Characterization of prosomes in human lymphocyte subpopulations and their presence as surface antigens.

Prosomes, also called "multicatalytic proteinase" (MCP) or "proteasomes," are a new type of ubiquitous RNP particle present in some archeobacteria and in all eukaryotic cells tested from yeast to human. They were discovered as subcomplexes of untranslated messenger-ribonucleoproteins (mRNP) and later found to have a MCP activity putatively involved in antigen processing. Being composed of variable sets of characteristic proteins and associating small RNAs (pRNA), families of individual "mosaic" prosome particles seem to characterize the differentiation type and physiological state of individual cells and tissues. Here, prosomes from human lymphocytes, isolated and characterized biochemically and by Western blot analysis, were found to differ in their subunit composition compared to other human prosomes. Surprisingly, prosomal antigens were discovered at the outer surface of blood cells monitored by flow cytometry with monoclonal antibodies to individual prosomal proteins. It was observed that human T and B lymphocytes have variable and characteristic prosomal antigens at their surface according to their CD classification. Interestingly, the lymphocyte subpopulations most strongly labeled by the anti-p25K and anti-p27K mAbs were the NK and B cells.

Localization and active transport of mRNA in axons of sympathetic neurons in culture.

In neurons, the establishment and maintenance of distinct somatic, dendritic, and axonal domains has long been known to rely on regulated traffic of organelles and proteins. More recently, the local targeting of specific mRNAs has also been demonstrated, at least in dendrites, to provide a local supply of specific proteins. Here we set out to test directly for the presence of mRNA in axons of cultured chick sympathetic neurons, to examine their distribution during axonal outgrowth, to determine the reliance of this distribution on specific cytoskeletal elements, and to assess whether the axonal and somatic mRNA complements differ. Using fluorescent in situ hybridization, we found that sympathetic axons do contain poly(A+) mRNA along their length in a pattern that changes gradually as axons elongate, from an evenly dispersed punctate distribution with strong growth cone staining to a distribution focused at branch points, varicosities, and some growth cones. Selective perturbations of the cytoskeleton revealed that the presence of axonal mRNA was dependent on microtubules (MTs), but not actin filaments, indicating that mRNA transport and/or anchoring within the axon are active processes involving MTs. Finally, reverse transcription-PCR amplification of RNAs from the axonal and somatic compartments showed that beta-actin mRNA was present in both compartments, whereas mRNA encoding alpha-tubulin was restricted to the somatic compartment and entirely absent from the axons. Thus, the mRNA populations in the soma versus the axon are both quantitatively and qualitatively different, and these neurons are able to direct specific mRNAs to the axon.

Cytolocation of prosome antigens on intermediate filament subnetworks of cytokeratin, vimentin and desmin type.

Analysis by double-label indirect immunofluorescence of PtK1 and HeLa cells had previously demonstrated that prosome* antigens form networks that superimpose on those of the intermediate filaments of the cytokeratin type. We show here that in PtK1 cells various prosomal antigens also reside to a variable extent on intermediate filaments subnetworks of the vimentin type. In proliferating human fibroblasts the prosome and vimentin networks were found to coincide, while in proliferating myoblasts of the C2.7 mouse myogenic cell line the prosomal antigens seem to superimpose on the intermediate filaments of the desmin type. Thus, the prosomes, which are RNP particles of variable composition and subcomplexes of untranslated mRNP, and carry a multicatalytic proteinase activity, seem to co-localize with the specific kind of cytoplasmic intermediate filament in relation to the cell type. These results, which generalize the previous data, are discussed in view of possible role(s) for prosomes in mRNA metabolism and/or intermediate filaments remodelling.

Distribution of prosome proteins and their relationship with the cytoskeleton in oogenesis of Xenopus laevis.

The presence of prosome proteins (p25K and p27K) was shown and their distribution was studied in oogenesis of Xenopus laevis using immunoblotting and immunofluorescence. These proteins form numerous granular clusters of variable size all over the cell. At previtellogenic stages, the prosome antibodies homogeneously stain the oocyte nucleus and the evenly distributed relatively large clusters in the cytoplasm. As the oocyte grows, the pattern of distribution of the prosome proteins undergoes changes: animal-vegetal and cortical gradients appear in the cytoplasm. In the course of oocyte maturation the size of clusters diminishes. Artificial activation of the egg leads to a dorso-ventral gradient in distribution of the prosome proteins. In this way, specific localization of prosome proteins is first visualized during formation of the dorso-ventral polarity. Co-localization of prosome proteins and actin and myosin was found in the oocyte by double staining. Small clusters of prosomes dispersed in the cytoplasm acquire capability of movement (after artificial activation) due, in all likelihood, to persisting connection with the acto-myosin complex of the egg.

Patterns of cytodistribution of prosomal antigens on the vimentin and cytokeratin networks of monkey kidney cells.

Prosomes were found as mRNA-associated ribonucleoprotein particles (RNP) and cofactors of untranslated (ribosome-) free mRNP. Previous data have shown the presence of prosomal networks in the cytoplasm of PtK1 and HeLa cells and their superposition onto the intermediate filaments (IF) of cytokeratin type but little if any of vimentin type. Here it is shown that in LLC-MK2 cells various prosomal antigens are present on both, vimentin and cytokeratin networks, individual prosomal antigens superposing to variable degrees onto the IF subnetworks. Some prosomal antigens in variable relative concentrations were also observed in the nuclei of these cells. We suggest the existence of prosomal subnetworks specific for each prosomal antigen superposing to a variable extent onto the IF of both types.

Specific types of prosomes are associated to subnetworks of the intermediate filaments in PtK1 cells.

Prosomes are small ribonucleoprotein (RNP) particles of unique morphology in the electron microscope but of variable protein and RNA composition, depending on the differentiation state of the cells studied. They were initially observed as subcomplexes of untranslated mRNP. In previous studies, we found that prosomes are associated to the intermediate filaments (IF) of cytokeratin type in HeLa and PtK1 cells. Here we have studied in detail the association of prosomal antigens with the IF networks in PtK1 cells. Contrary to our earlier conclusions, in these cells the vimentin fibers also carry prosomes which, thus, distribute in between the two types of networks. During the selective collapse of the IF induced by acrylamide, and upon recovery after the withdrawal of the drug, no dissociation of the prosome and IF networks of cytokeratin- and vimentin-type could be observed. These data show that even in a dynamic situation, prosome and IF antigens do not dissociate, indicating strongly that they are located on one and the same structure. Furthermore, the differential distribution of specific prosomal antigens between both types of intermediate filament networks indicates that prosomes do not ubiquitously populate the intermediate filaments but occupy subnetworks of either vimentin or cytokeratin type.

[The distribution and relation to the cytoskeleton of specific prosomal proteins in the oogenesis of the clawed toad]. / Raspredelenie i sviaz' s tsitoskeletom spetsificheskikh prosomnykh belkov v oogeneze shportsevoi liagushki.

Using immunoblotting and immunofluorescent microscopy, we showed the presence in Xenopus laevis oocytes of two prosomal proteins (27 and 31-33 kDa) and studied their distribution during oogenesis. In the ooplasm, both proteins are detected in prosomal clusters of various size. During previtellogenesis, prosomal proteins are diffusely distributed in the nucleoplasm and form evenly distributed clusters in the cytoplasm. During oocyte growth, prosomal proteins disappear from the nucleus and form animal-vegetal and cortical gradients in the cytoplasm. In the course of oocyte maturation prosomal clusters become smaller. After artificial activation of the egg, the dorso-ventral gradient of distribution of prosomal proteins is observed. Double immunohistochemical labeling revealed morphological association between prosomal clusters and fibril-like structures of the oocyte containing actin and myosin. The latter are then replaced by diffusely distributed actin and myosin. Thus, correlation is observed between localization of the acto-myosin complex of the oocyte and that of prosomal proteins.

Immunolocalization of a specific type of prosome close to the bile canaliculi in fetal and adult rat liver.

Prosomes are mRNA-associated RNP particles and cofactors of untranslated (ribosome-) free mRNP having a multicatalytic proteinase (MCP; proteasome) activity. The expression of prosomal proteins in fetal development of the rat liver was investigated by indirect immunofluorescence, using a panel of monoclonal antibodies to individual prosomal proteins (p-mAbs). In all fetal and adult stages tested, strong immunofluorescence staining was observed with the p31K-specific p-mAb exclusively, whilst Western blot analysis showed reactivity also with the p27K and p33K antigens. Double labeling with the 31K p-mAb and an anti-cytokeratin antibody showed that the prosome antigen superimposes partially onto this type of intermediate filaments (IF), confirming earlier observations made on cultured cell lines of various types. Most interestingly, the p31K antigen was found preferentially in the pericanalicular zone of hepatocytes in the developing liver, from day 17 onwards up to the adult state. This shows a preferential concentration of prosomes of a specific type, including the p31K antigen, in the morphologically and possibly functionally specialized apical domain of the hepatocyte, in a differentiation-related fashion.

The association of prosomes with some of the intermediate filament networks of the animal cell.

The small RNP complexes of defined morphology and biochemical composition termed prosomes, first isolated from the cytoplasm associated with repressed mRNA (Martins de Sa, C., M.-F. Grossi de Sa, O. Akhayat, F. Broders, and K. Scherrer. J. Mol. Biol. 1986. 187:47-493), were found also in the nucleus (Grossi de Sa, M.-F., C. Martins de Sa, F. Harper, O. Coux, O. Akhayat, P. Gounon, J. K. Pal, Y. Florentin, and K. Scherrer. 1988. J. Cell Sci. 89:151-165). Immunofluorescence, immunoelectron microscopy, and immunochemical studies using mAbs directed against some of the prosomal proteins of duck erythroblasts indicate that in the cytoplasm of HeLa and PtK cells, prosome antigens are associated with the intermediate filament network of the cytokeratin type.