The assembly and positioning of cytoskeletal elements in Tetrahymena

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 23-30
Author(s):  
NE Williams ◽  
JE Honts
Keyword(s):  
Membrane Skeleton ◽  
Basal Bodies ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Conditional Mutant ◽  
Correct Pattern ◽  
Feeding Structure ◽  
Filament Protein ◽  
Cytoskeletal Elements

The oral skeleton of Tetrahymena is a precisely arranged assemblage of basal bodies, microtubule bundles and connecting filaments found associated with the feeding structure in this cell type. Tubulin and filament proteins have been isolated but no actin has been recovered. The conditional mutant NP1 of Tetrahymena thermophila forms a normal oral skeleton at the permissive temperature (28 degrees C), but forms an abnormal one at the restrictive temperature (37 degrees C). Antibodies against tubulin and oral filament protein OF1 were used to visualize the abnormal oral skeleton and stages in its development, and ultrastructural comparisons of abnormal and wild-type oral skeletons were made. It was found that the overall pattern of organization was altered in the mutant, whereas the substructure appeared everywhere to be normal. Studies of cells in which the mutant phenotype was coming to expression revealed that normal basal bodies in the oral skeleton failed to move from the disordered state characteristic of early stages of development into the correct pattern of four organized clusters characteristic of later stages. Together, these results suggest that the lesion in NP1 does not affect cytoskeleton assembly per se, but instead affects a discrete mechanism responsible for the positioning of cytoskeletal elements with respect to each other after they have been formed (meta-assembly). Reasons for suspecting the involvement of the membrane skeleton are presented.

The Membrane Skeleton of Pseudomicrothorax

1991 ◽  
Vol 100 (4) ◽  
pp. 707-715 ◽  
Author(s):  
IRM HUTTENLAUCH ◽  
ROBERT K. PECK
Keyword(s):  
Spatial Organization ◽  
Membrane Skeleton ◽  
Basal Bodies ◽  
Structural Elements ◽  
Or Groups ◽  
Positive Immunoreactivity ◽  
Cortical Membranes ◽  
Cytoskeletal Elements

The membrane skeleton, or epiplasm, is part of the structurally complex ciliate cortex. It is thought to have skeletal functions concerning the spatial organization of cortical elements such as the basal bodies. Here we report the biochemical and immunological characterization of some components of the purified epiplasm of Pseudomicrothorax dubius. The epiplasm proteins consist of two quantitatively major groups of proteins, one of 76–80x103Mr, the other of 11–13x103Mr, which appear to be the principal structural elements of the epiplasm, and a series of minor components of 62–18x103Mr. Based upon lectin labeling and glycosidase treatment, some of the latter have been identified as glycoproteins. Using affinity-purified antibodies specific for individual glycoproteins or groups of glycoproteins, we were able to localize them in situ by immunoelectron microscopical methods. This in situ localization demonstrates that the glycosylated epitopes, unlike the glycoresidues of membrane proteins, are distributed throughout the entire epiplasmic layer rather than being restricted to regions adjacent to the cortical membranes. Thus, these proteins represent glycosylated, cytoskeletal elements. At least one of these glycoproteins (Mr 62x103) shows positive immunoreactivity with a monoclonal antibody (Pruss anti-IFA) recognizing most intermediate filament (IF) proteins, indicating that IF proteins might be present in protozoan cytoskeletons.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

scholarly journals Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Joseph C. Chen ◽  
Michael Minev ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Random Mutagenesis ◽  
Dominant Negative ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Negative Effects ◽  
Mutant Proteins ◽  
Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

scholarly journals Mitochondrial GrpE modulates the function of matrix Hsp70 in translocation and maturation of preproteins.

1995 ◽  
Vol 15 (12) ◽  
pp. 7098-7105 ◽  
Author(s):  
S Laloraya ◽  
P J Dekker ◽  
W Voos ◽  
E A Craig ◽  
N Pfanner
Keyword(s):  
Proteolytic Processing ◽  
Mitochondrial Proteins ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Conditional Mutant ◽  
The Matrix ◽  
Matrix Heat

Mitochondrial GrpE (Mge1p) is a mitochondrial cochaperone essential for viability of the yeast Saccharomyces cerevisiae. To study the role of Mge1p in the biogenesis of mitochondrial proteins, we isolated a conditional mutant allele of MGE1 which conferred a temperature-sensitive growth phenotype and led to the accumulation of mitochondrial preproteins after shifting of the cells to the restrictive temperature. The mutant Mge1 protein was impaired in its interaction with the matrix heat shock protein mt-Hsp70. The mutant mitochondria showed a delayed membrane translocation of preproteins, and the maturation of imported proteins was impaired, as evidenced by the retarded second proteolytic processing of a preprotein in the matrix. Moreover, the aggregation of imported proteins was decreased in the mutant mitochondria. The mutant Mge1p differentially modulated the interaction of mt-Hsp70 with preproteins compared with the wild type, resulting in decreased binding to preproteins in membrane transit and enhanced binding to fully imported proteins. We conclude that the interaction of Mge1p with mt-Hsp70 promotes the progress of the Hsp70 reaction cycle, which is essential for import and maturation of mitochondrial proteins.

A conditional mutant having paralyzed cilia and a block in cytokinesis is rescued by cytoplasmic exchange in Tetrahymena thermophila.

Genetics ◽  
1988 ◽  
Vol 120 (3) ◽  
pp. 697-705
Author(s):  
D G Pennock ◽  
T Thatcher ◽  
J Bowen ◽  
P J Bruns ◽  
M A Gorovsky
Keyword(s):  
Energy Production ◽  
Complementation Group ◽  
Restrictive Temperature ◽  
Single Mutation ◽  
Wild Type ◽  
Alpha Tubulin ◽  
Ciliary Function ◽  
Conditional Mutant ◽  
Complementation Groups ◽  
Group 1

Abstract Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis.

scholarly journals A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.

1988 ◽  
Vol 106 (4) ◽  
pp. 1171-1183 ◽  
Author(s):  
T Hirano ◽  
Y Hiraoka ◽  
M Yanagida
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Gradient Centrifugation ◽  
Metaphase Plate ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
In The Wild ◽  
Spindle Elongation ◽  
Mutant Cells

A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.

Suppression of a conditional mutation in alpha-tubulin by overexpression of two checkpoint genes

1995 ◽  
Vol 108 (3) ◽  
pp. 1195-1204 ◽  
Author(s):  
S. Guenette ◽  
M. Magendantz ◽  
F. Solomon
Keyword(s):  
Microtubule Assembly ◽  
Cold Sensitivity ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Checkpoint Gene ◽  
Genomic Libraries ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Checkpoint Pathway

To identify proteins that regulate microtubule assembly in Saccharomyces cerevisiae, we screened for multicopy suppressors of a conditional mutation in alpha-tubulin. Cells expressing the recessive allele tub1-729 as their sole alpha-tubulin gene grow normally at permissive temperature. However, at 15 degrees C the cells lose viability and arrest primarily with large buds and quantitatively diminished microtubule structures. Transformation of mutant cells with genomic libraries repeatedly identified three different suppressors: the two wild-type alpha-tubulin genes, TUB1 and TUB3; and BUB3. BUB3 is a checkpoint gene that permits entry into mitosis depending upon the assembly state of microtubules. Excess BUB3 rescues both the loss of viability and microtubule defects but not the benomyl supersensitivity associated with tub1-729. The suppression is specific for the mutation ALA422VAL in TUB1, and does not affect several other mutations in TUB1 that produce the ‘no microtubule’ phenotype. Overexpression of BUB1, which interacts genetically with BUB3 and which is involved in the same checkpoint pathway, also rescues the cold sensitivity of tub1-729, but another checkpoint gene, MAD2, does not. Overexpression of BUB3 in wild-type cells has no detectable growth or microtubule defect, but disruption of the BUB3 gene produces slow growth and benomyl supersensitivity. Our results suggest that BUB1 and BUB3 overexpression modulate an event required for mitotic spindle function which is rate limiting for tub1-729 cells at the restrictive temperature.

scholarly journals Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells.

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905 ◽  
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

F-actin ring formation and the role of F-actin cables in the fission yeastSchizosaccharomyces pombe

2002 ◽  
Vol 115 (5) ◽  
pp. 887-898 ◽  
Author(s):  
Ritsuko Arai ◽  
Issei Mabuchi
Keyword(s):  
Three Dimensional ◽  
Spindle Pole ◽  
Ring Formation ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Actin Ring ◽  
Medial Region ◽  
Dimensional Reconstruction ◽  
Actin Cables

Cells of the fission yeast Schizosaccharomyces pombe divide by the contraction of the F-actin ring formed at the medial region of the cell. We investigated the process of F-actin ring formation in detail using optical sectioning and three-dimensional reconstruction fluorescence microscopy. In wild-type cells, formation of an aster-like structure composed of F-actin cables and accumulation of F-actin cables were recognized at the medial cortex of the cell during prophase to metaphase. The formation of the aster-like structure seemed to initiate from branching of the longitudinal F-actin cables at a site near the spindle pole bodies, which had been duplicated but not yet separated. A single cable extended from the aster and encircled the cell at the equator to form a primary F-actin ring during metaphase. During anaphase,the accumulated F-actin cables were linked to the primary F-actin ring, and then all of these structures seemed to be packed to form the F-actin ring. These observations suggest that formation of the aster-like structure and the accumulation of the F-actin cables at the medial region of the cell during metaphase may be required to initiate the F-actin ring formation. In the nda3 mutant, which has a mutation in ß-tubulin and has been thought to be arrested at prophase, an F-actin ring with accumulated F-actin cables similar to that of anaphase wild-type cells was formed at a restrictive temperature. Immediately after shifting to a permissive temperature, this structure changed into a tightly packed ring. This suggests that the F-actin ring formation progresses beyond prophase in the nda3 cells once the cells enter prophase. We further examined F-actin structures in both cdc12 and cdc15 early cytokinesis mutants. As a result,Cdc12 seemed to be required for the primary F-actin ring formation during prophase, whereas Cdc15 may be involved in both packing the F-actin cables to form the F-actin ring and rearrangement of the F-actin after anaphase. In spg1, cdc7 and sid2 septum initiation mutants, the F-actin ring seemed to be formed in order.

scholarly journals Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

1984 ◽  
Vol 4 (9) ◽  
pp. 1815-1822 ◽  
Author(s):  
G C Zeng ◽  
J Donegan ◽  
H L Ozer ◽  
R Hand
Keyword(s):  
Dna Synthesis ◽  
Mutant Cell ◽  
Chain Elongation ◽  
Initial Increase ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
3T3 Cells ◽  
Mutant Cells

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.

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