[Novel splicing isoform of actin-binding protein alpha-actinin 4 in epidermoid carcinoma cells A431].

Alpha-actinin 4 (ACTN4) belongs to actin binding proteins of the spectrin superfamily. Structural organisation of actin fibres and focal contacts is considered to be its primary function in a cell. Besides that, nucleocytoplasmic shuffling of ACTN4 and its involvement in nuclear processes were demonstrated. Lately, additional isoforms of ACTN4 resulted from an alternative splicing has been described in various cell types and malignant tumours. In this study, we present investigation of a novel ACTN4 isoform of 80 kDa. The isoform was found in human epidermoid carcinoma cells A431, and it was not detected in human skin fibroblasts, normal human keratinocytes and transformed human embryonic cells HEK293T. Analysis of ACTN4 mRNA in A431 cells showed the presence of a splice variant that lacked the exons 2-8. The deleted exons code two calponin homology domains responsible for ACTN4 binding to F-actin. Intracellular distribution of the described ACTN4 isoform (ACTN4ISO) overexpressed in HEK293T cells differed from that of the full size protein. In the cytoplasm, ACTN4ISO was allocated diffusively with no colocalisation with actin cytoskeleton structures. Intranuclear distribution of ACTN4ISO also differed from that of the full size ACTN4. Nevertheless, immunochemical analysis demonstrated possibility of ACTN4ISO to form heterodimers with the full size protein. Additional investigations of novel isoform interactions with ACTN4 protein partners might clarify its functional features in A431 cells.

[Analysis of nuclear protein complexes comprising alpha-actinin-4 by 2D-electrophoresis and mass-spectrometry].

Actin-binding protein alpha-actinin-4 is a member of spectrin super family. It is located in the cytoplasm and in the nucleus. However, nuclear functions of alpha-actinin-4 are still not clear. In this study, we analyzed composition of nuclear protein complexes associated with alpha-actinin-4 in A431 cells. Using 2D electrophoresis, we have determined that about 50 different proteins may be associated with nuclear alpha-actinin-4. Using mass-spectrometry, we analyzed major proteins of these complexes. beta-Actin, alpha- and beta-tubulins, ribonucleoprotein A2/B1, which regulates splicing and is associated with beta-actin, peroxiredoxin-1, which is involved in oxidative stress, and glycolytic enzyme D-3-phosphoglycerate dehydrogenase were identified by MALDI-TOF. Detection of these proteins in nuclear complexes with alpha-actinin-4 may suggest that alpha-actinin-4 is involved in transcription and splicing. Presence of beta-actin in the investigated complexes was confirmed by tandem mass-spectrometry (MALDI-TOF-TOF). Immunoprecipitation of nuclear proteins with antibodies against alpha-tubulin confirmed association of alpha-actinin-4 with alpha-tubulin in the protein complex. Nuclear alpha-actinin-4 constitutes of 105 KDa fullsize isoform and two truncated isoforms of 65 and 75 kDa, whereas only the truncated isoform have been found in nuclear complexes with alpha-tubulin. These data suggest that alpha-actinin-4 is associated with a number of different nuclear protein complexes which may carry out different functions in the cell nucleus.

[Analysis of extracellular matrix proteins produced by cultured cells].

Extracellular matrix (ECM) is a highly organized multimolecular structure essential for vital function of any organism. Although a lot of data on the extracellular matrix components has been accumulated, an isolation of the entire set of these proteins still remains to be a complex procedure since ECM contains fibrillar proteins and proteoglycans, which form multidomain net-like structures. In the presented study, we developed a method for isolation of ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membrane. Following treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibrils significantly improved fractioning of ECM proteins. Extraction of the remained proteins from culture plate surface was preformed using a buffer developed on the basis of Laemmli probe buffer. With this method, we isolated ECM proteins synthesized by culturing cells and suitable for a future analysis by SDS PAGE and two-dimentional electrophoresis as well as for identification of individual proteins by mass-spectrometry. This study may allow comparing protein contents of ECMs isolated from different sources, and elucidate influences of various proteins on the protein and the properties of extracellular matrix of investigated cells.

[Dynamic of DNA-binding activity of transcription factors in A431 cells during their spreading on immobilized ligands].

Dynamics of actin cytoskeleton in A431 cells and specific NF-kappaB SRF and AP-1 DNA-binding activities were studied during a 2 h spreading of these cells on fibronectin, laminin-2/4 or an antibody to epidermal growth factor receptor. Cell spreading was shown to be accompanied by sequential formation of actin cytoskeleton structures, whose spatial organization depends on the type of immobilized ligand. We have determined the time intervals, within which certain forms of cytoskeleton do not change qualitatively and are specific for dominant part of cell population. It has been shown that DNA-binding activities of the above transcription factors studied oscillate during cell spreading. The cycles of DNA-binding activity were found to be equel to 15-40 min. The character of oscillations depends on both transcription factor and ligand type. The temporal comparison of presses of actin cytoskeleton formation and DNA-binding activity of NF-kappaB and SRF times suggest that actin cytoskeleton reorganization may be presumably associated with activation of NF-KppaB and SRF.

[SRE, NF-kappaB, and AP-1 DNA-binding activities induced by A431 cell adhesion correlate with actin cytoskeleton reorganization].

Cell adhesion to extracellular matrix proteins induces activation of different signal molecules and influences gene expression. As shown earlier, epidermoid carcinoma A431 cell adhesion to fibronectin, laminin-2/4 or antibodies to receptor EGF (ab EGFR) results in reorganization of specific cell shape and actin cytoskeleton in the majority of cells. This study resolves a question whether morphological changes are accompanied with some cell response at the level of gene expression in nuclei. We have shown that cell reattachment promotes a specific DNA-binding of nuclear extracts with consensus sequences SRE, NF-kappaB and AP-1, compared to the control. NF-kappaB and AP-1 activities were considerably reduced in spread cells, which did not show actin filament's structures typical for the ligands. SRE specific proteins demonstrated other peculiarities and depended on the type of immobilized ligands. Our results argue that actin cytoskeleton reorganization, induced by cell adhesion to immobilized ligands at the early period after cell reattachment, is correlated with a specific answer at the levels of DNA-binding activity of transcription factors SRE, NF-kappaB and AP-1.

[Morphology of epidermoid carcinoma A431 cells spread on immobilized ligands]. / Analiz morfologicheskikh osobennostei populiatsii kletok épidermoidnoi kartsinomy A431, rasplastannykh na immobilizovannykh ligandakh.

Cell interaction with extracellular matrix is a multi-step process characterized by cell attachment to substrata with subsequent cell spreading accompanied by actin cytoskeleton and cellular membrane receptor reorganization. It has been shown elsewhere that epidermoid carcinoma A431 cells, spread on solid substrata coated with fibronectin, laminin-2/4 or antibodies to EGF receptor, form specific actin filament structures typical for each particular ligand. Here quantitative analysis of heterogeneous A431 cell population spread on the above ligands has been reported. Cells were subdivided into morphological classes, according to their shape and actin filament structure, and the relationship among classes under various experimental conditions were quantitatively estimated for every ligand. We studied the influence of cell detachment pattern, short-term and long-term starvation, and cell incubation in suspended state in the medium before plating on the cell population composition. It was possible to recognize the modal morphological class of cells with typical actin cytoskeleton structure dominating for the ligand in the population. Long-term starvation and incubation in suspension before cell spreading are considered as the crucial experimental parameters leading to dramatic changes in cell population.

[Intracellular distribution of tyrosine-phosphorylated actin-binding proteins in A431 cells spread on different ligands]. / Vnutrikletochnoe raspredelenie aktinsviazyvaiushchikh belkov, fosforilirovannykh po tirozinu, pri rasplastyvanii kletok A431 na raznykh ligandakh.

Spreading A431 cells on extracellular matrix elements fibronectin, laminin 2/4 and antibody to EGF receptor (5A9 clone) leads to tyrosine phosphorylation of actin-binding proteins, which participate in focal adhesions formation. Tyrosine phosphorylation of the proteins is retained for 1 h of cell spreading. When cells interact with ligands, focal adhesion kinase (FAK) becomes tyrosine phosphorylated, and eventually phosphorylates the target proteins. The cooperative effect of integrins and EGF receptor in FAK autophosphorylation at cell spreading on antibody to EGF receptor is discussed.

[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.

[Fibrillar actin promotes assembly of dissociated 20S-proteasome complex]. / Fibrilliarny'i aktin sposobstvuetsborke dissotsiirovannogo kompleksa 20S-proteasomy.

20S-proteasome was purified from rat liver cells by ultracentrifugation, gel-chromatography and ultrafiltration. The ability of 20S-proteasome to interact with fibrillar actin (F-actin) was examined by rapid cosidementation of these dissociated particles with F-actin in isokinetic sucrose gradient. Proteasomes, which were dissociated with Zn2+ ions, can be assembled on the fibrillar actin once again (with the exception of protein 27 kDa) at deleting ions Zn2+ from the solution with the help of EDTA.

[26S-ribonucleoprotein complex (26S-proteasome) directly interacts with fibrillar actin]. / 26S-ribonukleoproteinovyi kompleks (26S-proteasoma) neposredstvenno vzaimodeistvuet s fibrilliarnym aktinom.

26S-complex was purified from rat liver cells by centrifugation in sucrose gradients and ion exchange chromatography. The ability of 26S-proteasome to interact with fibrillar actin (F-actin) was examined by rapid cosidementation of these particles with F-actin in isokinetic sucrose gradients. It was shown that direct interaction occurred only at a low ATP concentration.

[EGF-dependent association of 20S-proteasome and alpha-RNP particles with the epidermal growth factor receptor in A-431 cells]. / EFR-vyzvannaia assotsitsiia 20S-proteasom i alpha-RNP-chastits s retseptorom EFR v kletkakh A-431.

Here we demonstrate that the epidermal growth factor (EGF) induces association of prosomes (20S-proteasomes) with its receptor in A-431 cells. Additionally, ligand-dependent association of ribonucleoprotein particles (alpha-RNP), containing small ALU-like RNA, with the EGF receptor was demonstrated. A suggestion has been put forward on the involvement of prosomes and alpha-RNP in the EGF signal transmission to different stages of gene expression.

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. III. The DNA-binding activity of nuclear alpha-RNP. The Signal Transmission from Growth Factors Group of the Medical Faculty, F. Schiller University, Jena, Germany]. / Novyi klass malykh RNP (al'fa-RNP), soderzhashchikh antismyslovuiu RNK, v kletkakh linii K-562. III. DNK-sviazyvaiushchaia aktivnost' iadernykh al'fa-RNP. Gruppa "Peredacha signala s rostovykh faktorov" pri Meditsinskom fakul'tete Universiteta im. F. Shillera, Iena, Germaniia.

DNA-binding activity of small nuclear alpha-RNP identified in acid-soluble fraction of chromatin of human proerythroleukemic cell line K-562 was studied using the technique of gel retardation. We found that nuclear alpha-RNP isolated from K-562 cells through treatment with dimethylsulfoxide, an agent inducing differentiation, acquire a capacity to specific interaction with Alu repeats of DNA leading to the formation of alpha-RNP-Alu-DNA complexes; nuclear alpha-RNP from cells that were not treated with dimethylsulfoxide do not show such capacity, although they are tightly bound with chromatin in the cell. Thus, the capacity of nuclear alpha-RNP to direct interaction with DNA Alu repeats appearing after the induction of K-562 cells to differentiation along erythroid pathway is an inducible property. We discuss hypothesis about the involvement of nuclear alpha-RNP in the control of expression of inducible genes at the level of chromatin and interaction with DNA.

[The release and absorption of small ribonucleoprotein complexes (alpha RNPs) in cultures of transformed rat fibroblasts and human epidermoid carcinoma]. / Eksport i pogloshchenie malykh ribonukleoproteinovykh kompleksov (al'fa RNP) v kul'turakh transformirovannykh krysinykh fibroblastov i épidermoidnoi kartsinomy cheloveka.

The release of alpha RNPs and their absorbtion by the cells from culture medium were studied. The rat fibroblasts of parental serum-dependent cell line LRec-1, and of selected serum-free cell line LRec-1sf rapidly released and absorbed alpha RNPs. In the latter case, both auto- and heterologous alpha RNPs were seen to penetrate, whereas only autologous alpha RNPs entered LRec-1 cells. Besides, the ability to rapid export and absorbtion of autocrine alpha RNPs was demonstrated for human epidermoid carcinoma cell line A431. Both LRec-1 and LRec-1sf cells expressed mRNAs with homology to ID-like nucleotide sequences, the level of mRNA expression decreasing markedly when serum-dependent LRec-1 cells were grown in serum-free medium.

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. I. Their characteristics and changes during erythroid differentiation]. / Novyi klass malykh RNP (al'fa-RNO), soderzhashchokh antismyslovuiu RNK, v kletkakh linii K-562. I. Kharakteristika i izmeneniia pri differentsirovke po éritroidnomu puti.

A new class of small RNP (alpha-RNP) has been detected and identified in nuclei and cytoplasm of A-562 erythroid leukemia cell line; these RNPs have a characteristic spectrum of proteins containing conservative and specific components and a special RNA component, which contains a small antisense component (alpha-RNA), a homolog of short dispersed Alu repeats. alpha-RNP is highly stable, tightly associated with chromatin in the nucleus, and is found in the free state in cytoplasm. The composition of nuclear and cytoplasmic alpha-RNP differ and have a specific pattern of changes in response to dimethylsulfoxide, an agent causing differentiation.

[The DNA-binding activity of the membrane protein annexin II and its interaction with antibodies to the chromatin nuclear ribonucleoprotein (alpha-RNP)]. / DNK-sviazyvaiushchaia aktivnost' primembrannogo belka anneksina II i ego vzaimodeistvie s antitelami k iadernomu ribonukleoteinu khromatina (al'fa-RNP).

By screening with labeled Alu DNA, a clone was isolated from cDNA expression library, which appeared identical in sequence to the well-known Ca-phospholipid-binding protein annexin II. To evidence the DNA-binding activity of recombinant annexin II and its presence in the cell nucleus, we have expressed full-length mouse annexin II cDNA in bacteria with pGEMEX vector. The expressed protein was studied with electrophoretic mobility shift assay and for its reaction with polyclonal antibody to chromatin-associated ribonucleoprotein (alpha-RNP), which is one of the major acid-dissolvent components of the nucleus. The obtained results confirm the DNA-binding activity of recombinant annexin II. Annexin II reacts with polyclonal antibody to rat alpha-RNP. So, annexin II is a major nuclear DNA-binding protein in mammalian cells.

[A new class of RNP particles containing small RNA homologous to short dispersed DNA repetitive sequences]. / Novyi klass RNP chastits, soderzhashchikh malye RNK, gomologichnye korotkim dispergirovannym povtoriaiushchimsia posledovatel'nostiam DNK.

Here we show that small RNAs homologous to short interspersed repetitive DNA sequences: ID, B1, B2--in rat cells and Alu in human cells are complexed with specific proteins to form small nuclear and cytoplasmic RNP particles (alpha RNP) with common properties. alpha-RNP differ from other ribonucleoproteins by composition and properties. alpha RNA molecules are apparently transcribed by RNA polymerase III. alpha-RNAs are capable of stable antisense hybridization with specific messenger RNAs. Expression of alpha-RNA is specifically regulated by gene regulatory factors. The data obtained support the suggestion that alpha-RNA may belong to the group of regulatory eukaryotic RNAs and that alpha-RNP might be involved in the coordinative control of the expression of the sets of genes with SINE-homologous sequences in regulatory regions.