the epithelial foregut cells of controls that was no longer evident after CD exposure. This network seems to play an important role in foregut morphogenesis, since actin-filament disruption by CD causes loss of the normal shape.

We investigated the role of Galphaq, filamin, Rho, the RhoGEF Lbc, and the C terminus of calcium-sensing receptor (CasR) in CasR signaling. We found that Ca(2+), Mg(2+), or the calcimimetic R isomer of N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS-R568) stimulated serum response element (SRE) activity human embryonic kidney 293 cells transfected with CasR and an SRE-luciferase reporter construct. Coexpression of either the dominant negative Galphaq(305-359) minigene, regulators of G protein signaling (RGS)2 or RGS4, inhibited CasR-stimulated SRE activity, consistent with CasR activation of Galphaq. The cytoskeletal associated Rho protein is involved CasR activation of SRE, as evidenced by CasR-mediated increase in membrane-associated Rho A and by the ability of Clostridium botulinum C3 (C3) exoenzyme to inhibit both CasR and GalphaqQL-stimulated SRE activity. Overexpression of the RhoGEF Lbc, lacking either the Dbl-homology or Pleckstrin homology domain, as well as the filamin peptide (1530-1875) inhibited CasR-mediated activation of SRE. A carboxyl-terminal CasR minigene, CasR(906-980), encoding a filamin binding region, also blocked CasR- and GalphaqQL-stimulated SRE activity. Potential interactions between CasR, RhoGEF Lbc, Rho A, Galphaq, and filamin were demonstrated by reciprocal coimmunoprecipitation studies. Our results suggest that the C terminus of CasR may interact with filamin to create a cytoskeletal scaffold necessary for the spatial organization of Galphaq, RhoGEF Lbc, and Rho signaling pathways upstream of SRE activation.

Elastic fibers are composed of the protein elastin and a network of 10-12 nm microfibrils. The microfibrillar proteins include, among others, the fibrillins and microfibril-associated glycoproteins-1 and -2 (MAGP-1 and MAGP-2). Little is known about how microfibrillar proteins interact to support fiber assembly. We used the C-terminal half of MAGP-2 in a yeast two-hybrid library screen to identify relevant ligands. Six of 13 positive clones encoded known microfibrillar proteins, including fibrillin-1 and -2. Deletion analysis of partial fibrillin-1 and -2 clones revealed a calcium-binding epidermal growth factor repeat-containing region near the C terminus responsible for binding. This region is distinct from the region of fibrillin-1 reported by others to bind MAGP-1. The MAGP-2 bait was unable to interact productively with other epidermal growth factor repeats in fibrillin-1, demonstrating specificity of the interaction. Deletion analysis of the MAGP-2 bait demonstrated that binding occurred in a core region containing 48% identity and 7 conserved cysteine residues with MAGP-1. Immunoprecipitation of MAGP-2 from transfected COS-7 cells resulted in the coprecipitation of fibrillin. These results demonstrate that MAGP-2 specifically interacts with fibrillin-1 and -2 and suggest that MAGP-2 may help regulate microfibrillar assembly. The results also demonstrate the utility of the yeast two-hybrid system to study protein-protein interactions of the extracellular matrix.

The human meniscus is subject to injury that necessitates repair or removal. Many aspects of the cellular response to injury have not been well characterized. The purpose of this study was to describe the cellular distributions within the torn human meniscus. In addition to evaluating the cell density in selected regions, we investigated the cellular expression of a contractile actin isoform that has recently been found in the intact human meniscus. Included as a contemporaneous comparative group were torn human meniscal allografts. We hypothesized that a hypercellular surface zone would be found in the torn menisci, with a higher percentage of cells in this peripheral region expressing alpha-smooth muscle actin compared with other locations in the interior of the remnant. The rationale for this hypothesis was based on prior immunohistochemical investigations of the distribution of alpha-smooth muscle actin-containing cells in the torn human anterior cruciate ligament. Eighteen torn meniscal specimens were obtained from 17 patients, 0.5 to 84 months after injury, and four torn allograft meniscal samples were retrieved from three patients, 11 to 49 months after implantation. Microtomed sections of paraffin-embedded tissue were stained with hematoxylin and eosin and a monoclonal antibody to alpha-smooth muscle actin. The cell density and percentage of cells containing alpha-smooth muscle actin were determined in the following zones: synovial, vascular, hypercellular with loose collagen, hypocellular with dense collagen, and organized collagen. A cellular layer that resembled synovium was present on the surface of all but two of the specimens. Vascular regions were often continuous with the synovium abutting the more interior loose collagen zones. The total cell density was greatest in each of the zones closest to the periphery (synovium, vascular, and loose collagen; p < 0.001), when compared to the interior of the tissue. The synovium-like layer was found to have the highest percentage of alpha-smooth muscle actin-expressing cells and the highest alpha-smooth muscle actin-containing cell density (p < 0.05). Similar results were found for the torn allograft menisci. These findings confirm the working hypothesis and suggest that the torn human meniscus is capable of mounting a reparative response, including the proliferation of cells capable of contributing to wound closure. This underscores the importance of providing a bridging scaffold into which such cells can migrate.

Precisely regulated radial migration out of the ventricular zone is essential for corticogenesis. Here, we identify a mechanism that can tether ventricular zone cells in situ. FILIP interacts with Filamin A, an indispensable actin-binding protein that is required for cell motility, and induces its degradation in COS-7 cells. Degradation of Filamin A is identified in the cortical ventricular zone, where filip mRNA is localized. Furthermore, most ventricular zone cells that overexpress FILIP fail to migrate in explants. These results demonstrate that FILIP functions through a Filamin A F-actin axis to control the start of neocortical cell migration from the ventricular zone.

The etiology of adolescent idiopathic scoliosis (AIS), the most common form of scoliosis, is unclear. Researchers with divergent perspectives have tried to better define this etiology. Genetics, growth hormone secretion, connective tissue structure, muscle structure, vestibular dysfunction, melatonin secretion, and platelet microstructure are major areas of focus. In this article, we review the literature in these areas and present the consensus on proposed hypotheses. Studies that simplify the etiology to a single factor have been inconclusive or unsuccessful. Most likely, the etiology is multifactorial, and reported associations are links in pathogenesis rather than etiologic factors. Research is needed to better define the role of all factors in AIS development.

We have studied the contractile properties, structure, fiber-type composition, and myosin heavy chain (MyHC) expression pattern of regenerating and intact soleus muscles of adult CBA/J mice treated with cyclosporin A (CsA) or vehicle solutions (Cremophor, saline). A comparison of muscles after 4-7 weeks drug application with those receiving vehicle showed that the isometric contractile force of intact drug-treated muscles was reduced (tetanus, -21%; twitch, -34%) despite normal mass and muscle cross-sectional area. The frequency of fast-twitch fibers was increased, whereas no innervation deficits, histopathological alterations, or changes in fiber numbers were observed. Regeneration after cryolesion of the contralateral soleus proceeded more slowly in CsA-treated than in vehicle-treated animals. Despite this, when muscle properties reached mature levels (4-7 weeks), muscle mass recovery was better in CsA-treated animals (30% higher weight, 50% more fiber profiles in cross-sections). The force production per unit cross-sectional area was deficient, but not the maximum tension. Twitch time-to-peak and half-relaxation time were shorter than controls correlating with a predominance of fast-twitch fibers (98% Type II fibers versus 16%-18% in control muscles) and fast MyHC isoforms. Partial reversal of this fast phenotype and an increase in muscle force were observed when the animals were left to recover without treatment for 5-8 weeks after CsA application over 7 weeks. The high numbers of fiber profiles in CsA-treated regenerated muscles and increased mass remained unchanged after withdrawal. Thus, CsA treatment has a hyperplastic effect on regenerating muscles, and drug-induced phenotype alterations are much more prominent in regenerated muscles.

The contractile vacuole complex is a membrane-bound osmoregulatory organelle of fresh water protozoa such as Paramecium. In Paramecium it consists of a central vacuole (the contractile vacuole) and 5-10 arms that radially extend from the vacuole into the cytosol (the radial arms). Excess cytosolic water, acquired osmotically, is segregated by the radial arms and enters the vacuole, so that the vacuole swells (the fluid-filling phase). The vacuole then rounds (the rounding phase) and the radial arms sever from the vacuole. The vacuole membrane then fuses with the plasma membrane at the pore region and the pore opens. The vacuole shrinks as its fluid is discharged through the pore (the fluid-discharging phase). The pore closes when the fluid has been discharged. The radial arms then reattach to the vacuole, so that the vacuole swells again as the fluid enters from the arms (the next fluid-filling phase). We found that the vacuole continued to show rounding and slackening even after it together with a small amount of cytosol had been isolated from the cell. Using a microcantilever placed on the surface of the vacuole the tension of the in vitro vacuole increased to 5 x 10(-3)N m(-1) as the vacuole rounds, and its lowest value was 1 x 10(-4)N m(-1) during slackening. We propose a hypothesis that an increase in the spontaneous curvature of the organelle's membrane leads to an increase in membrane tension and thus to the vacuole's rounding, severing of the radial arms from the vacuole, and opening of the pore. Conversely, a decrease in the spontaneous curvature accompanied by a decrease in membrane tension could lead to the closing of the pore and reattachment of the radial arm at the start of the fluid-filling phase.

Objectives: The goal of this study was to identify genes upregulated in the heart in human patients with hypertrophic cardiomyopathy (HCM)., Background: Hypertrophic cardiomyopathy is a genetic disease caused by mutations in contractile sarcomeric proteins. The molecular basis of diverse clinical and pathologic phenotypes in HCM remains unknown., Methods: We performed polymerase chain reaction-select complementary DNA subtraction between normal hearts and hearts with HCM and screened subtracted libraries by Southern blotting. We sequenced the differentially expressed clones and performed Northern blotting to detect increased expression levels., Results: We screened 288 independent clones, and 76 clones had less than twofold increase in the signal intensity and were considered upregulated. Sequence analysis identified 36 genes including those encoding the markers of pressure overload-induced ("secondary") cardiac hypertrophy, cytoskeletal proteins, protein synthesis, redox system, ion channels and those with unknown function. Northern blotting confirmed increased expression of skeletal muscle alpha-actin (ACTA1), myosin light chain 2a (MLC2a), GTP-binding protein Gs-alpha subunit (GNAS1), NADH ubiquinone oxidoreductase (NDUFB10), voltage-dependent anion channel 1 (VDAC1), four-and-a-half LIM domain protein 1 (FHL1) (also known as SLIM1), sarcosin (SARCOSIN) and heat shock 70kD protein 8 (HSPA8) by less than twofold. Expression levels of ACTA1, MLC2a and GNAS1 were increased in six additional and FHL1 in four additional hearts with HCM., Conclusions: A diverse array of genes is upregulated in the heart in human patients with HCM, which could account for the diversity of clinical and pathologic phenotypes. Markers of secondary hypertrophy are also upregulated, suggesting commonality of pathways involved in HCM and the acquired forms of cardiac hypertrophy. Elucidation of the role of differentially expressed genes in HCM could provide for new therapeutic targets.

Unlabelled: This study was undertaken to test whether there is structural remodeling of lung parenchyma that could lead to tissue mechanical changes at an early phase of varying degrees of acute lung injury (ALI). Tissue resistance (R), dynamic elastance (E), and hysteresivity (eta) were analyzed during sinusoidal oscillations of rat lung parenchymal strips 24 h after intraperitoneal injection of saline (C) or paraquat (P [10, 15, 25, and 30 mg/kg]). These strips were also stained in order to quantify the amount of collagen and of three types of elastic fibers (elaunin, oxytalan, and fully developed elastic fibers) in the alveolar septa. E augmented progressively from C to P25, but the data from the P25 and P30 groups were not different (p < 0.0001). R and eta increased from C to P10 and from P15 to P25 (p < 0.001). Collagen fiber content increased exponentially with the severity of the injury. Elaunin and fully developed elastic fibers remained unchanged in the five groups, while oxytalan fibers increased only in the P25 and P30 groups. In conclusion, the pronounced mechanical changes at the tissue level and fibroelastogenesis happened at an early phase of the disease and even in mildly abnormal lung parenchyma., Keywords: elastance; collagen fibers; elastin; paraquat

In many cases, the biologic responses of cells to extracellular signals and the specificity of the responses cannot be explained solely on the basis of the interactions of known signaling proteins. Recently, scaffolding and adaptor proteins have been identified that organize signaling proteins in cells and that contribute to the nature and specificity of signaling pathways. In an effort to identify proteins that might organize the signaling system(s) activated by the extracellular Ca(2+) receptor (CaR), we used a bait construct representing the intracellular C terminus of the human CaR and the yeast two hybrid system to screen a human kidney cDNA library. We identified a clone representing the C-terminal 1042 amino acids (aa) of the cytoskeletal protein filamin (ABP-280). Analysis of truncation and deletion constructs of the CaR C terminus and the filamin cDNA clone demonstrated that the CaR and filamin interact via regions containing aa 907-997 of the CaR C terminus and aa 1566-1875 of filamin. Interaction of the two proteins in mammalian HEK-293 cells was demonstrated by co-immunoprecipitation and colocalization of them using immunofluorescence microscopy. The functional importance of their interaction was documented by transiently expressing the CaR in M2 melanoma cells that lack filamin, or in A7 melanoma cells that stably express filamin, and demonstrating that the CaR activated ERK only in the presence of filamin. Co-expression of the CaR with a peptide derived from the region of the CaR C terminus that interacts with filamin reduced the ability of the CaR to activate p42ERK in a dose-dependent manner, but did not inhibit the ability of the ET(A) receptor to activate ERK. The fact that filamin interacts with the CaR and other cell signaling proteins including mitogen-activated protein kinases and small GTPases, indicates that it may act as a scaffolding protein to organize cell signaling systems involving the CaR.

The contractile cycle of the cardiac myocyte is essentially controlled by the concentration of intracellular calcium ([Ca2+]i). Measurement of [Ca2+]i using Ca2+-dependent fluorescence and simultaneous monitoring of cell dynamics enable characterization of a variety of substances interacting with ion channels and contractile proteins. In this report we describe a novel method featuring up to 480 frames/s for monitoring rapid changes in cellular calcium and cell length, in which every individual cycle allows effective evaluation of major cell parameters. Computers aid in determination of time to peak (in ms), time to 50% decrease (ms), diastolic Ca2+ (relative fluorescence units, rfu), systolic Ca2+ (rfu), Ca2+ transients (rfu), DeltaCa2+/Delta(t) rise (rfu/s), and DeltaCa2+/Delta(t) fall (rfu/s). Contractile parameters are as follows: maximum cell length (microm), minimum cell length (microm), absolute cell shortening (microm), peak DeltaL/Delta(t) shortening (microm/s), and peak DeltaL/Delta(t) relaxation (microm/s). In summary, we succeeded in demonstrating that this system is a unique and valuable tool that allows simultaneous and accurate assessment of contractile parameters and of calcium movements of isolated adult cardiac myocytes.

Xenopus oocytes assemble an array of F-actin and myosin 2 around plasma membrane wounds. We analyzed this process in living oocytes using confocal time-lapse (four-dimensional) microscopy. Closure of wounds requires assembly and contraction of a classic "contractile ring" composed of F-actin and myosin 2. However, this ring works in concert with a 5-10-microm wide "zone" of localized actin and myosin 2 assembly. The zone forms before the ring and can be uncoupled from the ring by inhibition of cortical flow and contractility. However, contractility and the contractile ring are required for the stability and forward movement of the zone, as revealed by changes in zone dynamics after disruption of contractility and flow, or experimentally induced breakage of the contractile ring. We conclude that wound-induced contractile arrays are provided with their characteristic flexibility, speed, and strength by the combined input of two distinct components: a highly dynamic zone in which myosin 2 and actin preferentially assemble, and a stable contractile actomyosin ring.