Gene Knockouts Reveal Separate Functions for Two Cytoplasmic Dyneins in Tetrahymena thermophila

In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

Mutations in the gene encoding IFT dynein complex component WDR34 cause jeune asphyxiating thoracic dystrophy

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery.

Perspectives on the origin of microfilaments, microtubules, the relevant chaperonin system and cytoskeletal motors - a commentary on the spirochaete origin of flagella

The origin of cytoskeleton and the origin of relevant intracellular transportation system are big problems for understanding the emergence of eukaryotic cells. The present article summarized relevant information of evidences and molecular traces on the origin of actin, tubulin, the chaperonin system for folding them, myosins, kinesins, axonemal dyneins and cytoplasmic dyneins. On this basis the authors proposed a series of works, which should be done in the future, and indicated the ways for reaching the targets. These targets are mainly: 1) the reconstruction of evolutionary path from MreB protein of archaeal ancestor of eukaryotic cells to typical actin; 2) the finding of the MreB or MreB-related proteins in crenarchaea and using them to examine J. A. Lake's hypothesis on the origin of eukaryote from "eocytes" (crenarchaea); 3) the examinations of the existence and distribution of cytoskeleton made of MreB-related protein within coccoid archaea, especially in amoeboid archaeon Thermoplasm acidophilum; 4) using Thermoplasma as a model of archaeal ancestor of eukaryotic cells; 5) the searching for the homolog of ancestral dynein in present-day living archaea. During the writing of this article, Margulis' famous spirochaete hypothesis on the origin of flagella and cilia was unexpectedly involved and analyzed from aspects of tubulins, dyneins and spirochaetes. Actually, spirochaete cannot be reasonably assumed as the ectosymbiotic ancestor of eukaryotic flagella and cilia, since their swing depends upon large amount of bacterial flagella beneath the flexible outer wall, but not depends upon their intracellular tubules and the assumed dyneins. In this case, if they had "evolved" into cilia and lost their bacterial flagella, they would immediately become immobile! In fact, tubulin and dynein-like proteins have not been found in any spirochaete.

Evidence for Four Cytoplasmic Dynein Heavy Chain Isoforms in Rat Testis

Recent studies have revealed the expression of multiple putative cytoplasmic dynein heavy chain (DHC) genes in several organisms, with each gene encoding a separate protein isoform. This finding is consistent with the hypothesis that different isoforms do different things, as is the case for the axonemal dyneins. Furthermore, the large number of tasks ascribed to cytoplasmic dynein suggests that there may be additional isoforms not yet identified. Two of the mammalian cytoplasmic dynein heavy chains are DHC1a and DHC1b. DHC1a is conventional cytoplasmic dynein and is found in all organisms examined. DHC1b is expressed in organisms that have multiple dyneins, and has been implicated in the intracellular trafficking of molecules in unciliated and ciliated cells. In the present study, we examined the DHC1b protein from rat testis. Testis cytoplasmic dynein contains a large amount of dynein heavy chain reactive with an antibody raised against a peptide sequence of rat DHC1b. The testis anti-DHC1b immunoreactive protein is slightly smaller than testis DHC1a, as assessed by SDS-PAGE. In Northern blots, the DHC1b mRNA is smaller than the DHC1a mRNA. In sucrose gradients made in low ionic strength, DHC1a sedimented at approximately 20S, and the anti-1b immunoreactive heavy chains sedimented in a broad band centered at approximately 14S. The V1-photolysis reaction of individual sucrose gradient fractions revealed three distinct patterns of photolysis, suggesting that there are at least three separate 1b-like heavy chain isoforms in testis. Using a high-stringency Western blotting protocol, the anti-1b antibody and the anti-DHC2 antibody recognized the same heavy chain and specifically bound to one of the three 1b-like heavy chains. We conclude that rat testis contains three 1b-like dynein heavy chains, and one of these is the product of the DHC1b/DHC2 gene previously identified.

Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cellinvasion

The invasion of human malignant melanoma cells into the extracellular matrix (ECM) involves the accumulation of proteases at sites of ECM degradation where activation of matrix metalloproteases (MMP) occurs. Here, we show that when membrane type 1 MMP (MT-MMP) was overexpressed in RPMI7951 human melanoma cells, the cells made contact with the ECM, activated soluble and ECM-bound MMP-2, and degraded and invaded the ECM. Further experiments demonstrated the importance of localization of the MT-MMP to invadopodia. Overexpression of MT-MMP without invadopodial localization caused activation of soluble MMP-2, but did not facilitate ECM degradation or cell invasiveness. Up-regulation of endogenous MT-MMP with concanavalin A caused activation of MMP-2. However, concanavalin A treatment prevented invadopodial localization of MT-MMP and ECM degradation. Neither a truncated MT-MMP mutant lacking transmembrane (TM) and cytoplasmic domains (ΔTM(MT-MMP)), nor a chimeric MT-MMP containing the interleukin 2 receptor α chain (IL-2R) TM and cytoplasmic domains (ΔTM(MT-MMP)/TM(IL-2R)) were localized to invadopodia or exhibited ECM degradation. Furthermore, a chimera of the TM/cytoplasmic domain of MT-MMP (TM(MT-MMP)) with tissue inhibitor of MMP 1 (TIMP-1/TM(MT- MMP)) directed the TIMP-1 molecule to invadopodia. Thus, the MT-MMP TM/cytoplasmic domain mediates the spatial organization of MT-MMP into invadopodia and subsequent degradation of the ECM.

The Saccharomyces cerevisiae RNA-binding protein Rbp29 functions in cytoplasmic mRNA metabolism

Here we report that the Saccharomyces cerevisiae RBP29 (SGN1, YIR001C) gene encodes a 29-kDa cytoplasmic protein that binds to mRNA in vivo. Rbp29p can be co-immunoprecipitated with the poly(A) tail-binding protein Pab1p from crude yeast extracts in a dosageand RNA-dependent manner. In addition, recombinant Rbp29p binds preferentially to poly(A) with nanomolar binding affinity in vitro. Although RBP29 is not essential for cell viability, its deletion exacerbates the slow growth phenotype of yeast strains harboring mutations in the eIF4G genes TIF4631 and TIF4632. Furthermore, overexpression of RBP29 suppresses the temperaturesensitive growth phenotype of specific tif4631, tif4632, and pab1 alleles. These data suggest that Rbp29p is an mRNA-binding protein that plays a role in modulating the expression of cytoplasmic mRNA.

Some cytoplasmic details of yeast revealed by the electron microscope

Abstract is not available.

Biosynthesis of cytoplasmic subunits of cytochrome C oxidase in rat liver

The biosynthesis of the cytoplasmic subunits of cytochrome oxidase from rat liver has been studied in vitro by translating liver poly (A)-containing RNA in the wheat germ cell-free system and immunoprecipitating the products with anti-cytochrome oxidase antibody. Analysis of the labelled immunoprecipitate on SDS-gels does not reveal the presence of a polyprotein precursor. On the other hand discrete products which are either slightly bigger or closely similar to the mature subunits present in purified cytochrome oxidase have been detected.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

Summary We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

Dexamethasone negatively regulates phenobarbitone-activated transcription but synergistically enhances cytoplasmic levels of cytochrome P-450b/e messenger RNA

Dexamethasone has a potentiating effect on phenobarbitone mediated induction of cytochrome P-450b + e mRNAs in adult rat liver. However, the glucocorticoid inhibits phenobarbitone-activated transcription of cytochrome P-450b + e mRNAs by 60-70%. This inhibitory effect is evident in run-off transcription of the endogenous genes as well as in the transcription of an added cloned gene fragment. Dexamethasone inhibits the phenobarbitone-mediated increase in the binding of a transcription factor(s) to the upstream region of the gene as evidenced by gel retardation and Southwestern blot analysis. The glucocorticoid does not stabilize the phenobarbitone-induced polyribosomal cytochrome P-450b + e mRNAs but appears to stabilize the nuclear transcripts. It is proposed that a negative element may mediate the action of dexamethasone at the level of nuclear transcription and stabilization of the nuclear transcript may account for the potentiating effect of the glucocorticoid on phenobarbitone-mediated increase in cytochrome P-450b + e mRNAs in the cytoplasm of the adult rat liver. However, the cytochrome P-450b protein levels are slightly lower in phenobarbitone + dexamethasone treatment than in phenobarbitone-treated liver microsomes.

Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells

Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation.