THE EFFECT OF LYSOPHOSPHATIDIC ACID ON THE COMPOSITION OF MYOSIN-9 AND TROPOMYOSIN CONTAINING CYTOPLASMIC PROTEIN COMPLEXES.

In this paper we investigate the distribution of myosin-9 in the cytoplasm of human embryonic lung fibroblasts. Using immunofluorescence, immunoprecipitation and western blotting, we demonstrated that myosin-9 forms multimolecular complexes with high molecular weight tropomyosin isoforms and actin in cytoplasm of cultured cells. We explored the levels of myosin-9, tropomyosin and actin in cytosol and cytomatrix extracts at different time points after LPA addition (20 ng/ml) to culture medium. Cytosole extracts from human embryonic lung fibroblasts were subjected to co-immunoprecipitation assay with polyclonal antibodies specific to C-terminal peptide of human myosin-9, or with monoclonal antibodies recognizing high molecular weight tropomyosin isoforms. The cross-immunoprecipitation method revealed changes in the composition of myosin- 9/tropomyosin complexes under the action of LPA. We have observed the significant decrease in myosin-9 co-immunoprecipitated tropomyosin level immediately (1 min) after LPA addition. Additionally, we have found that LPA treatment during 1 h induces proteolytic degradation of myosin-9 with accumulation of 130 kDa and 40 kDa fragments in cytomatrix fraction. These results suggest that cytoplasmic multimolecular protein complexes containing myosin-9 and tropomyosin are involved in the regulation of cellular response to LPA.

[Isolation of tropomyosin particles from the cytosol of cultured cells and their protein composition analysis].

The presence of actin-binding protein, tropomyosin, shaped as particles or protein complexes that have no bonds with actin structures were found while the analisys of structural rearrangements of actin cytoskeleton. However, their functioning is still unknown. To study the composition and properties of these protein complexes a novel method of their separation from the cells without destroying the structures of the cytoskeleton have been developed. The protein composition of isolated tropomyosin particles has been analised by gel filtration, electrophoresis and Western blotting. They appeared to be a multimolecular complexes of about 700 kDa. Beside the tropomyosin and actin these complexes also contain the Hsp70, Hsp90 and myosin-9 identified by mass spectrometry analisys. Also, under inhibition of deacetylases by trichostatin A, changes in the number of particles and redistribution of tropomyosin between cytosol and cytoskeleton take place along with actin cytoskeleton rearrangements. The results obtained give a reason to assume that these multimolecular complexes may participate in the process of reorganization of the actin microfilaments.

[Effect of formaldehyde in low concentrations on the proliferation and organization of the cytoskeleton of cultured cells].

3-4% (1.07-1.42 M) formaldehyde is one of the most popular and well-known organs, tissues and cells fixer. In this manuscript we have shown that formaldehyde in concentrations of up to 60 microM (0.0002%) does not have any negative effect on the viability of cell lines A431, HEK293 and primary rat fibroblasts, but it is also increases the proliferative activity of A431. The influence on A431 cells might be explained by the activation of epidermal growth factor receptors as a result of their interaction with formaldehyde.

[alpha-Actinin-4 and p65/RelA subunit of NF-kappaB transcription factor are co-localized and migrate together into the nucleus in EGF-stimulated A431 cell]. / alpha-Aktinin-4 i p65-sub"edinitsa transkriptsionnogo faktora NF-kappaB solokalizuiutsia i sovmestno migriruiut v iadro v kletkakh A431 pod deistviem épidermal'nogo faktora rosta.

The NF-kappaB/Rel family of transcription factors in mammalian cells regulates inducible transcription of a large number of genes in response to diverse stimuli. Despite a great number of publications on this subject, little is known about precise NF-kappaB localization in the cytoplasm. As previously demonstrated, in normal rat fibroblast and human epidermoid carcinoma A431 cells p65/RelA subunit of NF-kappaB is co-localized in the cytoplasm with actin structures. However, the mechanism of NF-kappaB interaction with actin remains unclear. We have investigated localization of p65/RelA subunit NFkappaB and alpha-actinin isoforms during cell activation by epidermal growth factor (EGF). Using confocal microscopy, we have shown that alpha-actinin-4 and p65/RelA subunit of NF-kappaB transcription factor are co-localized in A431 cells. Cell treatment with EGF leads to translocation of the proteins to membrane ruffles, and eventually to migration into the nucleus. Pretreatment of A431 cells with cytochalasin D or wortmannin prior to EGF treatment increases p65/RelA and alpha-actinin-4 accumulation in nuclear extracts. Co-localization of alpha-actinin-4 with p65/RelA subunit of NF-kappaB was found in nuclei isolated from stimulated cells. These results support the notion that actin cytoskeleton reorganization and alpha-actinin-4 are involved in NF-kappaB signaling.