Specification of actin filament function and molecular composition by tropomyosin isoforms

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5(NM1)), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5(NM1) was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.     

Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: evidence for distinct microfilament populations

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the alpha Tm(fast) and beta-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and alpha(f)9d (detects specific Tms from the alpha Tm(fast) and beta-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by alpha(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and alpha(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the alpha Tm(fast) and beta-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to alpha Tm(fast) and beta-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo.     

Tropomyosin isoforms define distinct microfilament populations with different drug susceptibility

Two tropomyosin isoforms, human Tm5(NM1) and Tm3, were over-expressed in B35 rat neuro-epithelial cells to examine preferential associations between specific actin and tropomyosin isoforms and to determine the role tropomyosin isoforms play in regulating the drug susceptibility of actin filament populations. Immunofluorescence staining and Western blot analysis were used to study the organisation of specific filament populations and their response to treatment with two widely used actin-destabilising drugs, latrunculin A and cytochalasin D. In Tm5(NM1) cells, we observed large stress fibres which showed predominant co-localisation of beta-actin and low-molecular-weight gamma-tropomyosin isoforms. Tm3 cells had an abundance of cellular protrusions which contained both the beta- and gamma-actin isoforms, predominately populated by high-molecular-weight alpha- and beta-tropomyosin isoforms. The stress fibres observed in Tm5(NM1) cells were more resistant to both latrunculin A and cytochalasin D than filaments containing the high-molecular-weight tropomyosins observed in Tm3 cells. Knockdown of the over-expressed Tm5(NM1) isoform with a human-specific Tm5(NM1) siRNA reversed the phenotype and caused a reversal in the observed drug resistance. We conclude that there are preferential associations between specific actin and tropomyosin isoforms, which are cell type specific, but it is the tropomyosin composition of a filament population which determines the susceptibility to actin-targeting drugs.     

Cytoskeletal Tropomyosin Tm5NM1 Is Required for Normal Excitation–Contraction Coupling in Skeletal Muscle

The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments; the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation–contraction coupling in skeletal muscle.     

Divergent regulation of the sarcomere and the cytoskeleton

The existence of a feedback mechanism regulating the precise amounts of muscle structural proteins, such as actin and the actin-associated protein tropomyosin (Tm), in the sarcomeres of striated muscles is well established. However, the regulation of nonmuscle or cytoskeletal actin and Tms in nonmuscle cell structures has not been elucidated. Unlike the thin filaments of striated muscles, the actin cytoskeleton in nonmuscle cells is intrinsically dynamic. Given the differing requirements for the structural integrity of the actin thin filaments of the sarcomere compared with the requirement for dynamicity of the actin cytoskeleton in nonmuscle cells, we postulated that different regulatory mechanisms govern the expression of sarcomeric versus cytoskeletal Tms, as key regulators of the properties of the actin cytoskeleton. Comprehensive analyses of tissues from transgenic and knock-out mouse lines that overexpress the cytoskeletal Tms, Tm3 and Tm5NM1, and a comparison with sarcomeric Tms provide evidence for this. Moreover, we show that overexpression of a cytoskeletal Tm drives the amount of filamentous actin.     

Ultra short yeast tropomyosins show novel myosin regulation

Tropomyosin (Tm) is an alpha-helical coiled-coil actin-binding protein present in all eukaryotes from yeast to man. Its functional role has been best described in muscle regulation; however its much wider role in cytoskeletal actin regulation is still to be clarified. Isoforms vary in size from 284 or 248 amino acids in vertebrates, to 199 and 161 amino acids in yeast, spanning from 7 to 4 actin binding sites respectively. In Saccharomyces cerevisiae, the larger yTm1 protein is produced by an internal 38-amino acid duplication, corresponding to a single actin-binding site. We have produced an ultra-short Tm with only 125 amino acids by removing both of the 38 amino acid repeats from yTm1, with the addition of an Ala-Ser extension used to mimic the essential N-terminal acetylation. This short Tm, and an M1T mutant of it, bind to actin with a similar affinity to most Tms previously studied (K(50%) approximately 0.5 microm). However, an equilibrium fluorescence binding assay shows a much greater inhibition of myosin binding to actin than any previously studied Tm. Actin cosedimentation assays show this is caused by direct competition for binding to actin. The M1T mutant shows a reduced inhibition, probably due to weaker end-to-end interactions making it easier for myosin to displace Tm. All previously characterized Tms, although able to sterically block the myosin-binding site, are able to bind to actin along with myosin. By showing that Tm can compete directly with myosin for the same binding site these new Tms provide direct evidence for the steric blocking model.     

Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function

Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform. Periodic repeats in the sequence have been proposed to correspond to actin binding sites. To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5). Recombinant Tms (unacetylated) were expressed in Escherichia coli. Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn). dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition. Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding. dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1. In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding. The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding. The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA. In the presence of Ca(2+), relief of inhibition by these Tms was incomplete. We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different.     

From skeletal muscle to cancer: insights learned elucidating the function of tropomyosin

The tropomyosins (Tms) are a family of actin filament binding proteins that possess a simple dimeric α-helical coiled-coil structure along their entire length. Our knowledge of Tm structure and function has greatly expanded since they were first discovered in skeletal muscle almost 65 years ago. In multicellular organisms they exhibit extensive cell type specific isoform diversity. In this essay we discuss the genetic mechanisms by which this diversity is generated and its significance to actin-based cellular functions.     

Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells

Tropomyosin (Tm) is known to be an important gatekeeper of actin function. Tm isoforms are encoded by four genes, and each gene produces several variants by alternative splicing, which have been proposed to play roles in motility, proliferation, and apoptosis. Smooth muscle studies have focused on gizzard smooth muscle, where a heterodimer of Tm from the α-gene (Tmsm-α) and from the β-gene (Tmsm-β) is associated with contractile filaments. In this study we examined Tm in differentiated mammalian vascular smooth muscle (dVSM). Liquid chromatography-tandem mass spectrometry (LC MS/MS) analysis and Western blot screening with variant-specific antibodies revealed that at least five different Tm proteins are expressed in this tissue: Tm6 (Tmsm-α) and Tm2 from the α-gene, Tm1 (Tmsm-β) from the β-gene, Tm5NM1 from the γ-gene, and Tm4 from the δ-gene. Tm6 is by far most abundant in dVSM followed by Tm1, Tm2, Tm5NM1, and Tm4. Coimmunoprecipitation and coimmunofluorescence studies demonstrate that Tm1 and Tm6 coassociate with different actin isoforms and display different intracellular localizations. Using an antibody specific for cytoplasmic γ-actin, we report here the presence of a γ-actin cortical cytoskeleton in dVSM cells. Tm1 colocalizes with cortical cytoplasmic γ-actin and coprecipitates with γ-actin. Tm6, on the other hand, is located on contractile bundles. These data indicate that Tm1 and Tm6 do not form a classical heterodimer in dVSM but rather describe different functional cellular compartments.     

Regulatory properties of tropomyosin effects of length, isoform, and N-terminal sequence

The regulatory properties of naturally occurring tropomyosins (Tms) of differing lengths have been examined. These Tms span from 4 to 7 actin subunits. Native proteins have been used to study the common 7 actin-spanning skeletal and smooth muscle variants and expressed recombinant proteins to study the shorter fibroblast 5a, 5b, yeast Tm1 and yeast Tm2 Tms (6, 6, 5, and 4 actin-spanning variants, respectively). The yTm2 has been overexpressed in Escherichia coli with N-terminal constructs equivalent to those previously used for yTm1 [Maytum, R., et al. (2000) Biochemistry 39, 11913]. The regulation of myosin subfragment 1 (S1) binding to actin by Tm has been assessed using a sensitive S1 binding titration. The equilibrium between closed and open (C to M states, KT = 0.1-0.14) was similar for all vertebrate Tms. Apart from skTm where the apparent cooperative unit size (n) is the same as the structural size (n = 7 actin sites), the other vertebrate Tms that were studied exhibited large n values (n = 12-14). The yeast Tms also exhibited large values of n (6-9) in comparison to their structural sizes (4-5). The determined value of KT depended on the N-terminal sequence (KT = 0.15-1). These results are compared with the effect of S1 upon Tm's affinity for actin. The yeast Tms have regulatory parameters similar to those of skTm, but unlike skTm, S1 has little effect upon their actin affinity. This shows that an actin state with a high affinity for S1 and Tm is not necessary for regulation, and the higher affinity of S1 for actin in the presence of vertebrate Tms is probably the result of a direct interaction of S1 with Tm.     

Targeting of a tropomyosin isoform to short microfilaments associated with the Golgi complex

A growing body of evidence suggests that the Golgi complex contains an actin-based filament system. We have previously reported that one or more isoforms from the tropomyosin gene Tm5NM (also known as gamma-Tm), but not from either the alpha- or beta-Tm genes, are associated with Golgi-derived vesicles (Heimann et al., (1999). J. Biol. Chem. 274, 10743-10750). We now show that Tm5NM-2 is sorted specifically to the Golgi complex, whereas Tm5NM-1, which differs by a single alternatively spliced internal exon, is incorporated into stress fibers. Tm5NM-2 is localized to the Golgi complex consistently throughout the G1 phase of the cell cycle and it associates with Golgi membranes in a brefeldin A-sensitive and cytochalasin D-resistant manner. An actin antibody, which preferentially reacts with the ends of microfilaments, newly reveals a population of short actin filaments associated with the Golgi complex and particularly with Golgi-derived vesicles. Tm5NM-2 is also found on these short microfilaments. We conclude that an alternative splice choice can restrict the sorting of a tropomyosin isoform to short actin filaments associated with Golgi-derived vesicles. Our evidence points to a role for these Golgi-associated microfilaments in vesicle budding at the level of the Golgi complex.     

Tropomyosin isoform switching in tumorigenic human fibroblasts.

We identified six tropomyosin (Tm) isoforms in diploid human fibroblasts. We used computerized microdensitometry of 2-dimensional protein profiles to measure the relative rates of synthesis and abundance of the individual Tm isoforms and actin, the two major structural constituents of microfilaments. In carcinogen-transformed human fibroblasts (HuT-14), the rates of synthesis of three Tm isoforms (Tm1, Tm2, and Tm6) were greatly decreased relative to normal diploid parental fibroblasts and to actin. In contrast, related nontumorigenic HuT fibroblasts which are "immortalized" and anchorage independent exhibited both slight down-regulation of Tm1 and Tm6 and 3.5-fold up-regulation of Tm3. Thus, Tm isoform switching from the predominance of the larger more avid Tm isoforms (Tm1, Tm2, Tm3, and Tm6) to the smaller, less avid Tm isoforms (Tm4 and Tm5) in microfilaments was a transformation-induced change correlated with tumorigenicity in human fibroblasts.     

Specific features of neuronal size and shape are regulated by tropomyosin isoforms

Spatially distinct populations of microfilaments, characterized by different tropomyosin (Tm) isoforms, are present within a neuron. To investigate the impact of altered tropomyosin isoform expression on neuronal morphogenesis, embryonic cortical neurons from transgenic mice expressing the isoforms Tm3 and Tm5NM1, under the control of the beta-actin promoter, were cultured in vitro. Exogenously expressed Tm isoforms sorted to different subcellular compartments with Tm5NM1 enriched in filopodia and growth cones, whereas the Tm3 was more broadly localized. The Tm5NM1 neurons displayed significantly enlarged growth cones accompanied by an increase in the number of dendrites and axonal branching. In contrast, Tm3 neurons displayed inhibition of neurite outgrowth. Recruitment of Tm5a and myosin IIB was observed in the peripheral region of a significant number of Tm5NM1 growth cones. We propose that enrichment of myosin IIB increases filament stability, leading to the enlarged growth cones. Our observations support a role for different tropomyosin isoforms in regulating interactions with myosin and thereby regulating morphology in specific intracellular compartments.     

Mechanisms of spatial segregation of actin isoforms

Actin sequences are conserved to a much greater degree than those of almost any other proteins, such that two cytoplasmic isoforms differ by only 4 out of 374 amino acid residues. Nevertheless, the results of biochemical, immunocytochemical, and molecular biology experiments demonstrate that the appearance, amount, and localization of actin isoforms are strongly controlled by the cellular machinery. Although at the early stages of cell differentiation expression of any actin gene is potentially possible, under normal physiological conditions, while differentiation proceeds, synthesis of specific actin isoforms is temporally regulated and the produced proteins are segregated spatially. Pathological situations of a tissue injury or a mammalian disease correlate either with up-and down-regulation of distinct actin genes returning to a fetal gene program or with a failure to sort actin isoforms. Different actin isoforms cannot substitute for each other, and changes in the expression of specific actin genes are accompanied by alterations in cell structure and function, suggesting that specific actin isoforms perform unique cellular functions. This article summarizes the data on the segregation of actin isoforms in cell compartments and analyzes the mechanisms suggested to explain spatial segregation of cytoplasmic actin isoforms in the cell.     

Tropomyosin Tm5NM1 Spatially Restricts Src Kinase Activity through Perturbation of Rab11 Vesicle Trafficking

In order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules.     

Tropomyosin 4 regulates adhesion structures and resorptive capacity in osteoclasts

Tropomyosins (Tms) are alpha-helical dimers that bind and stabilize actin microfilaments while regulating their accessibility to other actin-associated proteins. Four genes encode expression of over forty Tms, most of which are expressed in nonmuscle cells. In recent years, it has become clear that individual Tm isoforms may regulate specific actin pools within cells. In this study, we examined how osteoclast function may be regulated by the tropomyosin isoform Tm-4, which we previously showed to be highly localized to podosomes and sealing zones of osteoclasts. RNAi-mediated knockdown of Tm-4, both in RAW264.7- and mouse marrow-derived osteoclasts, resulted in thinning of the actin ring of the sealing zone. Knockdown of Tm-4 also resulted in diminished bone resorptive capacity and altered resorption pit shape. In contrast, osteoclasts overexpressing Tm-4 demonstrated thickened podosomes on glass as well as thickened, aberrant actin structures on bone, and diminished motility and resorptive capacity. These results indicate that Tm-4 plays a role in regulating adhesion structures of osteoclasts, most likely by stabilizing the actin microfilaments present in podosomes and the sealing zone.     

Mechanisms of spatial segregation of actin isoforms

Actin sequences are conserved to a much greater degree than those in almost any other proteins, so that two cytoplasmic isoforms differ by only four of 374 amino acid residues. Nevertheless, the results of biochemical, immunocytochemical and molecular biology experiments demonstrate that appearance, amount and localization of actin isoforms are strongly controlled by cell machinery. Although at the early stages of cell differentiation expression of any actin gene is potentially possible, under normal physiological conditions, while differentiation proceeds, synthesis of specific actin isoforms is temporally regulated and the produced proteins are segregated spatially. Pathological situations of tissue injury or mammalian disease correlate either with up- and down-regulation of distinct actin genes returning to a fetal gene program or with a failure to sort actin isoforms. Different actin isoforms cannot substitute for each other, and changes in expression of specific actin genes are accompanied by alterations in cell structure and function suggesting that specific actin isoforms perform unique cellular functions. This article summarizes the data on segregation of actin isoforms in cell compartments and analyses the mechanisms suggested to explain spatial segregation of cytoplasmic actin isoforms within a cell.     

Functional significance of isoform diversification in the protocadherin gamma gene cluster

The mammalian Protocadherin (Pcdh) alpha, beta, and gamma gene clusters encode a large family of cadherin-like transmembrane proteins that are differentially expressed in individual neurons. The 22 isoforms of the Pcdhg gene cluster are diversified into A-, B-, and C-types, and the C-type isoforms differ from all other clustered Pcdhs in sequence and expression. Here, we show that mice lacking the three C-type isoforms are phenotypically indistinguishable from the Pcdhg null mutants, displaying virtually identical cellular and synaptic alterations resulting from neuronal apoptosis. By contrast, mice lacking three A-type isoforms exhibit no detectable phenotypes. Remarkably, however, genetically blocking apoptosis rescues the neonatal lethality of the C-type isoform knockouts, but not that of the Pcdhg null mutants. We conclude that the role of the Pcdhg gene cluster in neuronal survival is primarily, if not specifically, mediated by its C-type isoforms, whereas a separate role essential for postnatal development, likely in neuronal wiring, requires isoform diversity.