scholarly journals Distribution of acetylated alpha-tubulin in Physarum polycephalum.

1987 ◽  
Vol 104 (2) ◽  
pp. 303-309 ◽  
Author(s):  
M A Diggins ◽  
W F Dove
Keyword(s):  
Posttranslational Modification ◽  
Physarum Polycephalum ◽  
Two Dimensional ◽  
Microtubule Organizing Center ◽  
Western Blots ◽  
Alpha Tubulin ◽  
Cell Biol ◽  
Tubulin Isotype ◽  
Organizing Center ◽  
Double Label

The expression and cytological distribution of acetylated alpha-tubulin was investigated in Physarum polycephalum. A monoclonal antibody specific for acetylated alpha-tubulin, 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094), was used to screen for this protein during three different stages of the Physarum life cycle--the amoeba, the flagellate, and the plasmodium. Western blots of two-dimensional gels of amoebal and flagellate proteins reveal that this antibody recognizes the alpha 3 tubulin isotype, which was previously shown to be formed by posttranslational modification (Green, L. L., and W. F. Dove, 1984, Mol. Cell. Biol., 4:1706-1711). Double-label immunofluorescence demonstrates that, in the flagellate, acetylated alpha-tubulin is localized in the flagella and flagellar cone. Similar experiments with amoebae interestingly reveal that only within the microtubule organizing center (MTOC) are there detectable amounts of acetylated alpha-tubulin. In contrast, the plasmodial stage gives no evidence for acetylated alpha-tubulin by Western blotting or by immunofluorescence.

Variable pathways for developmental changes in composition and organization of microtubules in Physarum polycephalum

1990 ◽  
Vol 96 (3) ◽  
pp. 383-393
Author(s):  
L. Solnica-Krezel ◽  
M. Diggins-Gilicinski ◽  
T.G. Burland ◽  
W.F. Dove
Keyword(s):  
Physarum Polycephalum ◽  
Microtubule Organizing Center ◽  
Developmentally Regulated ◽  
Tubulin Isotypes ◽  
Cellular Events ◽  
Organizing Center ◽  
Cytoplasmic Microtubules ◽  
Beta 2 ◽  
Rare Cells ◽  
Microtubular Structures

The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cytoskeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodium-specific beta 2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit beta 2-tubulin at earlier or only at later stages of development. Amoeba-specific acetylated alpha 3-tubulin disappears gradually during development. Individual cells differ in the timing of loss of this isotype: alpha 3-tubulin is present in the majority of uninucleate cells, in a fraction of binucleate and quadrinucleate cells, and is absent from larger multinucleate cells. Cytoplasmic microtubules in uninucleate cells are organized by a single microtubule-organizing center (MTOC) juxtaposed to the nucleus. Binucleate cells and quadrinucleate cells exhibit variable numbers of MTOCs. Cytoplasmic microtubules persist during the APT until the stage of plasmodia containing at least 100 nuclei. The lack of a strict correlation between the changes in tubulin composition and changes in organization of microtubular structures indicates that accumulation of beta 2-tubulin and disappearance of alpha 3-tubulin isotypes are not sufficient to bring about reorganization of microtubules during development. Individual cells in a developing population differ not only in the succession of accumulation and loss of developmentally regulated tubulins, but also in the sequences of other cellular changes occurring during the APT.

scholarly journals Aggresomes Resemble Sites Specialized for Virus Assembly

2001 ◽  
Vol 153 (3) ◽  
pp. 449-456 ◽  
Author(s):  
Colin M. Heath ◽  
Miriam Windsor ◽  
Thomas Wileman
Keyword(s):  
Cellular Response ◽  
Virus Assembly ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Microtubule Organizing Center ◽  
Dna Viruses ◽  
Cytoplasmic Dna Viruses ◽  
Cell Biol ◽  
Cytoplasmic Dna ◽  
Organizing Center

The large cytoplasmic DNA viruses such as poxviruses, iridoviruses, and African swine fever virus (ASFV) assemble in discrete perinuclear foci called viral factories. Factories exclude host proteins, suggesting that they are novel subcellular structures induced by viruses. Novel perinuclear structures, called aggresomes are also formed by cells in response to misfolded protein (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883–1898; García-Mata, R., Z. Bebök, E.J. Sorscher, and E.S. Sztul. 1999. J. Cell Biol. 146:1239–1254). In this study, we have investigated whether aggresomes and viral factories are related structures. Aggresomes were compared with viral factories produced by ASFV. Aggresomes and viral factories were located close to the microtubule organizing center and required an intact microtubular network for assembly. Both structures caused rearrangement of intermediate filaments and the collapse of vimentin into characteristic cages, and both recruited mitochondria and cellular chaperones. Given that ASFV factories resemble aggresomes, it is possible that a cellular response originally designed to reduce the toxicity of misfolded proteins is exploited by cytoplasmic DNA viruses to concentrate structural proteins at virus assembly sites.

scholarly journals Reversal of the posttranslational modification on Chlamydomonas flagellar alpha-tubulin occurs during flagellar resorption.

1985 ◽  
Vol 100 (2) ◽  
pp. 457-462 ◽  
Author(s):  
S W L'Hernault ◽  
J L Rosenbaum
Keyword(s):  
Posttranslational Modification ◽  
Alpha Tubulin ◽  
Cell Biol ◽  
Control Step ◽  
Flagellar Assembly

We previously have shown that a posttranslational modification of alpha-tubulin takes place in the flagellum during Chlamydomonas flagellar assembly (L'Hernault, S. W., and J. L. Rosenbaum, 1983, J. Cell Biol., 97:258-263). In this report, we show that the posttranslationally modified alpha-3 tubulin is changed back to its unmodified alpha-1 precursor form during the microtubular disassembly that takes place during flagellar resorption. These data indicate that the addition and removal of a posttranslational modification on alpha-tubulin might be a control step in the assembly and disassembly of flagella.

Developmental regulation and identification of an isotype encoded by altB, an alpha-tubulin locus in Physarum polycephalum

1987 ◽  
Vol 7 (9) ◽  
pp. 3337-3340
Author(s):  
L L Green ◽  
M M Schroeder ◽  
M A Diggins ◽  
W F Dove
Keyword(s):  
Physarum Polycephalum ◽  
Developmental Regulation ◽  
Alpha Tubulin ◽  
Tubulin Isotype

A subcloned portion of the 5' nontranslated sequence from a Physarum alpha-tubulin cDNA is specific for a single alpha-tubulin locus, altB, of Physarum polycephalum. We find that this locus is expressed only in the plasmodium and encodes at least an alpha 1-tubulin isotype, which we have designated alpha 1B. Hybridization patterns of other subclones of this cDNA reveal two sequences for alpha-tubulin at the altB locus.

scholarly journals Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum.

1984 ◽  
Vol 4 (9) ◽  
pp. 1706-1711 ◽  
Author(s):  
L L Green ◽  
W F Dove
Keyword(s):  
Posttranslational Modification ◽  
Physarum Polycephalum ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
New Form ◽  
Rna Levels

Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, alpha 3, that is not found in nonflagellated cells. Evidence is presented that suggests that alpha 3 tubulin arises through posttranslational modification of a preexisting alpha tubulin. Pulse-chase experiments showed that labeled alpha 3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no alpha 3 tubulin, also consistent with alpha 3 being made by posttranslational modification. Levels of alpha and beta tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.

Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum

1984 ◽  
Vol 4 (9) ◽  
pp. 1706-1711
Author(s):  
L L Green ◽  
W F Dove
Keyword(s):  
Posttranslational Modification ◽  
Physarum Polycephalum ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
New Form ◽  
Rna Levels

Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, alpha 3, that is not found in nonflagellated cells. Evidence is presented that suggests that alpha 3 tubulin arises through posttranslational modification of a preexisting alpha tubulin. Pulse-chase experiments showed that labeled alpha 3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no alpha 3 tubulin, also consistent with alpha 3 being made by posttranslational modification. Levels of alpha and beta tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.

scholarly journals Developmental regulation and identification of an isotype encoded by altB, an alpha-tubulin locus in Physarum polycephalum.

1987 ◽  
Vol 7 (9) ◽  
pp. 3337-3340 ◽  
Author(s):  
L L Green ◽  
M M Schroeder ◽  
M A Diggins ◽  
W F Dove
Keyword(s):  
Physarum Polycephalum ◽  
Developmental Regulation ◽  
Alpha Tubulin ◽  
Tubulin Isotype

A subcloned portion of the 5' nontranslated sequence from a Physarum alpha-tubulin cDNA is specific for a single alpha-tubulin locus, altB, of Physarum polycephalum. We find that this locus is expressed only in the plasmodium and encodes at least an alpha 1-tubulin isotype, which we have designated alpha 1B. Hybridization patterns of other subclones of this cDNA reveal two sequences for alpha-tubulin at the altB locus.

Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

1994 ◽  
Vol 52 ◽  
pp. 310-311
Author(s):  
M.B. Braunfeld ◽  
M. Moritz ◽  
B.M. Alberts ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Vital Role ◽  
Complex Nature ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Nucleation Sites ◽  
Dimensional Reconstruction ◽  
Organizing Center ◽  
Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

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