Search

The actin cytoskeleton plays a central role in many essential cellular processes. Its involvement requires actin filaments to form multiple populations with different structural and therefore functional properties in specific subcellular locations. This diversity is facilitated through the interaction between actin and a number of actin binding proteins. One family of proteins, the tropomyosins, are absolutely essential in regulating actin's ability to form such diverse structures. In this review we integrate studies from different organisms and cell types in an attempt to provide a unifying view of tropomyosin dependent regulation of the actin cytoskeleton.

Tropomyosins are rodlike coiled coil dimers that form continuous polymers along the major groove of most actin filaments. In striated muscle, tropomyosin regulates the actin-myosin interaction and, hence, contraction of muscle. Tropomyosin also contributes to most, if not all, functions of the actin cytoskeleton, and its role is essential for the viability of a wide range of organisms. The ability of tropomyosin to contribute to the many functions of the actin cytoskeleton is related to the temporal and spatial regulation of expression of tropomyosin isoforms. Qualitative and quantitative changes in tropomyosin isoform expression accompany morphogenesis in a range of cell types. The isoforms are segregated to different intracellular pools of actin filaments and confer different properties to these filaments. Mutations in tropomyosins are directly involved in cardiac and skeletal muscle diseases. Alterations in tropomyosin expression directly contribute to the growth and spread of cancer. The functional specificity of tropomyosins is related to the collaborative interactions of the isoforms with different actin binding proteins such as cofilin, gelsolin, Arp 2/3, myosin, caldesmon, and tropomodulin. It is proposed that local changes in signaling activity may be sufficient to drive the assembly of isoform-specific complexes at different intracellular sites.

Tropomyosin 1 (TM1) is downregulated in a number of transformed cell types, and exogenous expression of TM1 can restore actin organisation and reverse cellular transformation. We find that TM1 is also downregulated in human neuroblastoma cell lines, correlating with increasing malignancy. However, exogenous TM1 does not restore actin cytoskeleton organisation in neuroblastoma cells.

The generation of isoforms via gene duplication and alternative splicing has been a valuable evolutionary tool for the creation of biological diversity. In addition to the formation of molecules with related but different functional characteristics, it is now apparent that isoforms can be segregated into different intracellular sites within the same cell. Sorting has been observed in a wide range of genes, including those encoding structural molecules, receptors, channels, enzymes, and signaling molecules. This results in the creation of intracellular compartments that (a) can be independently controlled and (b) have different functional properties. The sorting mechanisms are likely to operate at the level of both proteins and mRNAs. Isoform sorting may be an important consequence of the evolution of isoforms and is likely to have contributed to the diversity of functional properties within groups of isoforms.

Malignant transformation is frequently accompanied by obvious changes in cytoarchitecture, but the importance of these changes has been difficult to assess in view of the large number of other cellular changes that also occur. In this study, we transfected the SV40-immortalized human bronchial epithelial cell line, BEAS-2B, with human wild-type beta or gamma actin gene expression plasmids to induce cytoskeletal changes and to determine whether this was associated with altered cellular growth properties. Cells expressing the exogenous full-length actin genes underwent a fibroblastoid change in morphology which was reflected in changes in their pattern of actin cable organization, and acquired both the ability to grow under anchorage-independent conditions and resistance to the normal growth inhibitory effects of fetal bovine serum. These phenotypic changes correlated with changes in actin mRNA levels, but not with changes in actin protein levels. The phenotypically altered cells were not tumorigenic when injected subcutaneously in athymic nude mice, and they retained the ability to suppress the tumorigenic potential of a lung carcinoma cell line, HuT-292. Therefore, alteration of the cytoskeleton of immortalized human bronchial epithelial cells resulted in the acquisition of some properties commonly found in malignant cells, but did not result in tumorigenicity.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.

A cloned human cardiac actin gene, introduced into mouse Ltk- cells, is expressed in several thymidine kinase (tk)-positive cotransfectants. The clones not only produce authentic polyadenylated human cardiac actin mRNA but also synthesize human cardiac actin protein. The cardiac actin protein, normally found only in myofibrils, is stably accumulated at a high level, about one-third that of the endogenous mouse beta-actin. Furthermore, this sarcomeric protein partitions between the Triton X-100 insoluble and soluble phases to the same extent as the endogenous beta-actin. This suggests that a sarcomeric actin can participate in the formation of Triton X-100-insoluble cytoskeletal structures.

Membranous nephropathy (MN) continues to be a leading cause of nephrotic syndrome in non-diabetic adults. As a unique subtype in the serology-based classification of MN, thrombospondin type 1 domain containing 7A (THSD7A)-associated MN has attracted increasing interest, because, unlike other autoantigens, THSD7A is also expressed in preclinical species, facilitating the study of its role in MN. A heterologous mouse model of THSD7A-associated MN was previously established using a proprietary in-house antibody that was unfortunately not available to the research community. Here, we developed a mouse model of THSD7A-associated MN by administering a commercially available antibody targeting the most N-terminal part of THSD7A. Our model was characterized by heavy proteinuria and pathological features of human MN without sex differences. Complement depletion with cobra venom factor only partially attenuated proteinuria and glomerular injury in this model, entailing that complement-independent pathomechanisms also contribute. Consistently, in vitro in primary podocytes, exposure to the anti-THSD7A antibody caused evident podocytopathic changes, including disruption of actin cytoskeleton integrity, podocyte hypermobility, oxidative stress, and apoptotic cell death. These signs of podocytopathy were preserved, albeit to a lesser extent, after complement inactivation, indicating autonomous podocyte injury. Furthermore, as the first FDA-approved treatment for primary MN, adrenocorticotropic hormone therapy with repository corticotropin injection (Purified Cortrophin Gel®) appeared to be beneficial and significantly attenuated proteinuria and glomerular injury, suggesting that this model may be useful for developing novel treatments or understanding the pathogenesis of MN. Collectively, our model, based on the use of a commercially available anti-THSD7A antibody, will be an important tool for MN research.

Cell migration is an integral component of metastatic disease. The ability of cells to transit between mesenchymal and amoeboid modes of migration has complicated the development of successful therapies designed to target cell migration as a means of inhibiting metastasis. Therefore, investigations of the mechanisms that regulate cell migration and render cells stationary are necessary. Tropomyosins are actin-associating proteins that regulate the activity of several effectors of actin filament dynamics. Previously, we have shown that the tropomyosin isoform Tm5NM1 stabilizes actin filaments and inhibits cell migration in a two-dimensional culture system. Here, we show that Tm5NM1 inhibits the mesenchymal migration of multiple cell lines in an isoform-specific manner. Tm5NM1 stimulates the downregulation of Src kinase activity and a rounded or elliptical morphology in three-dimensional collagen gels, and cells have dramatically reduced capacity to form pseudopodia. Importantly, we find that Tm5NM1 inhibits both the mesenchymal to amoeboid and amoeboid to mesenchymal transitions. Collectively, our data suggest that mimicking the action of Tm5NM1 overexpression represents an approach for effectively inhibiting the mesenchymal mode of migration. [ABSTRACT FROM AUTHOR]