Galina Schevzov

A Molecular Pathway for Myosin II Recruitment to Stress Fibers

BACKGROUND Cell migration and morphogenesis are driven by both protrusive and contractile actin filament structures. The assembly mechanisms of lamellipodial and filopodial actin filament arrays, which provide the force for plasma membrane protrusions through actin filament treadmilling, are relatively well understood. In contrast, the mechanisms by which contractile actomyosin arrays such as stress fibers are generated in cells, and how myosin II is specifically recruited to these structures, are not known. RESULTS We demonstrate that four functionally distinct tropomyosins are required for assembly of stress fibers in cultured osteosarcoma cells. Tm1, Tm2/3, and Tm5NM1/2 stabilize actin filaments at distinct stress fiber regions. In contrast, Tm4 promotes stress fiber assembly by recruiting myosin II to stress fiber precursors. Elimination of any one of the tropomyosins fatally compromises stress fiber formation. Importantly, Dia2 formin is critical to stress fiber assembly by nucleating Tm4-decorated actin filaments at the cell cortex. Myosin II is specifically recruited through a Tm4-dependent mechanism to the Dia2-nucleated filaments, which subsequently assemble endwise with Arp2/3-nucleated actin filament structures to yield contractile stress fibers. CONCLUSIONS These experiments identified a pathway, involving Dia2- and Arp2/3-promoted actin filament nucleation and several functionally distinct tropomyosins, that is required for generation of contractile actomyosin arrays in cells.

Tissue-specific tropomyosin isoform composition.

Four distinct genes encode tropomyosin (Tm) proteins, integral components of the actin microfilament system. In non-muscle cells, over 40 Tm isoforms are derived using alternative splicing. Distinct populations of actin filaments characterized by the composition of these Tm isoforms are found differentially sorted within cells (Gunning et al. 1998b). We hypothesized that these distinct intracellular compartments defined by the association of Tm isoforms may allow for independent regulation of microfilament function. Consequently, to understand the molecular mechanisms that give rise to these different microfilaments and their regulation, a cohort of fully characterized isoform-specific Tm antibodies was required. The characterization protocol initially involved testing the specificity of the antibodies on bacterially produced Tm proteins. We then confirmed that these Tm antibodies can be used to probe the expression and subcellular localization of different Tm isoforms by Western blot analysis, immunofluorescence staining of cells in culture, and immunohistochemistry of paraffin wax-embedded mouse tissues. These Tm antibodies, therefore, have the capacity to monitor specific actin filament populations in a range of experimental systems.

Tropomyosin Localization Reveals Distinct Populations of Microfilaments in Neurites and Growth Cones

The functional and structural differences between neurites and growth cones suggests the possibility that distinct microfilament populations may exist in each domain. Tropomyosins are integral components of the actin-based microfilament system. Using antibodies which detect three different sets of tropomyosin isoforms, we found that the vast majority of tropomyosin was found in a microfilament-enriched fraction of cultured cortical neurons, therefore enabling us to use the antisera to evaluate compositional differences in neuritic and growth cone microfilaments. An antibody which reacts with all known nonmuscle isoforms of the alpha Tms gene (Tm5NM1-4) stains both neurites and growth cones, whereas a second antibody against the isoform subset, Tm5NM1-2, reacts only with the neurite. A third antibody which reacts with the Tm5a/5b isoforms encoded by a separate gene from alpha Tms was strongly reactive with both neurites and growth cones in 16-h cultures but only with the neurite shaft in 40-h cultures. Treatment of neurons with cytochalasin B allowed neuritic Tm5NM1-2 to spread into growth cones. Removal of the drug resulted in the disappearance of Tm5NM1-2 from the growth cone, indicating that isoform segregation is an active process dependent on intact microfilaments. Treatment of 40-h cultures with nocodazole resulted in the removal of Tm5NM1-2 from the neurite whereas Tm5a/5b now spread back into the growth cone. We conclude that the organization of Tm5NM1-2 and Tm5a/5b in the neurite is at least partially dependent on microtubule integrity. These results indicate that tropomyosin isoforms Tm5NM1-2, Tm5NM3-4, and Tm5a/5b mark three distinct populations of actin filaments in neurites and growth cones. Further, the composition of microfilaments differs between neurites and growth cones and is subject to temporal regulation.

Tropomyosin isoforms and reagents

Tropomyosins are rod-like dimers which form head-to-tail polymers along the length of actin filaments and regulate the access of actin binding proteins to the filaments. The diversity of tropomyosin isoforms, over 40 in mammals, and their role in an increasing number of biological processes presents a challenge both to experienced researchers and those new to this field. The increased appreciation that the role of these isoforms expands beyond that of simply stabilizing actin filaments has lead to a surge of reagents and techniques to study their function and mechanisms of action. This report is designed to provide a basic guide to the genes and proteins and the availability of reagents which allow effective study of this family of proteins. We highlight the value of combining multiple techniques to better evaluate the function of different Tm isoforms and discuss the limitations of selected reagents. Brief background material is included to demystify some of the unfortunate complexity regarding this multi-gene family of proteins including the unconventional nomenclature of the isoforms and the evolutionary relationships of isoforms between species. Additionally, we present step-by-step detailed experimental protocols used in our laboratory to assist new comers to the field and experts alike.

Specialisation of the Tropomyosin Composition of Actin Filaments Provides New Potential Targets for Chemotherapy

The actin microfilament network is important in maintaining cell shape and function in eukaryotic cells. It has a multitude of roles in cellular processes such as cell adhesion, motility, cellular signalling, intracellular trafficking and cytokinesis. Alterations in the organisation of the cytoskeleton and changes in cellular morphology, motility and adhesiveness are characteristic features of transformed cancer cells. For this reason cytoskeletal microfilaments have become promising targets for chemotherapy. In contrast to the microtubules, which have been targeted successfully with anti-tumour drugs such as Taxol-like compounds and the Vinca alkaloids, very few actin targeting drugs have been characterised. To date, no actin targeting drugs have been used in clinical trials due to their severe cytotoxicity. One reason for this cytotoxicity is that drugs such as the cytochalasins and latrunculins disrupt actin microfilaments in both non-tumour and tumour cells. To circumvent this problem, actin filament populations need to be targeted more specifically. Not all actin filaments are the same and there is growing evidence that within a cell there are different populations of actin filaments which are spatially organised into distinct cellular compartments each with a unique function. The structure and function of the actin cytoskeleton is primarily regulated by the associated actin binding proteins. Tropomyosin is an intrinsic component of most actin filaments and over 40 isoforms have been identified in non-muscle cells. Tm isoforms are spatially segregated and current evidence suggests that they can specify the functional capacity of the actin microfilaments. Therefore the composition of these functionally distinct actin filaments may be important in determining their stability and function within the cell. If actin filament populations can be discriminated and targeted based on their tropomyosin composition then this becomes a powerful approach for anticancer therapy.

Intracellular Localization of Tropomyosin mRNA and Protein Is Associated with Development of Neuronal Polarity

Neuronal differentiation involves extensive rearrangement of the cytoskeleton, including the actin-based microfilament system, and establishment of molecular compartments within the neuron. The intracellular distribution of tropomyosin (Tm) mRNA in vivo and in vitro has been examined and correlated with protein targetting. The mRNAs encoding two Tm isoforms were found to be differentially localized in developing neurons. Tm-5 mRNA is localized to the axonal pole of differentiating embryonic rat neurons, in contrast to TmBr-2 mRNA distribution throughout the cell body. Tm-5 mRNA is transported into the axon of differentiating primary cultured neurons. This mRNA localization is developmentally regulated and correlates with the targeting of Tm-5 protein to growing axons. Tm-5 colocalizes with a subset of neuronal microfilaments associated with the initiation and maintenance of outgrowth. The segregation of Tm-5 is the earliest known marker of neuronal polarity and may play a role in the establishment of polarity.

A Novel Class of Anticancer Compounds Targets the Actin Cytoskeleton in Tumor Cells

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.

Tropomyosin isoform expression regulates the transition of adhesions to determine cell speed and direction.

ABSTRACT The balance of transition between distinct adhesion types contributes to the regulation of mesenchymal cell migration, and the characteristic association of adhesions with actin filaments led us to question the role of actin filament-associating proteins in the transition between adhesive states. Tropomyosin isoform association with actin filaments imparts distinct filament structures, and we have thus investigated the role for tropomyosins in determining the formation of distinct adhesion structures. Using combinations of overexpression, knockdown, and knockout approaches, we establish that Tm5NM1 preferentially stabilizes focal adhesions and drives the transition to fibrillar adhesions via stabilization of actin filaments. Moreover, our data suggest that the expression of Tm5NM1 is a critical determinant of paxillin phosphorylation, a signaling event that is necessary for focal adhesion disassembly. Thus, we propose that Tm5NM1 can regulate the feedback loop between focal adhesion disassembly and focal complex formation at the leading edge that is required for productive and directed cell movement.

Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy

Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

Distinct localizations of tropomyosin isoforms in LLC‐PK1 epithelial cells suggests specialized function at cell–cell adhesions

At least eight nonmuscle, nonbrain tropomyosin isoforms have been described. We used antibodies, microinjection, and transfection to characterize their expression and localization in LLC-PK1 kidney epithelial cells and compared them with other cells. Similar to primary enterocytes, LLC-PK1 cells exhibited predominantly TM-1 and TM-3 of the high-molecular-weight (HMW) isoforms; TM-5 and TM-5b of the low-molecular-weight (LMW) isoforms. Neither TM-4 nor TM-5a was detectable in the LLC-PKI cells. Immunofluorescence studies revealed that HMW isoforms were localized only on stress fibers, not adhesion belts, whereas the adhesion belts were stained by LMW isoform antibodies. When exogenous proteins are introduced either by transfection or microinjection, the HMW isoforms do not incorporate into the adhesion belt, whereas the LMW isoforms can incorporate into the stress fibers, thus indicating there are different mechanisms at work for the selective localization. Temporal changes in the microfilament system of the LLC-PK1 cells were studied during differentiation in culture as defined by spectrin expression and F-actin architecture. Western blot analysis indicated that TM-5b is only expressed in the LLC-PK1 cells after a certain degree of maturation in culture, which suggests isoform switching after the cell-cell contacts are developed. Collectively these results demonstrate that epithelial cells express a complex pattern of TM isoforms, which exhibit differential localizations within the cells and different patterns of expression depending on their origin and stage of differentiation. The implication of differential localization of TM isoforms on their specific functions is discussed.