The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. We have identified a 1-tubulin cDNA clone, 13105, which is shown to correspond to the trnscript of the beiC P-tubulin locus and to encode 12 tubulin, the 1 tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that (2 tubulin is only 83% identical to the two ,3 tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum P12 tubulin and the 1 tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum 12 tubulin is no more similar to, for example, Aspergillus 1 tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged 1 tubulin as well as one or more 1 tubulins that conform more closely to a consensus 1-tubulin sequence. We suggest that ,B-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among P tubulins could have resulted through neutral drift. For example, exclusive use of Physarum P2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the 1 tubulins that are utilized in multiple microtubular organelles.Alternatively, restricted use of P tubulins may allow positive selection to operate more freely to refine 3-tubulin function.Most eucaryotes express multiple genes each for a and K tubulins, the principal protein components of microtubules. The products of these genes usually have distinct sequences. For 13 tubulins, identity between multiple polypeptides within a single organism varies from 100% for Chlamydomonas reinhardtii (38) to 78% for mammals (34). The varied functions of microtubules in the cytoskeleton, centrioles, spindles, and flagella, together with the observed tubulin sequence differences, have inspired the conclusion that the sequence differences between multiple tubulins within an organism have been positively selected during evolution (32). The murine i tubulin M,1l is only 78% identical to several other murine i tubulins and is expressed specifically in hematopoietic tissues, prompting the speculation that at least some of the sequence differences reflect functional specialization of individual 1 tubulins (34).In the myxomycete Physarum polycephalum, threetubulin genes are known; one gene is expressed specifically in the amoebal cell type, another gene is expressed specifically in the plasmodial cell type, and the third gene, betB, is expressed in both amoebae and plasmodia (4, 28). The multinucleate plasmodium of P. polycephalum is unusual among eucaryotic cells in that it uses tubulins o...
To investigate the role of post-translational events in intestinal cell differentiation we have studied the effects of heat shock on processing and cell surface delivery of sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV) and aminopeptidase N (APN) in Caco-2 cells. In cells cultured at 42.5 degrees C there was a rapid decline in sucrase activity, while DPPIV and APN were unaffected over a 3-day period. Immunofluorescence staining confirmed the selective disappearance of SI from the surface membrane after only 1 day of culture at 42.5 degrees C. Cell-surface biotinylation of cells metabolically labelled with [35S]methionine 4 h after a switch from 37 degrees C to 42.5 degrees C demonstrated that newly synthesized APN and DPPIV were associated with the surface membrane, while SI was almost completely retained intracellularly. Pulse-chase experiments confirmed that, in these cells, DPPIV and APN were normally processed and vectorially delivered to the cell surface; in contrast, conversion between the two conformationally distinct high-mannose precursor forms of SI (hmP1 and hmP2) was markedly inhibited, a significant fraction of newly synthesized enzyme was degraded, probably in the ER, and an immature form of complex-glycosylated SI precursor (cP) was produced and mostly retained intracellularly. Double labelling of Caco-2 cells for SI and cathepsin D excluded an accumulation of SI in the lysosomes, suggesting that this organelle was not involved in the degradation of SI. These results indicate that the ER may play an important role in intestinal cell differentiation by regulating the conformational maturation, degradation and eventual cellular localization of some digestive enzymes.
Clonal cell lines have been established from primary fetal rat intestinal epithelial cells by stable transfection with plasmids containing either the simian virus 40 (SV40) large T-antigen gene under the control of the heavy metal inducible metallothionein promoter (pMTWt) or the thermolabile SV40 T-antigen gene under the control of the SV40 early promoter (pZipSVtsa58). pMTWt-transfected cells produced sufficient T-antigen to allow them to proliferate both when the metallothionein promoter was induced and uninduced. No differences were observed in the pattern of intestinal epithelial markers expressed when the cells were cultured in the presence or absence of inducing agent (zinc). In contrast, fetal rat intestinal epithelial cells transfected with pZipSVtsa58 were immortalized conditionally; cells proliferated at 32 degrees C but ceased to proliferate between 48 and 72 h of culture at 39 degrees C. Four of these cell lines were characterized in detail; they showed microvilli and tight junctions as well as dome formation and expressed functional and biochemical markers of intestinal epithelial cells, including keratins 8, 19, and 21, aminopeptidase N, and dipeptidyl peptidase IV. One cell line, 2/4/A1, expressed in addition a low level of lactase and sucrase-isomaltase. The amount and/or activity of some of these markers changed during the switch from the proliferative to the nonproliferative state (switch from culture at 32 to 39 degrees C), resulting in a more differentiated phenotype and mimicking similar changes taking place during intestinal epithelial cell differentiation in vivo.
Cytocentrin is a cytosolic protein that transiently associates with the mitotic spindle poles in early prophase, and dissociates from them after completion of mitosis. Cloning of its cDNA demonstrated a high degree of homology with three proteins known to specifically interact with an activated form of Ral. Herein we demonstrate that overexpression of cytocentrin inhibits assembly of the mitotic spindle without affecting polymerization or distribution of interphase microtubules. Conversely, loss of cytocentrin expression leads to formation of monopolar spindles. These results indicate that association of cytocentrin with the centrosome may be essential for a timely separation of the diplosomes. They also implicate Ral GTPases and their related pathways in the assembly and function of the mitotic apparatus.
The microbial eukaryote Physarum pofycephahm displays several distinct cell types in its life cycle, including amoebae, flagellates and plasmodia. Despite its relative simplicity, Physarum has a tubulin gene family of complexity comparable to that of Drosophifa. We have identified P-tubulin cDNAs from Physarum that are derived from the betA P-tubulin locus and encode P1A tubulin. We have also identified a partial cDNA for the unlinked betB P-tubulin gene, which encodes P1B tubulin. The polypeptide sequences encoded by betA and betB show 99 % identity, but the nucleotide sequences show only 85 % identity, consistent with an ancient duplication of these genes. The betB gene is expressed in amoebae, flagellates and plasmodia, whereas betA is expressed only in amoebae and flagellates. During the amoeba-flagellate transition the level of betA transcript increases over 100-fold, while the level of betB transcript changes very little. Thus Physarum has a mechanism for regulating the level of discrete P-tubulin transcripts differentially during flagellate development. A need for this differential regulation could account for the maintenance of the virtually isocoding betA and betB P-tubulin genes.
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