Talin concentrates to the midbody region during mammalian cell cytokinesis.

In this study we investigated the cellular distribution of talin, a cytoskeletal protein, during mammalian cell cytokinesis. Immunohistochemical experiments on various carcinoma cell lines and mesenchyme-derived cells reveal that talin displays a cell cycle-dependent cellular localization. During metaphase, talin is located in the centromeric region of the chromosome, like the TD-60 protein and intrinsic centromere components detected by a CREST serum. From anaphase to telophase, talin is present in the cleavage furrow. As the cells progress to cytokinesis, when the furrow is complete, talin is concentrated in the midbody structures, as assessed by immunofluorescence and confirmed by Western blot experiments on purified midbodies. Double staining experiments reveal that alpha-tubulin, TD-60 protein, and talin co-localize in the midbodies. These results suggest that talin, in addition to its implication in focal adhesion organization and signaling, may play a critical role in cytokinesis. (J Histochem Cytochem 47:1357-1367, 1999)     

Targeting of dystroglycan to the cleavage furrow and midbody in cytokinesis

Dystroglycan is a cell adhesion molecule that interacts with ezrin family proteins and also components of the extracellular signal-regulated kinase pathway. Ezrin and extracellular signal-regulated kinase are both involved in aspects of the cell division cycle. We therefore examined the role of dystroglycan during cytokinesis. Endogenous dystroglycan colocalised with ezrin at the cleavage furrow and midbody during cytokinesis in REF52 cells. Live cell imaging of green fluorescent protein-tagged dystroglycan in Swiss 3T3 and Hela cells revealed a similar localisation. Live cell imaging of a dystroglycan lacking its cytoplasmic domain revealed an even membrane localisation but no cleavage furrow or midbody localisation. Deletion of a previously identified ezrin-binding site in the dystroglycan cytoplasmic domain however only resulted in a slight reduction in cleavage furrow localisation but loss of midbody staining. There was no apparent cytokinetic defect in cells depleted for dystroglycan, however apoptosis levels were considerably higher in dystroglycan knockdown cells. Cell cycle analysis showed a delay in G2/M transition, possibly caused by a more than 50% reduction in extracellular signal-regulated kinase levels in the knockdown cells. Dystroglycan may therefore not only have a role in organising the contractile ring through direct or indirect associations with actin, but can also modulate the cell cycle by affecting extracellular signal-regulated kinase levels.     

Two forms of human Inscuteable-related protein that links Par3 to the Pins homologues LGN and AGS3

In cell polarization of Drosophila neuroblasts, Inscuteable (Insc) functions via tethering Partner of Insc (Pins) to Bazooka, homologous to human cell polarity protein Par3. However, little has been known about mammalian homologues of Insc. Here we describe cloning of two distinct cDNAs from human Insc gene, which is differentially expressed from alternative first exons: one encodes 579 amino acids, whereas the other lacks the N-terminal 47 amino acids. In contrast to human homologues for Pins and Par3, human Insc exhibits a weak homology with the Drosophila counterpart. Nevertheless, human Insc proteins bind to the human Pins homologues LGN and AGS3, and also to human Par3 and its related protein Par3beta. Although LGN by itself is incapable of interacting with Par3, coexpression of human Insc leads to the interaction between LGN and Par3, indicating that human Insc plays an evolutionarily conserved role as an adaptor protein that links Pins to Par3.     

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis.

Abbreviations: CSN, COP9 Signalosome; MKLP1, mitotic kinesin-like protein 1; NEDD8, Neural precursor cell Expressed, Developmentally Down-regulated 8.     

Cep55, a microtubule-bundling protein, associates with centralspindlin to control the midbody integrity and cell abscission during cytokinesis

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.     

Localization and subcellular distribution of prolyl oligopeptidase in the mouse placenta

Prolyl oligopeptidase (POP) is a serine endopeptidase which selectively digests a -Pro-X- peptide bond. Our previous study showed that POP mRNA was strongly expressed in the spongiotrophoblast of the mouse placenta at E17.5, suggesting its importance in development. To gain more insight into POP’s role during gestation, we investigated its expression using different developmental stages of placenta. As a result of in situ hybridization, we found that localization of POP mRNA changed at E12.5. POP mRNA was strongly expressed in the spongiotrophoblast and labyrinth at E10.5 and E11.5 but thereafter only in the spongiotrophoblast. Immunohistochemistry revealed that POP was present in the parietal trophoblast giant cell, the spongiotrophoblast cell, and the labyrinth at E11.5 but the strong expression in the labyrinth was maintained only in the canal-associated and sinusoidal trophoblast giant cells at E16.5 and E18.5. To determine subcellular distribution of the POP protein, we fractionated the placental extract into cytoplasmic, membrane, and nuclear subfractions. By Western blot analysis, POP was detected in the cytoplasmic and membrane fractions but not in the nuclear fraction at E11.5 and E16.5. Interestingly, the cytoplasmic POP exhibited higher enzymatic activity than the membrane-associated type. These data suggest that the cytoplasmic and membrane-associated POP have distinct roles in different types of placental cells.     

Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct

Background  

Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS).     

Quantitation of the distribution and flux of myosin-II during cytokinesis

Background  

During cytokinesis, the cell's equator contracts against the cell's global stiffness. Identifying the biochemical basis for these mechanical parameters is essential for understanding how cells divide. To achieve this goal, the distribution and flux of the cell division machinery must be quantified. Here we report the first quantitative analysis of the distribution and flux of myosin-II, an essential element of the contractile ring.     

Interactions of the cbbII promoter-operator region with CbbR and RegA (PrrA) regulators indicate distinct mechanisms to control expression of the two cbb operons of Rhodobacter sphaeroides

In a previous study (Dubbs, J. M., Bird, T. H., Bauer, C. E., and Tabita, F. R. (2000) J. Biol. Chem. 275, 19224-19230), it was demonstrated that the regulators CbbR and RegA (PrrA) interacted with both promoter proximal and promoter distal regions of the form I (cbb(I)) promoter operon specifying genes of the Calvin-Benson-Bassham cycle of Rhodobacter sphaeroides. To determine how these regulators interact with the form II (cbb(II)) promoter, three cbbF(II)::lacZ translational fusion plasmids were constructed containing various lengths of sequence 5' to the cbb(II) operon of R. sphaeroides CAC. Expression of beta-galactosidase was monitored under a variety of growth conditions in both the parental strain and knock-out strains that contain mutations that affect synthesis of CbbR and RegA. The binding sites for both CbbR and RegA were determined by DNase I footprinting. A region of the cbb(II) promoter from +38 to -227 bp contained a CbbR binding site and conferred low level regulated cbb(II) expression. The region from -227 to -1025 bp contained six RegA binding sites and conferred enhanced cbb(II) expression under all growth conditions. Unlike the cbb(I) operon, the region between -227 and -545 bp that contains one RegA binding site, was responsible for the majority of the observed enhancement. Both RegA and CbbR were required for maximal cbb(II) expression. Two potentially novel and specific cbb(II) promoter-binding proteins that did not interact with the cbb(I) promoter region were detected in crude extracts of R. sphaeroides. These results, combined with the observation that chemoautotrophic expression of the cbb(I) operon is RegA independent, indicated that the mechanisms controlling cbb(I) and cbb(II) operon expression during chemoautotrophic growth are quite different.     

Genetic approaches to dissect the mechanisms of two distinct pathways of cell cycle-coupled cytokinesis in Dictyostelium

Dictyostelium discoideum is a unique experimental organism which allows genetic analysis of the mechanism of cytokinesis of the animal type, and a number of mutations which affect cytokinesis in one way or other have been identified. Myosin II filaments accumulate in the equatorial region, and myosin II-null cells cannot divide in suspension, indicating that active, myosin II-dependent constriction of the cleavage furrow contributes to bisection of the cell. We refer to this method of cytokinesis as cytokinesis A. On substrates, however, myosin II-null cells divide efficiently in a cell cycle-coupled manner. This adhesion-dependent but myosin II-independent division method, which we termed cytokinesis B, is carried out by a pathway that is genetically distinct from that of cytokinesis A. Morphological analyses suggested that cytokinesis B is driven by radial traction forces generated along polar peripheries, which indirectly cause furrow ingression. Identification of two redundant pathways have allowed us to search genes involved in either pathway by mutagenizing cells which are already defective in one of the pathways. This approach enabled us to identify a number of novel cytokinesis-related genes, as well as to reclassify known genes as cytokinesis-related.     

BRCA2 localization to the midbody by filamin A regulates cep55 signaling and completion of cytokinesis

Disruption of the BRCA2 tumor suppressor is associated with structural and numerical chromosomal defects. The numerical abnormalities in BRCA2-deficient cells may partly result from aberrations in cell division caused by disruption of BRCA2 during cytokinesis. Here we show that BRCA2 is a component of the midbody that is recruited through an interaction with Filamin A actin-binding protein. At the midbody, BRCA2 influences the recruitment of endosomal sorting complex required for transport (ESCRT)-associated proteins, Alix and Tsg101, and formation of CEP55-Alix and CEP55-Tsg101 complexes during abscission. Disruption of these BRCA2 interactions by cancer-associated mutations results in increased cytokinetic defects but has no effect on BRCA2-dependent homologous recombination repair of DNA damage. These findings identify a specific role for BRCA2 in the regulation of midbody structure and function, separate from DNA damage repair, that may explain in part the whole-chromosomal instability in BRCA2-deficient tumors.     

Role of the midbody matrix in cytokinesis: RNAi and genetic rescue analysis of the mammalian motor protein CHO1

CHO1 is a kinesin-like motor protein essential for cytokinesis in mammalian cells. To analyze how CHO1 functions, we established RNAi and genetic rescue assays. CHO1-depleted cells reached a late stage of cytokinesis but fused back to form binucleate cells because of the absence of the midbody matrix in the middle of the intercellular bridge. Expression of exogenous CHO1 restored the formation of the midbody matrix and rescued cytokinesis in siRNA-treated cells. By analyzing phenotypes rescued with different constructs, it was shown that both motor and stalk domains function in midbody formation, whereas the tail is essential for completion of cytokinesis after the midbody matrix has formed. During the terminal stage of cytokinesis, different subregions of the tail play distinctive roles in stabilizing the midbody matrix and maintaining an association between the midbody and cell cortex. These results demonstrate that CHO1 consists of functionally differentiated subregions that act in concert to ensure complete cell separation.     

NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated α-tubulin, suggesting that it plays a role in maintaining or enhancing stability of α-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated α-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.     

Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine. The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.     

Localization and activation of the ARF6 GTPase during cleavage furrow ingression and cytokinesis

The ARF6 GTPase mediates cell shape changes in interphase cells through its effects on membrane cycling and actin remodeling. In this study, we focus our attention on the dynamics of cell division and present evidence supporting a novel role for ARF6 during cleavage furrow ingression and cytokinesis. We demonstrate that endogenous ARF6 redistributes during mitosis and concentrates near the cleavage furrow during telophase. Constitutively activated ARF6 localizes to the plasma membrane at the site of cleavage furrow ingression and midbody formation, and dominant negative ARF6 remains cytoplasmic. By using a novel pull-down assay for ARF6-GTP, we find an abrupt, but transient, increase in ARF6-GTP levels as cells progress through cytokinesis. Whereas high levels of expression of a GTPase-defective ARF6 mutant induce aberrant phenotypes in cells at cytokinesis, cells expressing low levels of ARF6 mutants do not display a significant mitotic delay or cytokinesis defect, presumably due to compensatory or redundant mechanisms that allow cytokinesis to proceed when the ARF6 GTPase cycle is disrupted. Finally, actin accumulation and phospholipid metabolism at the cleavage furrow are unchanged in cells expressing ARF6 mutants, suggesting that ARF6 may be involved in membrane remodeling during cytokinesis via effector pathways that are distinct from those operative in interphase cells.     

Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity

The pheromone signal in the yeastSaccharomyces cerevisiae is transmitted by the and subunits of the mating response G-protein. TheSTE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G mutation. The same genetic screen identifiedBEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designatedMDG1 (multicopy suppressor ofdefectiveG-protein). TheMDG1 gene was independently isolated in a search for multicopy suppressors of abem1 mutation. TheMDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein fromAequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion ofMDG1 causes sterility in cells in which the wild-type G has been replaced by partly defective G derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted forSTE20 is partially suppressed by multiple copies ofBEM1 andCDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels ofSTE20 andBEM1 are capable of suppressing a temperature-sensitive mutation inCDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.     

Accumulation of F-actin during cytokinesis inAllium. Correlation with microtubule distribution and the effects of drugs

Summary The distribution of F-actin in the phragmoplast/cell plate complex of formaldehyde-fixedAllium root cells was visualized with rhodaminephalloidin (RP). Increased RP fluorescence appears in late anaphase in a broad zone between separating chromosomes. The fluorescence is mostly amorphous in appearance and does not resemble the distinct actin fibers seen in interphase cells. The actin becomes more concentrated near the midplane by telophase and takes the form of a relatively bright layer of fluorescence adjacent to the forming cell plate. This distribution differs markedly from that of phragmoplast microtubules (MTs) which extend back from the plate toward the daughter nuclei. F-actin continues to accumulate in new parts of the expanding phragmoplast, while RP fluorescence gradually decreases near older portions of the plate. It disappears completely near the new wall in most interphase cells. Treatment of root tips with cytochalasin B or D before fixation markedly reduces RP fluorescence, but phragmoplast MTs remain. Colchicine or oryzalin treatment leads to the disappearance of both phragmoplast actin and MTs. The possible function of actin in the phragmoplast/cell plate complex is discussed.Abbreviations CB cytochalasin B - CD cytochalasin D - CIPC isopropyl N-(3-chlorophenyl-)carbamate - DIC differential interference contrast - MT microtubule - PBS phosphate buffered saline - PM plasmalemma - RP rhodamine-phalloidin