PubMed · 15701920
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Steven J Winder, Kathryn R Ayscough. 2005-02-15. Actin-binding proteins.. https://doi.org/10.1242/jcs.01670
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The molecular mechanisms leading to high-altitude pulmonary hypertension (HAPH) remains poorly understood. We previously analyzed the whole genome sequence of Kyrgyz highland population and identified eight genomic intervals having a potential role in HAPH. Tropomodulin 3 gene (TMOD3), which encodes a protein that binds and caps the pointed ends of actin filaments and inhibits cell migration, was one of the top candidates. Here we systematically sought additional evidence to validate the functional role of TMOD3. In-silico analysis reveals that some of the SNPs in HAPH associated genomic intervals were positioned in a regulatory region that could result in alternative splicing of TMOD3. In order to functionally validate the role of TMOD3 in HAPH, we exposed Tmod3-/+ mice to 4 weeks of constant hypoxia, i.e. 10% O2 and analyzed both functional (hemodynamic measurements) and structural (angiography) parameters related to HAPH. The hemodynamic measurements, such as right ventricular systolic pressure, a surrogate measure for pulmonary arterial systolic pressure, and right ventricular contractility (RV- ± dP/dt), increases with hypoxia did not separate between Tmod3-/+ and control mice. Remarkably, there was a significant increase in the number of lung vascular branches and total length of pulmonary vascular branches (P < 0.001) in Tmod3-/+ after 4 weeks of constant hypoxia as compared with controls. Notably, the Tmod3-/+ endothelial cells migration was also significantly higher than that from the wild-type littermates. Our results indicate that, under chronic hypoxia, lower levels of Tmod3 play an important role in the maintenance or neo-vascularization of pulmonary arteries.
BACKGROUND INFORMATION: Protein kinase pEg3 belongs to the evolutionarily conserved KIN1/PAR-1/MARK family, whose members are involved in a variety of functions, including cell polarity, microtubule stability, intracellular signalling and the cell cycle. Activity and phosphorylation of pEg3 are cell-cycle dependent and rise to maximum levels during mitosis. pEg3 was shown to interact with and phosphorylate phosphatase CDC25B, and to potentially control cell-cycle progression. Subcellular localization of pEg3 was investigated in Xenopus and human cultured cells. RESULTS: By expression of GFP (green fluorescent protein)-tagged pEg3 and indirect immunofluorescence with specific antibodies, pEg3 was found to be localized in the cytoplasm and the nucleus in interphase cells. During mitosis pEg3 was also found in the cytoplasm. From anaphase to telophase, a proportion of the protein was detected at the cell cortex. The cortical distribution in mitotic cells was dependent on F-actin, because the actin-depolymerization-inducing drugs cytochalasin D or latrunculin A prevented pEg3 cortical localization. The protein lacking the conserved C-terminal domain was not detected at the cell cortex, whereas the C-terminal domain was targeted to the cell periphery. In contrast with full-length pEg3, the cortical localization of the C-terminal domain and construct lacking the N-terminal domain was cell-cycle independent, and these constructs were found at the cell periphery in interphase cells. CONCLUSIONS: pEg3 is localized at the cell periphery specifically during mitosis. The C-terminal domain is the only pEg3 domain found to be necessary and sufficient for cortical targeting. Cortical distribution of pEg3 also requires the F-actin cytoskeleton. The cell-cycle-independent cortical localization of the pEg3 C-terminal domain and a construct lacking the N-terminal domain indicates that a negative control mechanism involving the pEg3 catalytic N-terminal domain probably acts to prevent pEg3 cortical distribution during interphase. These results suggest that pEg3 might play a role at the cell cortex during mitosis.
Two most common properties of malignant cells are the presence of aberrant actin cytoskeleton and resistance to anoikis. Suppression of several key cytoskeletal proteins, including tropomyosin-1 (TM1), during neoplastic transformation is hypothesized to contribute to the altered cytoskeleton and neoplastic phenotype. Using TM1 as a paradigm, we have shown that cytoskeletal proteins induce anoikis in breast cancer (MCF-7 and MDA MB 231) cells. Here, we have tested the hypothesis that TM1-mediated cytoskeletal changes regulate integrin activity and the sensitivity to anoikis. TM1 expression in MDA MB 231 cells promotes the assembly of stress fibers, induces rapid anoikis via caspase-dependent pathways involving the release of cytochrome c. Further, TM1 inhibits binding of MDA MB 231 cells to collagen I, but promotes adhesion to laminin. Inhibition of Rho kinase disrupts TM1-mediated cytoskeletal reorganization and adhesion to the extracellular matrix components, whereas the parental cells attach to collagen I, spread and form extensive actin meshwork in the presence of Rho kinase inhibitor, underscoring the differences in parental and TM1-transduced breast cancer cells. Further, treatment with the cytoskeletal disrupting drugs rescues the cells from TM1-induced anoikis. These new findings demonstrate that the aberrant cytoskeleton contributes to neoplastic transformation by conferring resistance to anoikis. Restoration of stress fiber network through enhanced expression of key cytoskeletal proteins may modulate the activity of focal adhesions and sensitize the neoplastic cells to anoikis.