Ras Transformation Overrides a Proliferation Defect Induced by Tpm3.1 Knockout.

Extensive re-organisation of the actin cytoskeleton and changes in the expression of its binding proteins is a characteristic feature of cancer cells. Previously we have shown that the tropomyosin isoform Tpm3.1, an integral component of the actin cytoskeleton in tumor cells, is required for tumor cell survival. Our objective was to determine whether cancer cells devoid of Tpm3.1 would evade the tumorgenic effects induced by H-Ras transformation. The tropomyosin isoform (Tpm) expression profile of a range of cancer cell lines (21) demonstrates that Tpm3.1 is one of the most broadly expressed Tpm isoform. Consequently, the contribution of Tpm3.1 to the transformation process was functionally evaluated. Primary embryonic fibroblasts isolated from wild type (WT) and Tpm3.1 knockout (KO) mice were transduced with retroviral vectors expressing SV40 large T antigen and an oncogenic allele of the H-Ras gene, H-RasV12, to generate immortalized and transformed WT and KO MEFs respectively. We show that Tpm3.1 is required for growth factor-independent proliferation in the SV40 large T antigen immortalized MEFs, but this requirement is overcome by H-Ras transformation. Consistent with those findings, we found that Tpm3.1 was not required for anchorage independent growth or growth of H-Ras-driven tumors in a mouse model. Finally, we show that pERK and Importin 7 protein interactions are significantly decreased in the SV40 large T antigen immortalized KO MEFs but not in the H-Ras transformed KO cells, relative to control MEFs. The data demonstrate that H-Ras transformation overrides a requirement for Tpm3.1 in growth factor-independent proliferation of immortalized MEFs. We propose that in the SV40 large T antigen immortalized MEFs, Tpm3.1 is partly responsible for the efficient interaction between pERK and Imp7 resulting in cell proliferation, but this is overidden by Ras transformation.

Tropomyosins induce neuritogenesis and determine neurite branching patterns in B35 neuroblastoma cells.

BACKGROUND: The actin cytoskeleton is critically involved in the regulation of neurite outgrowth. RESULTS: The actin cytoskeleton-associated protein tropomyosin induces neurite outgrowth in B35 neuroblastoma cells and regulates neurite branching in an isoform-dependent manner. CONCLUSIONS: Our data indicate that tropomyosins are key regulators of the actin cytoskeleton during neurite outgrowth. SIGNIFICANCE: Revealing the molecular machinery that regulates the actin cytoskeleton during neurite outgrowth may provide new therapeutic strategies to promote neurite regeneration after nerve injury. SUMMARY: The formation of a branched network of neurites between communicating neurons is required for all higher functions in the nervous system. The dynamics of the actin cytoskeleton is fundamental to morphological changes in cell shape and the establishment of these branched networks. The actin-associated proteins tropomyosins have previously been shown to impact on different aspects of neurite formation. Here we demonstrate that an increased expression of tropomyosins is sufficient to induce the formation of neurites in B35 neuroblastoma cells. Furthermore, our data highlight the functional diversity of different tropomyosin isoforms during neuritogenesis. Tropomyosins differentially impact on the expression levels of the actin filament bundling protein fascin and increase the formation of filopodia along the length of neurites. Our data suggest that tropomyosins are central regulators of actin filament populations which drive distinct aspects of neuronal morphogenesis.

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 ß-gene (Tmsm-ß) 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-ß) from the ß-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.

Alternatively spliced N-terminal exons in tropomyosin isoforms do not act as autonomous targeting signals.

Tropomyosin (Tm) polymerises head-to-tail to form a continuous polymer located in the major groove of the actin filament. Multiple Tm isoforms are generated by alternative splicing of four genes, and individual isoforms show specific localisation patterns in many cell types, and can have differing effects on the actin cytoskeleton. Fluorescently-tagged Tm isoforms and mutants were expressed in C2C12 cells to investigate the mechanisms of alternative localisation of high molecular weight (HMW) and low molecular weight (LMW) Tms. Fluorescently-tagged Tm constructs show similar localisation to endogenous Tms as observed by antibodies, with the HMW Tm3 relatively diminished at the periphery of cells compared to LMW isoforms Tm5b or Tm5NM1. Tm3 and Tm5b only differ in their N-terminal exons, but these N-terminal exons do not independently direct localisation within the cell, as chimeric mutants Tm3-Tm5NM1 and Tm5b-Tm5NM1 show an increased peripheral localisation similar to Tm5NM1. The lower abundance of Tm3 at the periphery of the cell is not a result of different protein dynamics, as Tm3 and Tm5b show similar recovery after photobleaching. The relative exclusion of Tm3 from the periphery of cells does, however, require interaction with the actin filament, as mutants with truncations at either the N-terminus or the C-terminus are unable to localise to actin stress fibres, and are present in the most peripheral regions of the cell. We conclude that it is the entire Tm molecule which is the unit of sorting, and that the alternatively spliced N-terminal exons do not act as autonomous targeting signals.

Methylguanine DNA methyltransferase-mediated drug resistance-based selective enrichment and engraftment of transplanted stem cells in skeletal muscle.

Cell replacement therapy using stem cell transplantation holds much promise in the field of regenerative medicine. In the area of hematopoietic stem cell transplantation, O(6)-methylguanine-DNA methyltransferase MGMT (P140K) gene-mediated drug resistance-based in vivo enrichment strategy of donor stem cells has been shown to achieve up to 75%-100% donor cell engraftment in the host's hematopoietic stem cell compartment following repeated rounds of selection. This strategy, however, has not been applied in any other organ system. We tested the feasibility of using this MGMT (P140K)-mediated enrichment strategy for cell transplantation in skeletal muscles of mice. We demonstrate that muscle cells expressing an MGMT (P140K) drug resistance gene can be protected and selectively enriched in response to alkylating chemotherapy both in vitro and in vivo. Upon transplantation of MGMT (P140K)-expressing male CD34(+ve) donor stem cells isolated from regenerating skeletal muscle into injured female muscle treated with alkylating chemotherapy, donor cells showed enhanced engraftment in the recipient muscle 7 days following transplantation as examined by quantitative-polymerase chain reaction using Y-chromosome specific primers. Fluorescent in situ hybridization analysis using a Y-chromosome paint probe revealed donor-derived de novo muscle fiber formation in the recipient muscle 14 days following transplantation, with approximately 12.5% of total nuclei within the regenerated recipient muscle being of donor origin. Following engraftment, the chemo-protected donor CD34(+ve) cells induced substantial endogenous regeneration of the chemo-ablated host muscle that is otherwise unable to self-regenerate. We conclude that the MGMT (P140K)-mediated enrichment strategy can be successfully implemented in muscle.

Tropomyosin isoforms: divining rods for actin cytoskeleton function.

Actin filament functional diversity is paralleled by variation in the composition of isoforms of tropomyosin in these filaments. Although the role of tropomyosin is well understood in skeletal muscle, where it regulates the actin-myosin interaction, its role in the cytoskeleton has been obscure. The intracellular sorting of tropomyosin isoforms indicated a role in spatial specialization of actin filament function. Genetic manipulation and protein chemistry studies have confirmed that these isoforms are functionally distinct. Tropomyosins differ in their recruitment of myosin motors and their interaction with actin filament regulators such as ADF-cofilin. Tropomyosin isoforms have therefore provided a powerful mechanism to diversify actin filament function in different intracellular compartments.

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.

Myosin II governs intracellular pressure and traction by distinct tropomyosin-dependent mechanisms.

Two-dimensional (2D) substrate rigidity promotes myosin II activity to increase traction force in a process negatively regulated by tropomyosin (Tpm) 2.1. We recently discovered that actomyosin contractility can increase intracellular pressure and switch tumor cells from low-pressure lamellipodia to high-pressure lobopodial protrusions during three-dimensional (3D) migration. However, it remains unclear whether these myosin II-generated cellular forces are produced simultaneously, and by the same molecular machinery. Here we identify Tpm 1.6 as a positive regulator of intracellular pressure and confirm that Tpm 2.1 is a negative regulator of traction force. We find that Tpm 1.6 and 2.1 can control intracellular pressure and traction independently, suggesting these myosin II-dependent forces are generated by distinct mechanisms. Further, these tropomyosin-regulated mechanisms can be integrated to control complex cell behaviors on 2D and in 3D environments.

Tropomyosin gene expression in vivo and in vitro.

The evolution from unicellular to multicellular organisms of increasing complexity is paralleled by increased numbers of tropomyosin (Tm) genes and increasing numbers ofisoforms encoded by each gene. The regulation of Tm isoform expression is intimately associated with the morphological changes that take place during development and cell differentiation. The tissue- and cell-specific Tm expression patterns are regulated at multiple levels, allowing precise spatial and temporal regulation of Tm expression. In this chapter, we review the Tm isoform expression pattern during differentiation of different tissue types and from this data infer some general principles regarding Tm expression patterns during differentiation. Finally, we review the mechanisms that account for the highly regulated repertoire ofTm isoform expression.

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.

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.

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.

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.

Loss of Tpm4.1 leads to disruption of cell-cell adhesions and invasive behavior in breast epithelial cells via increased Rac1 signaling.

Here we report the identification and characterization of a novel high molecular weight isoform of tropomyosin, Tpm4.1, expressed from the human TPM4 gene. Tpm4.1 expression is down-regulated in a subset of breast cancer cells compared with untransformed MCF10A breast epithelial cells and in highly metastatic breast cancer cell lines derived from poorly metastatic MDA-MD-231 cells. In addition, patients with invasive ductal breast carcinoma show decreased TPM4 expression compared with patients with ductal breast carcinoma in situ, and low TPM4 expression is associated with poor prognosis. Loss of Tpm4.1 using siRNA in MCF10A cells increases cell migration in wound-healing and Boyden chamber assays and invasion out of spheroids as well as disruption of cell-cell adhesions. Down-regulation of Tpm4.1 in MDA-MB-231 cells leads to disruption of actin organization and increased cell invasion and dissemination from spheroids into collagen gels. The down-regulation of Tpm4.1 induces Rac1-mediated alteration of myosin IIB localization, and pharmacologic inhibition of Rac1 or down-regulation of myosin IIB using siRNA inhibits the invasive phenotypes in MCF10A cells. Thus Tpm4.1 plays an important role in blocking invasive behaviors through Rac1-myosin IIB signaling and our findings suggest that decreased expression of Tpm4.1 might play a crucial role during tumor progression.

ER/Golgi trafficking is facilitated by unbranched actin filaments containing Tpm4.2.

We have identified novel actin filaments defined by tropomyosin Tpm4.2 at the ER. EM analysis of mouse embryo fibroblasts (MEFs) isolated from mice expressing a mutant Tpm4.2 (Tpm4Plt53/Plt53 ), incapable of incorporating into actin filaments, revealed swollen ER structures compared with wild-type (WT) MEFs (Tpm4+/+ ). ER-to-Golgi, but not Golgi-to-ER trafficking was altered in the Tpm4Plt53/Plt53 MEFs following the transfection of the temperature sensitive ER-associated ts045-VSVg construct. Exogenous Tpm4.2 was able to rescue the ER-to-Golgi trafficking defect in the Tpm4Plt53/Plt53 cells. The treatment of WT MEFs with the myosin II inhibitor, blebbistatin, blocked the Tpm4.2-dependent ER-to-Golgi trafficking. The lack of an effect on ER-to-Golgi trafficking following treatment of MEFs with CK666 indicates that branched Arp2/3-containing actin filaments are not involved in anterograde vesicle trafficking. We propose that unbranched, Tpm4.2-containing filaments have an important role in maintaining ER/Golgi structure and that these structures, in conjunction with myosin II motors, mediate ER-to-Golgi trafficking.

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia.

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea-induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage-dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.

Tropomyosin Promotes Lamellipodial Persistence by Collaborating with Arp2/3 at the Leading Edge.

At the leading edge of migrating cells, protrusion of the lamellipodium is driven by Arp2/3-mediated polymerization of actin filaments [1]. This dense, branched actin network is promoted and stabilized by cortactin [2, 3]. In order to drive filament turnover, Arp2/3 networks are remodeled by proteins such as GMF, which blocks the actin-Arp2/3 interaction [4, 5], and coronin 1B, which acts by directing SSH1L to the lamellipodium where it activates the actin-severing protein cofilin [6, 7]. It has been shown in vitro that cofilin-mediated severing of Arp2/3 actin networks results in the generation of new pointed ends to which the actin-stabilizing protein tropomyosin (Tpm) can bind [8]. The presence of Tpm in lamellipodia, however, is disputed in the literature [9-19]. Here, we report that the Tpm isoforms 1.8/9 are enriched in the lamellipodium of fibroblasts as detected with a novel isoform-specific monoclonal antibody. RNAi-mediated silencing of Tpm1.8/9 led to an increase of Arp2/3 accumulation at the cell periphery and a decrease in the persistence of lamellipodia and cell motility, a phenotype consistent with cortactin- and coronin 1B-deficient cells [2, 7]. In the absence of coronin 1B or cofilin, Tpm1.8/9 protein levels are reduced while, conversely, inhibition of Arp2/3 with CK666 leads to an increase in Tpm1.8/9 protein. These findings establish a novel regulatory mechanism within the lamellipodium whereby Tpm collaborates with Arp2/3 to promote lamellipodial-based cell migration.

Smooth muscle-specific alpha tropomyosin is a marker of fully differentiated smooth muscle in lung.

Tropomyosin (Tm) is one of the major components of smooth muscle. Currently it is impossible to easily distinguish the two major smooth muscle (sm) forms of Tm at a protein level by immunohistochemistry due to lack of specific antibodies. Alpha-sm Tm contains a unique 2a exon not found in any other Tm. We have produced a polyclonal antibody to this exon that specifically detects alpha-sm Tm. We demonstrate here the utility of this antibody for the study of smooth muscle. The tissue distribution of alpha-sm Tm was shown to be highly specific to smooth muscle. Alpha-sm Tm showed an identical profile and tissue colocalization with alpha-sm actin both by Western blotting and immunohistochemistry. Using lung as a model organ system, we examined the developmental appearance of alpha-sm Tm in comparison to alpha-sm actin in both the mouse and human. Alpha-sm Tm is a late-onset protein, appearing much later than actin in both species. There were some differences in onset of appearance in vascular and airway smooth muscle with airway appearing earlier. Alpha-sm Tm can therefore be used as a good marker of mature differentiated smooth muscle cells. Along with alpha-sm actin and sm-myosin antibodies, alpha-sm Tm is a valuable tool for the study of smooth muscle.

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.

Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.