Cytoskeletal elements in arthropod sensilla and mammalian photoreceptors.

Ciliary receptor cells, typified by cilia or modified cilia, are very common in the animal kingdom. In addition to the cytoskeleton of their ciliary processes these receptors possess other specific prominent cytoskeletal elements. Two representative systems are presented: i) scolopidia, mechanosensitive sensilla of various arthropod species; and ii) photoreceptor cells of the retina of the bovine eye. Two cytoskeletal structures are characteristic for arthropod scolopidia: a scolopale typifies the innermost auxiliary cell, and long ciliary rootlets are extending well into the sensory cells. The latter element is also characteristic for the inner segment of the photoreceptor cells in bovine. The scolopale of scolopidia is mainly composed of actin filaments. In the absence of myosin, the uniform polarity of the actin filaments and their association with tropomyosin all indicate a stabilizing role of the filament bundles within the scolopale. This function and a certain elasticity of actin filament bundles may be important during stimulation of the sensilla. The ciliary rootlets of both systems originate at the basal bodies at the ciliary base of the sensory cells and project proximally. These rootlets are composed of longitudinally oriented, fine filaments forming a characteristic regular cross-striation. An alpha-actinin immunoreactivity was detected within the ciliary rootlets of scolopidia. In addition, antibodies to centrin react with the rootlets of both types of receptors. Since centrin is largely responsible for the contraction of the flagellar rootlets in green algae, contraction may also occur in the ciliary rootlets of insect sensilla and vertebrate photoreceptors. In both systems, contraction or relaxation of the ciliary rootlets could serve in sensory transduction or adaptation.     

Actin filaments: the main components of the scolopale in insect sensilla

Summary Two types of insect sensilla, mechanosensitive scolopidia and thermo-/hygrosensitive poreless sensilla contain a scolopale, which consists of numerous microtubules embedded in bundles of filaments (7–10 nm in diameter). The bundles are readily seen in the electron microscope in cryofixed (high-pressure freezing and rapid injection) and substituted samples. The filaments can be identified as actin filaments by using fluorescent phalloidins. Both electron microscopy and Triton-extraction exeriments reveal mechanical linkage between the main components in both types of sensilla. Since myosin appears to be absent in the scolopale, the actin filaments are unlikely to be involved in any contraction mechanism; these filaments more probably provide mechanical stability. The functional properties of the scolopale are discussed.     

Centrin- and α-actinin-like immunoreactivity in the ciliary rootlets of insect sensilla

Summary Long ciliary rootlets are a characteristic feature of the dendritic inner segments of the sensory cells in insect sensilla. These rootlets are composed of highly ordered filaments and are regularly cross-striated. Collagenase digestion and immunohistochemistry reveal that the rootlets are probably not composed of collagen fibers. However, double-labeling experiments with phalloidin and anti--actinins show that antibodies to -actinin react with the ciliary rootlets of the sensilla, but do not stain the scolopale, which is composed of actin filaments as visualized by phalloidin. Antibodies to centrin, a contractile protein isolated from flagellar rootlets of green algae, also stain the ciliary rootlets. Within the ciliary rootlets of insect sensilla, -actinin may be associated with filaments other than actin filaments. The immunohistochemical localization of a centrin-like protein suggests that contractions probably occur within the rootlets. The centrin-like protein may play a role during the mechanical transduction or adaptation of the sensilla.     

Morphological, Biochemical and Immunohistochemical Studies on Heart Development in Cardiac Mutant Axolotls, Ambystoma mexicanum

SYNOPSIS. A naturally-occurring genetic mutation, designatedc for "cardiac lethal" in axolotls, Ambystoma mexicanum, isproving to be a useful model for studying myofibrillogenesisin differentiating heart cells. In this paper I describe morphological,biochemical and immunofluorescence studies which compare thecontractile proteins in normal and mutant hearts. In addition,morphological studies on anterior endoderm, an important heartinductor tissue in salamanders, are reviewed. Detailed electronmicroscopic studies show that normal heart myocytes containnumerous well-organized myofibrils. Although mutant heart cellscontain a few myosin and actin filaments, there are no organizedmyofibrils. Instead, amorphous proteinaceous collections areprominent in the peripheral cytoplasm of the cell where myofibrilswould be expected to first form. SDS-polyacrylamide gel electrophoresisshows that actin is present in almost normal amounts in mutanthearts, myosin heavy chain is reduced and tropomyosin is virtuallyabsent. Immunofluorescence studies reveal that myosin, -actininand tropomyosin are located prominently in theorganized myofibrilsof normal heart cells. In mutant hearts myosin is localizedalmost exclusively in the amorphous collections at the cellperipheries, -actinin also is distributed mainly in the peripheralcell cytoplasm. There is almost no staining for tropomyosin.Heavy meromyosin (HMM) binding experiments demonstrate thatthe actin in mutant heart cells is contained within the amorphouscollections in a non-filamentous state and the addition of HMMcauses its polymerization into filaments. In view of these findings,we undertook studies to determine whether there might be a causalrelationship between theabsence of tropomyosin in mutants andthe failure of actin to form into filaments. Our results indeedshow that addition of tropomyosin to glycerinated mutant heartsor homogenates of mutant hearts causes the amorphous actin toform into filaments. Thus, this single gene mutation resultsin mutant heart cells having reduced, but significant, amountsof myosin and actin, even though non-filamentous, and substantialamounts of -actinin. There is almost no tropomyosin. It is impliedthat the drastic reduction of tropomyosin in mutant cells issomehow related to the failure of normal myofilament formation,which in turn would seem to be an essential step in the normalorganization of myofibrils.     

Submembrane cytoskeleton of pigmented glial cells,primary pigment cells and crystalline cone cells in the honeybee compound eye

Summary The organization of the submembrane cytoskeleton of non-photoreceptive, accessory cells in the honeybee compound eye was examined using light-microscopic (phallotoxin labeling, immunohistochemistry) and electron-microscopic (decoration with myosin fragments) techniques. The crystalline cone cells contain numerous peripheral actin filaments oriented longitudinally with antiparallel polarity. Bundles of microtubules lie under the plasma membrane of primary pigment cells, in close apposition to the crystalline cone; they are interspersed with only a few actin filaments. Pigmented glial cells (secondary pigment cells) contain a two-dimensional filament/particle web lining their entire plasma membranes. Both filamentous actin and -spectrin are localized within the cortex of these cells, indicating that they are web components. The results demonstrate that the three cell types contain different cortical cytoskeletons, implying different functional properties.     

Leiomodin and tropomodulin in smooth muscle

Evidence isaccumulating to suggest that actin filament remodeling is critical forsmooth muscle contraction, which implicates actin filament ends asimportant sites for regulation of contraction. Tropomodulin (Tmod) andsmooth muscle leiomodin (SM-Lmod) have been found in many tissuescontaining smooth muscle by protein immunoblot and immunofluorescencemicroscopy. Both proteins cofractionate with tropomyosin in theTriton-insoluble cytoskeleton of rabbit stomach smooth muscle and aresolubilized by high salt. SM-Lmod binds muscle tropomyosin, abiochemical activity characteristic of Tmod proteins. SM-Lmod stainingis present along the length of actin filaments in rat intestinal smoothmuscle, while Tmod stains in a punctate pattern distinct from that ofactin filaments or the dense body marker -actinin. After smoothmuscle is hypercontracted by treatment with 10 mM Ca²⁺,both SM-Lmod and Tmod are found near -actinin at the periphery ofactin-rich contraction bands. These data suggest that SM-Lmod is anovel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth musclemay be capped by Tmod in localized clusters.

     

Regulation of myosin sliding alongChara actin bundles by native skeletal muscle tropomyosin

Summary Native tropomyosin from rabbit skeletal muscle introduced by intracellular perfusion intoChara cells inhibited the cytoplasmic streaming irrespective of the Ca²⁺ concentration. To find the action site of native tropomyosin inChara, the cytoplasmic streaming was reconstituted by introducing isolated endoplasm into actin donorChara cells from which native endoplasm had been removed. The reconstituted streaming was inhibited by pretreatment of the actin donor cells with native tropomyosin but not by that of the endoplasm, suggesting that the native tropomyosin inhibited the cytoplasmic streaming by binding toChara actin bundles. Staining of the actin bundles with FITC-labeled native tropomyosin also showed that the native tropomyosin could bind to the actin bundles. Streaming reconstituted fromChara actin bundles and skeletal muscle myosin was insensitive to Ca²⁺, but became sensitive on application of the native tropomyosin.Abbrevations APW artificial pond water - ATP adenosine 5-triphosphoric acid - BSA bovine serum albumin - EDTA ethylene diamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - FITC-NTM fluorescein isothiocyanate-labeled native tropomyosin - NTM native tropomyosin     

Myosin and Tropomyosin Stabilize the Conformation of Formin-nucleated Actin Filaments

The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.     

The Effect of Tropomyosin Mutations on Actin-Tropomyosin Binding: In Search of Lost Time

The initial binding of tropomyosin onto actin filaments and then its polymerization into continuous cables on the filament surface must be precisely tuned to overall thin-filament structure, function, and performance. Low-affinity interaction of tropomyosin with actin has to be sufficiently strong to localize the tropomyosin on actin, yet not so tight that regulatory movement on filaments is curtailed. Likewise, head-to-tail association of tropomyosin molecules must be favorable enough to promote tropomyosin cable formation but not so tenacious that polymerization precedes filament binding. Arguably, little molecular detail on early tropomyosin binding steps has been revealed since Wegner’s seminal studies on filament assembly almost 40 years ago. Thus, interpretation of mutation-based actin-tropomyosin binding anomalies leading to cardiomyopathies cannot be described fully. In vitro, tropomyosin binding is masked by explosive tropomyosin polymerization once cable formation is initiated on actin filaments. In contrast, in silico analysis, characterizing molecular dynamics simulations of single wild-type and mutant tropomyosin molecules on F-actin, is not complicated by tropomyosin polymerization at all. In fact, molecular dynamics performed here demonstrates that a midpiece tropomyosin domain is essential for normal actin-tropomyosin interaction and that this interaction is strictly conserved in a number of tropomyosin mutant species. Elsewhere along these mutant molecules, twisting and bending corrupts the tropomyosin superhelices as they “lose their grip” on F-actin. We propose that residual interactions displayed by these mutant tropomyosin structures with actin mimic ones that occur in early stages of thin-filament generation, as if the mutants are recapitulating the assembly process but in reverse. We conclude therefore that an initial binding step in tropomyosin assembly onto actin involves interaction of the essential centrally located domain.     

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

Summary Studies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cell's long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a marking pin-like configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 M caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 M, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.Abbreviations APM amiprophos-methyl - CD cytochalasin D - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - DMSO dimethyl sulfoxide - Rh-Ph rhodamine-phalloidin     

The effect of caldesmon on dynamic properties of F-actin alone and bound to heavy meromyosin and/or tropomyosin

The effect of caldesmon on the rotational dynamics of actin filaments alone or conjugated with heavy meromyosin and/or tropomyosin has been measured by the electron paramagnetic resonance (EPR) technique using a maleimide spin label rigidly bound to Cys374 of actin. The rotation of actin protomers in filaments and the angular distribution of spin probes on actin were determined by conventional EPR spectroscopy, while torsional motions within actin filaments were detected by saturation transfer EPR measurements. Binding of caldesmon to F-actin resulted in the reduction of torsional mobility of actin filaments. The maximum effect was produced at a ratio of about one molecule of caldesmon/seven actin protomers. Smooth muscle tropomyosin enhanced the effect of caldesmon, i.e. caused further slowing down of internal motions within actin filaments. Caldesmon increased the degree of order of spin labels on F-actin in macroscopically oriented pellets in the presence of tropomyosin but not in its absence. Computer analysis of the spectra revealed that caldesmon alone slightly changed the orientation of spin probes relative to the long axis of the filament. In the presence of tropomyosin this effect of caldesmon was potentiated and then approximately every twentieth protomer along the actin filament was affected. Caldesmon weakened the effect of heavy meromyosin both on the polarity of environment of the spin label attached to F-actin and on the degree of order of labels on actin in macroscopically oriented pellets. Whereas the former effect of caldesmon was independent of tropomyosin, the latter one was observed only in the absence of tropomyosin.     

Rate of binding of tropomyosin to actin filaments

The decrease of the rate of actin polymerization by tropomyosin molecules which bind near the ends of actin filaments was analyzed in terms of the rate of binding of tropomyosin to actin filaments. Monomeric actin was polymerized onto actin filaments in the presence of various concentrations of tropomyosin. At high concentrations of monomeric actin (c1) and low tropomyosin concentrations (ct) (c1/ct greater than 10), actin polymerization was not retarded by tropomyosin because actin polymerization was faster than binding of tropomyosin to actin filaments. At low actin concentrations and high tropomyosin concentrations (c1/ct less than 5), the rate of elongation of actin filaments was decreased because actin polymerization was slower than binding of tropomyosin at the ends of actin filaments. The results were quantitatively analyzed by a model in which it was assumed that actin-bound tropomyosin molecules which extend beyond the ends of actin filaments retard association of actin monomers with filament ends. Under the experimental conditions (100 mM KCl, 1 mM MgCl2, pH 7.5, 25 degrees C), the rate constant for binding of tropomyosin to actin filaments turned out to be about 2.5 X 10(6) to 4 X 10(6) M-1 S-1.     

Interior decoration: Tropomyosin in actin dynamics and cell migration

Cell migration and invasion requires the precise temporal and spatial orchestration of a variety of biological processes. Filaments of polymerized actin are critical players in these diverse processes, including the regulation of cell anchorage points (both cell-cell and cell-extracellular matrix), the uptake and delivery of molecules via endocytic pathways and the generation of force for both membrane protrusion and retraction. How the actin filaments are specialized for each of these discrete functions is yet to be comprehensively elucidated. The cytoskeletal tropomyosins are a family of actin associating proteins that form head-to-tail polymers which lay in the major groove of polymerized actin filaments. In the present review we summarize the emerging isoform-specific functions of tropomyosins in cell migration and invasion and discuss their potential roles in the specialization of actin filaments for the diverse cellular processes that together regulate cell migration and invasion.Key words: tropomyosin, actin, migration, invasion, cytoskeleton, actin dynamics, adhesionActin is the most abundant protein in eukaryotic cells and is critical for maintaining structural integrity. The polymerization of globular (G)-actin monomers forms actin filaments (F-actin),¹ which play a role in diverse and complex cellular functions including intercellular transport of organelles and vesicles,^2,3 cytokinesis,⁴ apoptosis⁵ and cell motility.⁶ Intricate details describing the molecular scale interactions between regulatory proteins and actin have been extensively investigated but the mechanistic control of diverse actin filament functions remain largely unclear. Recent improvements in analysis techniques⁷ and the use of physiologically relevant models of 3D cell culturing⁸ have now begun to reveal mechanisms of actin cytoskeleton regulation. Accruing evidence suggests that the actin decorating protein tropomyosin is a key regulator of actin filament specialization. Of particular interest is the impact that tropomyosin regulation has on actin filament activity during cell migration and invasion that underpins immunological cell homing, development, wound healing and metastasis.     

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 -type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.Hiroshi Yasuda and Katsuhiro Kanda contributed equally.     

Effect of the state of oxidation of cysteine 190 of tropomyosin on the assembly of the actin-tropomyosin complex

Tropomyosin, cross-linked at cysteine 190, was found to bind more weakly to actin filaments than uncross-linked tropomyosin. Cross-linking of tropomyosin can cause actin filaments nearly completely covered with tropomyosin to be uncovered almost completely. The critical monomer concentration of actin is not significantly changed by binding of cross-linked or uncross-linked tropomyosin to actin filaments. The binding curves were analyzed quantitatively, thereby taking into account the polar end-to-end contact of tropomyosin molecules bound by actin and the overlap of the seven subunit binding sites along the actin filament. Under the conditions of the experiment (80 mM KCl, 1 mM MgCl2, pH 7.5, 38-42 degrees C), the equilibrium constant for isolated binding of tropomyosin to actin filaments is in the range 1 x 10(3)-3 x 10(3) M-1. The equilibrium constants for binding of tropomyosin to binding sites along the actin filament with one or two neighbouring tropomyosin molecules are in the range of 10(6) or 10(8) to 10(9) M-1, respectively. The equilibrium constants for binding of tropomyosin to binding sites along the actin filament with one or two neighbouring tropomyosin molecules are in the range of 10(6) or 10(8) to 10(9) M-1, respectively. The equilibrium constants for cross-linked and uncross-linked tropomyosin differ by a factor of only about two. Owing to the highly cooperative binding, these differences are sufficient so that actin filaments nearly completely covered with uncross-linked tropomyosin are uncovered almost completely by cross-linking tropomyosin at cysteine 190.     

Effect of tropomyosin on formin-bound actin filaments

Formins are conservative proteins with important roles in the regulation of the microfilament system in eukaryotic cells. Previous studies showed that the binding of formins to actin made the structure of actin filaments more flexible. Here, the effects of tropomyosin on formin-induced changes in actin filaments were investigated using fluorescence spectroscopic methods. The temperature dependence of the Förster-type resonance energy transfer showed that the formin-induced increase of flexibility of actin filaments was diminished by the binding of tropomyosin to actin. Fluorescence anisotropy decay measurements also revealed that the structure of flexible formin-bound actin filaments was stabilized by the binding of tropomyosin. The stabilizing effect reached its maximum when all binding sites on actin were occupied by tropomyosin. The effect of tropomyosin on actin filaments was independent of ionic strength, but became stronger as the magnesium concentration increased. Based on these observations, we propose that in cells there is a molecular mechanism in which tropomyosin binding to actin plays an important role in forming mechanically stable actin filaments, even in the case of formin-induced rapid filament assembly.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Two general classes of cytoplasmic actin filaments in tissue culture cells: The role of tropomyosin

In the assembly of actin filaments that takes place during the spreading of a polulation of human lung cells, after trypsin detachment off the substratum and replating, tropomyosin exhibits a considrable lag in its association with the newly forming filament bundles; it begins to associate with them during the later stages of cell spreading as the actin filament bundles normally seen in interphase cells begin to organize. This lag is evident in a number of cell types that are spreading onto a substratum; it does not appear to be due to a selective degradation of this molecule during rounding up of the cells, since tropmyosin associates with the actin filament bundles after this lag even under conditions where the protein synthetic activity of the cell is inhibited to more than 95% by cycloheximide. The preferential binding of tropomyosin to fully assembled filament bundles but not to newly formed bundles of actin filaments suggests therefore the existence of two classes of action filaments: those that bind tropomyosin and those that do not. This selective localization of tropomyosin and those that do not. This selective localization of tropomyosin on actin filaments was further pursued by examining the localization of this molecule in membrane ruffles. The immunofluorescent results indicate that ruffling is an actin-filament-dependent, microtubule-independent phenomenon. Tropomyosin is absent from membrane ruffles under a variety of circumstances where ruffling is expressed and, more generally, from any other cellular activity where actin filaments are expected to be in a dynamic state of reorganization or are required to be in a flexible configuraion. It is concluded that in tissue culture cells tropomyosin binds preferentially to actin filaments involved in structural support to confer rigidity upon them as well as aid them in maintaining a stretched phenotype. The absence of tropomyosin from certain motile phenomena where actin filaments are involved indicates that these classes of actin filaments are regulated by cytoplasmic mechanisms distinct from that by which tropomyosin (and troponin) mediates contractility in skeletal mulscle; it opens the possibility that different types of actin filaments enagaged in different cellular motile phenomenon in tissue culture cells may be regulated by a host of coexisting regulatory mechanisms, some as yet undetermined.     

Dual roles of tropomyosin as an F-actin stabilizer and a regulator of muscle contraction in Caenorhabditis elegans body wall muscle

Tropomyosin is a well-characterized regulator of muscle contraction. It also stabilizes actin filaments in a variety of muscle and non-muscle cells. Although these two functions of tropomyosin could have different impacts on actin cytoskeletal organization, their functional relationship has not been studied in the same experimental system. Here, we investigated how tropomyosin stabilizes actin filaments and how this function is influenced by muscle contraction in Caenorhabditis elegans body wall muscle. We confirmed the antagonistic role of tropomyosin against UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament organization using multiple UNC-60B mutant alleles. Tropomyosin was also antagonistic to UNC-78 (AIP1) in vivo and protected actin filaments from disassembly by UNC-60B and UNC-78 in vitro, suggesting that tropomyosin protects actin filaments from the ADF/cofilin-AIP1 actin disassembly system in muscle cells. A mutation in the myosin heavy chain caused greater reduction in contractility than tropomyosin depletion. However, the myosin mutation showed much weaker suppression of the phenotypes of ADF/cofilin or AIP1 mutants than tropomyosin depletion. These results suggest that muscle contraction has only minor influence on the tropomyosin's protective role against ADF/cofilin and AIP1, and that the two functions of tropomyosin in actin stability and muscle contraction are independent of each other.     

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

Summary In the primitive red algaCyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study.C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules ofC. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against aC. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin immature contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments.Abbreviations ORF open reading frame - IPTG isopropyl--D(–)-thiogalactopyranoside - SDS-PAGE sodium dodecyl sulphate-poly-acrylamide gel electrophoresis - DAPI 4,6-diamidino-2-phenylindole