The carboxy terminus of Tub4p is required for gamma-tubulin function in budding yeast

2000 ◽  
Vol 113 (21) ◽  
pp. 3871-3882 ◽  
Author(s):  
J. Vogel ◽  
M. Snyder
Keyword(s):  
Temperature Sensitive ◽  
Carboxy Terminus ◽  
Tail Region ◽  
Microtubule Organization ◽  
Electron Microscopic ◽  
Bud Neck ◽  
Cytoplasmic Microtubules ◽  
Gamma Tubulin ◽  
Carboxy Terminal

The role of gamma-tubulin in microtubule nucleation is well established, however, its function in other aspects of microtubule organization is unknown. The carboxy termini of alpha/beta-tubulins influence the assembly and stability of microtubules. We investigated the role of the carboxy terminus of yeast gamma-tubulin (Tub4p) in microtubule organization. This region consists of a conserved domain (DSYLD), and acidic tail. Cells expressing truncations lacking the DSYLD domain, tail or both regions are temperature sensitive for growth. Growth defects of tub4 mutants lacking either or both carboxy-terminal domains are suppressed by the microtubule destabilizing drug benomyl. tub4 carboxy-terminal mutants arrest as large budded cells with short bipolar spindles positioned at the bud neck. Electron microscopic analysis of wild-type and CTR mutant cells reveals that SPBs are tightly associated with the bud neck/cortex by cytoplasmic microtubules in mutants lacking the tail region (tub4-delta 444, tub4-delta 448). Mutants lacking the DSYLD residues (tub4-delta 444, tub4-delta DSYLD) form many cytoplasmic microtubules. We propose that the carboxy terminus of Tub4p is required for re-organization of the microtubules upon completion of nuclear migration, and facilitates spindle elongation into the bud.

Mutations in the bimC box of Cut7 indicate divergence of regulation within the bimC family of kinesin related proteins

1998 ◽  
Vol 111 (7) ◽  
pp. 853-865 ◽  
Author(s):  
D.R. Drummond ◽  
I.M. Hagan
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Temperature Sensitive ◽  
Sequence Motif ◽  
Tail Region ◽  
Amino Acid Homology ◽  
Carboxy Terminal ◽  
Related Proteins ◽  
P34 Cdc2

Members of the bimC family of kinesin related proteins (KRPs) play vital roles in the formation and function of the mitotic spindle. Although they share little amino acid homology outside the highly conserved microtubule motor domain, several family members do contain a ‘bimC box’, a sequence motif around a p34(cdc2) consensus phosphorylation site in their carboxy-terminal ‘tail’ region. One family member, Eg5, requires phosphorylation at this site for association with the mitotic spindle. We show that mutations in the Schizosaccharomyces pombe cut7+ gene that change the bimC box p34(cdc2) consensus phosphorylation site at position 1,011 and a neighbouring MAP kinase consensus phosphorylation site at position 1,020 to non-phosphorylatable residues did not affect the ability of S. pombe cut7 genes to complement temperature sensitive cut7 mutants. Phosphorylation site mutants expressed as fusions to green fluorescent protein associated with the mitotic spindle with a localisation indistinguishable from similarly expressed wild-type Cut7. Cells in which cut7.T1011A replaced the genomic copy of cut7+ were viable and formed normal spindles. Deletion of the entire carboxy-terminal tail region did not affect the ability of Cut7 to associate with the mitotic spindle but did inhibit normal spindle formation. Thus, unlike Eg5, neither the p34(cdc2) consensus phosphorylation site in the bimC box nor the entire tail region of Cut7 are required for association with the mitotic spindle.

scholarly journals Role of the Carboxy Terminus of Escherichia coli FtsA in Self-Interaction and Cell Division

2000 ◽  
Vol 182 (22) ◽  
pp. 6366-6373 ◽  
Author(s):  
Lucía Yim ◽  
Guy Vandenbussche ◽  
Jesús Mingorance ◽  
Sonsoles Rueda ◽  
Mercedes Casanova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Biological Function ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Carboxy Terminal ◽  
Two Hybrid

ABSTRACT The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of theftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362–5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsAΔ1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsAΔ27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsAΔ27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.

scholarly journals B1-type cyclins control microtubule organization during cell division in Arabidopsis

2021 ◽  
Author(s):  
Mariana Romeiro Motta ◽  
Xin'Ai Zhao ◽  
Martine Pastuglia ◽  
Katia Belcram ◽  
Farshad Roodbarkelari ◽  
...  
Keyword(s):  
Double Mutant ◽  
Cell Cycle Control ◽  
Flowering Plants ◽  
Microtubule Organization ◽  
Complex Protein ◽  
Dividing Cells ◽  
Gamma Tubulin ◽  
Microtubule Networks

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support an important role in the regulation of GIP1 by CYCB1s.

scholarly journals Autonomous Function of the Amino-Terminal Inhibitory Domain of TAF1 in Transcriptional Regulation

2004 ◽  
Vol 24 (8) ◽  
pp. 3089-3099 ◽  
Author(s):  
Shinya Takahata ◽  
Koji Kasahara ◽  
Masashi Kawaichi ◽  
Tetsuro Kokubo
Keyword(s):  
Transcriptional Regulation ◽  
Transcriptional Activation ◽  
Carboxy Terminus ◽  
Amino Terminal ◽  
Autonomous Function ◽  
General Transcription Factor ◽  
Inhibitory Domain ◽  
Carboxy Terminal ◽  
Tfiid Complex

ABSTRACT The general transcription factor TFIID is composed of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TFIID mediates the transcriptional activation of a subset of eukaryotic promoters. The N-terminal domain (TAND) of TAF1 protein (Taf1p) inhibits TBP by binding to its concave and convex surfaces. This study examines the role of the TAND in transcriptional regulation and tests whether the TAND is an autonomous regulator of TBP. The TAND binds to and regulates TBP function when it is fused to the amino or carboxy terminus of Taf1p, the amino or carboxy terminus of Taf5p, or the amino terminus of Taf11p. However, a carboxy-terminal fusion of the TAND and Taf11p is not compatible with several other TAF proteins, including Taf1p, in the TFIID complex. These results indicate that there is no or minimal geometric constraint on the ability of the TAND to function normally in transcriptional regulation as long as TFIID assembly is secured.

scholarly journals Dynamics of Multiple Nuclei in Ashbya gossypii Hyphae Depend on the Control of Cytoplasmic Microtubules Length by Bik1, Kip2, Kip3, and Not on a Capture/Shrinkage Mechanism

2010 ◽  
Vol 21 (21) ◽  
pp. 3680-3692 ◽  
Author(s):  
Sandrine Grava ◽  
Peter Philippsen
Keyword(s):  
Budding Yeast ◽  
Ashbya Gossypii ◽  
Wild Type ◽  
Major Function ◽  
Bud Neck ◽  
Pulling Forces ◽  
Cytoplasmic Microtubules ◽  
Hyphal Tip

Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus (+)end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.

scholarly journals The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast

2003 ◽  
Vol 160 (3) ◽  
pp. 355-364 ◽  
Author(s):  
Wei-Lih Lee ◽  
Jessica R. Oberle ◽  
John A. Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Mitotic Spindle ◽  
Genetic Interactions ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
Bud Neck ◽  
Cytoplasmic Microtubules ◽  
In Cells

During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother–bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.

Stannous fluoride treatment of acid-etched caries-like lesions of enamel: A scanning electron microscopic study

1984 ◽  
Vol 42 ◽  
pp. 720-721
Author(s):  
M. John Hicks ◽  
Leon M. Silverstone ◽  
David G. Gantt ◽  
Catherine M. Flaitz
Keyword(s):  
Acid Etching ◽  
Surface Zone ◽  
Electron Microscopic ◽  
Fluoride Treatment ◽  
Stannous Fluoride ◽  
Scanning Electron Microscopic Study ◽  
Demineralized Enamel ◽  
Intact Surface ◽  
Topical Fluoride

Although fluoride levels become elevated in sound enamel following a topical fluoride treatment, the caries-preventive effect of fluoride is thought to be due primarily to the role of fluoride in remineralization of clinically undetectable enamel lesions and hypomineralized enamel. During lesion formation, redistribution of fluoride from the enamel surface to the subsurface demineralized enamel occurs. This results in a surface zone with a relatively low fluoride content. In order to maintain an intact surface zone over a carious lesion, it may be necessary to replenish the fluoride levels with an exogenous fluoride source. By acid-etching the lesion surface, a more reactive surface is made available for fluoride interaction. In addition, porosities and etching patterns may be created, allowing for bonding of a caries-resistant resin material to the lesion surface. The purpose of this study was to determine the integrity of the caries-like lesion surface following acid-etching and subsequent stannous fluoride treatment (SnF2).

Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

1986 ◽  
Vol 44 ◽  
pp. 208-209
Author(s):  
Grace C.H. Yang
Keyword(s):  
Chick Embryo ◽  
Extracellular Space ◽  
Fibroblast Cell ◽  
Collagen Fibrils ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
Scanning Electron Microscopic Study ◽  
Microscopic Studies ◽  
And Function

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

The Ability of Fibrinogens, FI and FII, to Support ADP- Induced Platelet Aggregation

1983 ◽  
Vol 50 (02) ◽  
pp. 527-529 ◽  
Author(s):  
H M Phillips ◽  
A Mansouri ◽  
C A Perry
Keyword(s):  
Platelet Aggregation ◽  
Terminal Portion ◽  
Hepes Buffer ◽  
Carboxy Terminal

SummaryFibrinogen plays an integral part in ADP-induced platelet aggregation. Controversy exists in regard to the role of the carboxy termini of fibrinogen Aa chains in this reaction. We have attempted to clarify this problem in view of the availability of a highly purified FII fibrinogen fraction. Kabi fibrinogen or its purified fractions FI, FII and FIII-IV-V were added to washed platelets in the presence of Tyrode-HEPES buffer pH 7.4. Aggregation was initiated by the addition of calcium and ADP. These fibrinogen fractions equally promoted ADP-induced platelet aggregation. The major difference among these fractions is in their Aα chains. The FI fraction contains intact Aα chains while FII and FIH-IV-V fractions have one and two partially degraded Aα chains at the carboxy terminal portion respectively. We conclude that the carboxy terminal portion of the Aα chain does not play an important role in promoting ADP-induced platelet aggregation.

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