Interactions between the Translation Machinery and Microtubules.

Microtubules are components of eukaryotic cytoskeleton that are involved in the transport of various components from the nucleus to the cell periphery and back. They also act as a platform for assembly of complex molecular ensembles. Ribonucleoprotein (RNP) complexes, such as ribosomes and mRNPs, are transported over significant distances (e.g. to neuronal processes) along microtubules. The association of RNPs with microtubules and their transport along these structures are essential for compartmentalization of protein biosynthesis in cells. Microtubules greatly facilitate assembly of stress RNP granules formed by accumulation of translation machinery components during cell stress response. Microtubules are necessary for the cytoplasm-to-nucleus transport of proteins, including ribosomal proteins. At the same time, ribosomal proteins and RNA-binding proteins can influence cell mobility and cytoplasm organization by regulating microtubule dynamics. The molecular mechanisms underlying the association between the translation machinery components and microtubules have not been studied systematically; the results of such studies are mostly fragmentary. In this review, we attempt to fill this gap by summarizing and discussing the data on protein and RNA components of the translation machinery that directly interact with microtubules or microtubule motor proteins.

[Stress-protective and cross action of the extracellular reactivating factor of the microorganisms of the domains Bacteria, Archaea, and Eukaryota].

Cross protection of members of the domains Bacteria, Archaea, and lower Eukaryota from stress factors due to the action of extracellular low-molecular metabolites with adaptogenic functions was shown. The adaptogen produced by Luteococcus japonicus subsp. casei and described previously as a reactivating factor (RF) was shown to protect the yeasts Saccharomyces cerevisiae, archaea Haloarcula marismorti, and the cells of higher eukaryotes (HeLa) against weak stressor impacts. Production of an archaeal extracellular metabolite with a weak adaptogenic effect of the producer cells and capable of a threefold increase in survival of heat-inactivated yeast cells was discovered. Our results confirm the similarity of the compensatory adaptive reactions in prokaryotes (bacteria and archaea) and eukaryotes.

[Antistress cross-effects of extracellular metabolites of bacteria, archaea, and yeasts: a review].

This paper reviews examples of specific and global responses of microorganisms and the characteristics of stress responses involving extracellular signaling metabolites. Information regarding the protective and reactivating effects produced by active exometabolites of representatives of domains of bacteria, archaea, and eukaryotes is summarized, and interdomain cross-responses to stressors are demonstrated.

Cellular acidosis inhibits assembly, disassembly, and motility of stress granules.

Stress granules (SGs) are large ribonucleoprotein (RNP)-containing particles that form in cytoplasm in response to a variety of acute changes in the cellular environment. One of the general parameters of the cell environment is pH. In some diseases, as well as in muscle fatigue, tissue acidosis occurs, leading to decrease in intracellular pH. Here we studied whether decrease in pH causes the formation of SGs in cultured animal cells, whether it affects the formation of the SGs under the action of arsenite and, if such effects occur, what are the mechanisms of the influence of acidosis. Acidosis was simulated by decreasing the pH of the culture medium, which acidified the cytoplasm. We found that medium acidification to pH 6.0 in itself did not cause formation of SGs in cells. Moreover, acidification prevented the formation of SGs under treatment with sodium arsenite or sodium arsenite together with the proteasome inhibitor MG132, and it inhibited the dissociation of preformed SGs under the influence of cycloheximide. We established that pH decrease did not affect the phosphorylation of eIF2α that occurs under the action of sodium arsenite, and even caused such phosphorylation by itself. We also found that the velocity of SG motion in cytoplasm at acidic pH was very low, and the mobile fraction of SG-incorporated PABP protein revealed by FRAP was decreased. We suppose that acidic pH impairs biochemical processes favoring assembly of RNPs in stress conditions and RNP dissociation on the termination of stress. Thus, in acidosis the reaction of the cellular translation apparatus to stress is modified.

[Stress granules in the cells with intact and discrupted microtubules: analysis with new algorithm of image processing].

Stress granules--temporary RNP structures that are formed in cells under stress. They are studied mainly by means of fluorescence microscopy with the quantitative analysis of cell images. We have developed a new algorithm for automatic detection of stress granules in the cytoplasm of cultured animal cells having non-uniform cytoplasmic background. Using this approach, we have found that visible stress granules are formed in cells as "all or nothing", and their number in cells is rather constant. We also show that disruption of cellular microtubules lead to a decrease in the average size of stress granules and an increase in their number in the cell.

[Is the microtubule disruption-induced alteration of peroxide concentration a factor inhibiting the assembly of ribonucleoprotein stress granules?].

It has been examined whether the destruction of cell microtubules affects the increase in the intracellular hydrogen peroxide concentration caused by sodium arsenite, which induces the formation of stress ribonucleoprotein granules. As expected, sodium arsenite caused a 50% increase in hydrogen peroxide concentration in HeLa cells; on the other hand, another stress granule inducer tert-buthylhydroquinone did not affect the peroxide concentration. The disruption of microtubules by nocodazole or vinblastine also resulted in some increase in the intracellular peroxide concentration,y taxol did not affect it. The combined treatment of cells with and the microtubule stabilization by taxol did not affect it. The combined treatment of cells with arsenite and antimicrotubule drugs caused an additive effect, and the peroxide concentration increased twice or more. Thus, the inhibition of stress granule formation after microtubule disruption cannot be explained by a decrease in peroxide concentration as compared with the affect of arsenite.

[Large subunit of translation initiation factor--3 p170 contains potentially functional nuclear localization signals]. / Signaly iadernoi lokalizatsii v bol'shoi sub''edintse faktora initsiatsii transliatsii 3--belke p170.

Eukaryotic translation factors and their subunits can have independent cellular functions, including regulation of nuclear events. We analyzed primary structure of p170 large subunit of human translation initiation factor eIF3 and found four potential bipartite nuclear localization signals (NLS). Then we studied whether these NLS were functional, that is were able to direct protein to cell nucleus. Complementary DNA of p170 fragments were expressed in cultured CV-1 and Cos-1 green monkey cells, and localization of fused with GFP proteins was determined by fluorescent microscopy. We established that p170 molecule possessed at least two functional NLS which determined nuclear localization of p170 fragments. At the same time more long p170 fragments containing the same functional NLS could be retained in cytoplasm. We speculate that either using specific factors or after limited proteolysis p170 can enter cell nucleus and participate in genome expression regulation. Also we do not exclude the possibility that functioning of p170 in cytoplasm can be regulated by reversible binding of importins to its NLS.

[Characteristics of Actinomycin D-induced segregation of a "noncanonical" nucleolus of the sea urchin Paracentrotus lividus]. / Osobennosti segregatsii "nekanonicheskogo" iadryshka ootsitov morskogo ezha Paracentrotus lividus, indytsirovannoi deistviem Aktinomitsina D.

The structure of a "noncanonical" nucleolus of vitellogenic oocytes in the sea urchin Paracentrotus lividus was studied using the inhibitor of transcription actinomycin D. In the control cells, the nucleolus consists of two separated structural subdomains: the dense fibrillar-granular peripheral area and the fibrillar central area. The nucleolus did not contain subdomains corresponding to the fibrillar center and dense fibrillar component of "typical" nucleoli. After treatment with actinomycin D, numerous argyrophilic granules appeared in the karyoplasm, the intranucleolar DNA became compact, and the nucleolar material was segregated into two or three separated zones, the residual peripheral area being the densest and largest. Lesser zones had a decreased electron density and contained argyrophilic proteins and, apparently, the nucleolar organizer material. These results suggest that, for normal rRNA expression and processing, the presence of structural subdomains in the nucleolus, such as fibrillar complexes and a dense fibrillar component, is not essential.

[Translation initiation factor eIF3 is able to bind with microtubules in mammalian cells]. / Faktor initsiatsii transliatsii eIF3 mozhet sviazyvat'sia s mikrotrubochkami v kletkakh mlekopitaiushchikh.

Association of the translation apparatus with the cytoskeleton is essential for its transportation within the cell and probably also for translation regulation. Very little is known about the involvement of particular proteins of this association. A polypeptide homologous with the heavy chain of translation initiation factor eIF3 p170 was found earlier in a microtubule preparation from adrenal cells. Antibody A167 directed against the recombinant fragment of p170 has been generated to study eIF3 interaction with microtubules in mammalian cells. This antibody was shown to recognize a single 170 kDa polypeptide in eIF3 preparations as well as in homogenates of various cell types. A167 allowed detection of the 170 kDa polypeptide in microtubule preparation from bovine brain and confirmation of its presence in microtubule preparations from adrenal cells. As shown by immunofluorescence microscopy using A167, the 170 kDa polypeptide is mainly located in the endoplasm within numerous small and some large granules. Cell treatment with cycloheximide resulted in growth and clustering of the large granules, and partial antigen redistribution along cellular microtubules. These new experimental data indicate that mammalian translation factor eIF3 may bind with microtubules.

Noncanonical structural-functional organization of nucleoli in maturing oocytes of sea urchin Paracentrotus lividus.

The dynamics of structural and functional organization of the nucleolus in the oocytes of P. lividus is described. At the late stages of oogenesis the nucleolus is composed of two main components, namely the peripheral zone (PZ) and the central zone (CZ) which are spatially separated. This two-component structure of the nucleolus is formed, at early stages of oogenesis, by stepwise segregation of the fibro-granular component and by its migration to the nucleolar periphery. Absence of morphologically distinct fibrillar centers and dense fibrillar component in nucleoli of both somatic cells and oocytes makes it possible to classify the nucleoli of P. lividus as 'noncanonical' type. Based on detailed morphological and cytochemical analysis the following molecular interpretation of nucleolar ultrastructure in oocytes of P. lividus is proposed: 1) the PZ, containing RNP-positive granules 15 nm in size, but lacking Ag-NOR proteins and BrU incorporation, can be considered a structural equivalent of the granular component of 'typical' nucleoli; 2) the CZ, which is the site of incorporation of RNA precursors, contains intranuclear DNA, RNP-fibers and accumulates Ag-NOR proteins, corresponds to both FC and DFC of 'typical' nucleoli; 3) nucleolar growth during oogenesis, leading to the 1000-fold increase of nucleolar volume, seems to be correlated with the stockpiling of nonfunctioning mature preribosomal particles which will be utilized during embryogenesis.

DNA localization in the nucleoli of sea urchin Paracentrotus lividus oocytes.

It is known that in oocytes of P. lividus the nucleus contains a single giant nucleolus of unusual structure where intensive rRNA synthesis occurs. However, the questions of structural and functional relationships between the nucleolar compartments and the sites of active rRNA transcription still remain open. In the present work, we studied the chromatin organization in the nucleoli of P. lividus oocytes using Feulgen's and osmium amine staining procedures. Our results indicate that nucleolar chromatin of small (immature) oocytes differs from that in large mature oocytes. At the early stage of development, the DNA filamentous network 0.2-0.5 microm in diameter is formed in the nucleoli. Profound changes in the structure of the nucleolar DNA are observed in the course of oogenesis. Thus, at the late stage of development, the nucleolar chromatin forms characteristic ring-like structures, which indicates a non-uniform distribution of active ribosomal genes. A model of structural organization of the P. lividus nucleolus is discussed.