scholarly journals Distribution of tyrosinated and nontyrosinated alpha-tubulin during mitosis.

1986 ◽  
Vol 102 (3) ◽  
pp. 1118-1126 ◽  
Author(s):  
G G Gundersen ◽  
J C Bulinski
Keyword(s):  
Mitotic Spindle ◽  
Cultured Cells ◽  
Alpha Tubulin ◽  
Restricted Distribution ◽  
C Terminus ◽  
Major Species ◽  
A Minor ◽  
Spindle Function ◽  
Mitotic Cells ◽  
Spindle Fibers

The C-terminus of alpha-tubulin undergoes a reversible posttranslational tyrosination/detyrosination. The distributions of the tyrosinated (Tyr) and nontyrosinated (Glu) species during mitosis of cultured cells have been investigated by immunofluorescence using antibodies directed against the C-terminus of either Tyr or Glu tubulin. The distribution of Tyr tubulin differed from that of Glu tubulin at each stage of mitosis; in general, the distribution of Tyr tubulin was similar to that of total tubulin, whereas Glu tubulin had a more restricted distribution. The Glu species was found in half-spindle fibers but was not detected in astral fibers at any stage and was seen in the interzone only during telophase. These results were confirmed by a direct comparison of the distributions of Tyr and Glu tubulin in cells double-labeled with the two antibodies. Evidence for the occurrence of Tyr and Glu tubulin in each class of half-spindle fibers (kinetochore and polar) was obtained from the staining patterns of the two antibodies in cold-treated cells. Immunoblots of extracts prepared from synchronous mitotic cells showed that Glu tubulin was a minor species of the total tubulin in the spindle; no changes in the amount of either Tyr or Glu tubulin were detected at any stage of mitosis. These results show that Tyr tubulin is the major species in the mitotic spindle and is found in all classes of spindle fibers, whereas Glu tubulin is present in small amounts and shows a more restricted distribution. The presence of two biochemically distinct forms of alpha-tubulin in the spindle may be important for spindle function.

Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells

2001 ◽  
Vol 114 (4) ◽  
pp. 775-784 ◽  
Author(s):  
T. Kato ◽  
N. Watanabe ◽  
Y. Morishima ◽  
A. Fujita ◽  
T. Ishizaki ◽  
...  
Keyword(s):  
Hela Cells ◽  
Mitotic Spindle ◽  
Green Fluorescence Protein ◽  
Small Gtpase ◽  
Binding Domain ◽  
Fixation Method ◽  
C Terminus ◽  
Gfp Fluorescence ◽  
Fluorescence Protein ◽  
Mitotic Cells

mDia1 is a mammalian homolog of Drosophila diaphanous and works as an effector of the small GTPase Rho. It is a member of the formin homology (FH) proteins and contains the Rho-binding domain and an FH3 region in its N terminus, an FH1 region containing polyproline stretches in the middle and an FH2 region in the C terminus. Several lines of evidence indicate that mDia1 and diaphanous are essential in cytokinesis. mDia1 is present in a large amount in the cytoplasm of both interphase and mitotic cells. Using the instantaneous fixation method that preferentially extracts soluble components, we have analyzed localization of mDia1 in mitotic HeLa cells. Immunocytochemistry using polyclonal anti-mDia1 antibody revealed specific immunofluorescence localized to the mitotic spindle. This localization was seen from prophase to telophase. Western blot analysis also detected anti-mDia1 immunoreactivity in the mitotic spindle fraction isolated from mitotic HeLa cells. Consistently, expression of full-length mDia1 as a fusion protein with green fluorescence protein (GFP) revealed the GFP fluorescence again in the mitotic spindle in HeLa cells. Expression of GFP fusions of various truncated mutants of mDia1 identified that this localization is determined by a 173 amino acid-long sequence between the Rho-binding domain and the FH1 region, which contains the C-terminal part of the FH3 region. Point mutation analysis revealed that Leu(434) and Leu(455) in the FH3 region are essential in localization to the mitotic spindle. Neither electroporation of botulinum C3 exoenzyme nor microinjection of Val14RhoA into mitotic cells affected the localization of endogenous mDia1 to the mitotic spindle, suggesting that mDia1 localizes to the mitotic spindle independent of Rho activity. The present study has thus established the mDia1 localization in the mitotic spindle. This localization suggests a role of mDia1 in the spindle-cleavage furrow interaction during cell division.

scholarly journals Posttranslational modifications of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells.

1988 ◽  
Vol 106 (4) ◽  
pp. 1213-1220 ◽  
Author(s):  
J C Bulinski ◽  
J E Richards ◽  
G Piperno
Keyword(s):  
Posttranslational Modifications ◽  
Cultured Cells ◽  
Cell Types ◽  
African Green Monkey Kidney ◽  
Alpha Tubulin ◽  
Acetylated Tubulin ◽  
Minor Population ◽  
A Minor ◽  
Kinetics Of

Subsets of microtubules enriched in posttranslationally detyrosinated (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779) or acetylated (Piperno, G., M. Le Dizet, and X. Chang. 1987. J. Cell Biol. 104:298), alpha tubulin have previously been described in interphase cultured cells. In this study an immunofluorescence comparison of these minor populations of microtubules revealed that, in African green monkey kidney epithelial cells (TC-7 line), the population of microtubules enriched in detyrosinated tubulin was virtually coincident with the population enriched in acetylated alpha tubulin. In some cell types, however, such as human HeLa or marsupial PtK-2 cells, only one posttranslationally modified form of tubulin, i.e., acetylated or detyrosinated, respectively, was detectable in microtubules. In TC-7 cells, although both modifications were present, dissimilar patterns and kinetics of reappearance of microtubules enriched in detyrosinated and acetylated tubulin were observed after recovery of cells from microtubule-depolymerizing treatments or from mitosis. Thus, a minor population of microtubules exists in cultured cells that contains an elevated level of tubulin modified in either one or two ways. While these two modifications occur primarily on the same subset of microtubules, they differ in their patterns of formation in vivo.

Filaments and membranes in the mitotic apparatus

1983 ◽  
Vol 41 ◽  
pp. 544-547
Author(s):  
Kent McDonald
Keyword(s):  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mitotic Apparatus ◽  
Light Microscope Level ◽  
Microtubule Associated Proteins ◽  
Recent Developments ◽  
Associated Proteins ◽  
Force Generator ◽  
Spindle Function ◽  
Antibody Technology

At the light microscope level the recent developments and interest in antibody technology have permitted the localization of certain non-microtubule proteins within the mitotic spindle, e.g., calmodulin, actin, intermediate filaments, protein kinases and various microtubule associated proteins. Also, the use of fluorescent probes like chlorotetracycline suggest the presence of membranes in the spindle. Localization of non-microtubule structures in the spindle at the EM level has been less rewarding. Some mitosis researchers, e.g., Rarer, have maintained that actin is involved in mitosis movements though the bulk of evidence argues against this interpretation. Others suggest that a microtrabecular network such as found in chromatophore granule movement might be a possible force generator but there is little evidence for or against this view. At the level of regulation of spindle function, Harris and more recently Hepler have argued for the importance of studying spindle membranes. Hepler also believes that membranes might play a structural or mechanical role in moving chromosomes.

scholarly journals RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains

2014 ◽  
Vol 42 (15) ◽  
pp. 10185-10195 ◽  
Author(s):  
Constanze Schelhorn ◽  
James M.B. Gordon ◽  
Lidia Ruiz ◽  
Javier Alguacil ◽  
Enrique Pedroso ◽  
...  
Keyword(s):  
Rna Binding ◽  
Titration Calorimetry ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Mobility Shift ◽  
Cytoplasmic Polyadenylation ◽  
C Terminus ◽  
Self Association ◽  
Mobility Shift Assay ◽  
A Minor

Abstract Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1–4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1–RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins’ ability to interact with RNA as demonstrated by ion mobility–mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1–RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1–RRM2–CPE complex that illustrates the experimental data.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

Microtubules rich in modified alpha-tubulin characterize the tail processes of motile fibroblasts

1989 ◽  
Vol 94 (2) ◽  
pp. 227-236
Author(s):  
A.R. Prescott ◽  
M. Vestberg ◽  
R.M. Warn
Keyword(s):  
Cell Motility ◽  
Immunofluorescence Microscopy ◽  
Fibroblast Cell ◽  
Microscopy Study ◽  
Post Translational Modifications ◽  
Alpha Tubulin ◽  
Major Species ◽  
Cytoplasmic Tails ◽  
Nih 3T3

The organisation of microtubules rich in post-translationally modified alpha-tubulin has been investigated in a fibroblast cell line (NIH-3T3-T15) that can be reversibly transformed. An immunofluorescence microscopy study of the static non-transformed cells has revealed a central distribution of wavy microtubules showing post-translational modifications. When transformed there is a marked increase in cell motility and the appearance of long thin cytoplasmic ‘tails’. These tails have been found to contain conspicuous bundles of post-translationally modified microtubules that run down the length of the processes and terminate close to the plasmalemma. Both detyrosinated and acetylated alpha-tubulin are present as major species in these modified microtubules. Such a pattern of modified microtubules is only occasionally seen in the untransformed NIH-3T3-T15 cells. We have also found them to be present in other transformed fibroblast lines. The presence of bundles of microtubules rich in modified alpha-tubulin in the cell tails is correlated with a marked reduction in the numbers of F-actin stress fibres. The possible role of these modified stable microtubules in cell motility is discussed.

scholarly journals A Minus-End–directed Kinesin with Plus-End Tracking Protein Activity Is Involved in Spindle Morphogenesis

2005 ◽  
Vol 16 (4) ◽  
pp. 1584-1592 ◽  
Author(s):  
J. Christian Ambrose ◽  
Wuxing Li ◽  
Adam Marcus ◽  
Hong Ma ◽  
Richard Cyr
Keyword(s):  
Motor Activity ◽  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Null Mutant ◽  
Mitotic Spindles ◽  
Protein Activity ◽  
Interphase Cells ◽  
Motor Activities ◽  
Spindle Function

Diverse kinesin motor proteins are involved in spindle function; however, the mechanisms by which they are targeted to specific sites within spindles are not well understood. Here, we show that a fusion between yellow fluorescent protein (YFP) and a minus-end–directed Kinesin-14 (C-terminal family) from Arabidopsis, ATK5, localizes to mitotic spindle midzones and regions rich in growing plus-ends within phragmoplasts. Notably, in Arabidopsis interphase cells, YFP::ATK5 localizes to microtubules with a preferential enrichment at growing plus-ends; indicating ATK5 is a plus-end tracking protein (+TIP). This +TIP activity is conferred by regions outside of the C-terminal motor domain, which reveals the presence of independent plus-end tracking and minus-end motor activities within ATK5. Furthermore, mitotic spindles of atk5 null mutant plants are abnormally broadened. Based on these data, we propose a model in which ATK5 uses plus-end tracking to reach spindle midzones, where it then organizes microtubules via minus-end–directed motor activity.

scholarly journals Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX

2002 ◽  
Vol 76 (14) ◽  
pp. 6893-6899 ◽  
Author(s):  
Igor P. Dmitriev ◽  
Elena A. Kashentseva ◽  
David T. Curiel
Keyword(s):  
Heparan Sulfate ◽  
Capsid Protein ◽  
Minor Component ◽  
Cell Types ◽  
Binding Motif ◽  
Flag Epitope ◽  
C Terminus ◽  
Protein Ix ◽  
A Minor ◽  
Carboxy Terminal

ABSTRACT The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

scholarly journals Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells.

1992 ◽  
Vol 119 (6) ◽  
pp. 1559-1572 ◽  
Author(s):  
V M Fowler ◽  
E J Adam
Keyword(s):  
Cell Body ◽  
Hela Cells ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Beta Subunit ◽  
Actin Binding ◽  
Culture Dish ◽  
Cell Rounding ◽  
Interphase Cells ◽  
Mitotic Cells

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)

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