scholarly journals Ase1 domains dynamically slow anaphase spindle elongation and recruit Bim1 to the midzone

2020 ◽  
Author(s):  
Ezekiel C. Thomas ◽  
Amber Ismael ◽  
Jeffrey K. Moore
Keyword(s):  
Elongation Rate ◽  
Modular System ◽  
Binding Domains ◽  
Anaphase Onset ◽  
Carboxy Terminal Domain ◽  
Late Anaphase ◽  
Early Anaphase ◽  
Spindle Elongation ◽  
Carboxy Terminal ◽  
And Control

ABSTRACTHow cells regulate microtubule crosslinking activity to control the rate and duration of spindle elongation during anaphase is poorly understood. In this study, we test the hypothesis that PRC1/Ase1 proteins use distinct microtubule-binding domains to control spindle elongation rate. Using budding-yeast Ase1, we identify unique contributions for the spectrin and carboxy-terminal domains during different phases of spindle elongation. We show that the spectrin domain uses conserved, basic residues to promote the recruitment of Ase1 to the midzone before anaphase onset and slow spindle elongation during early anaphase. In contrast, a partial Ase1 carboxy-terminal truncation fails to form a stable midzone in late anaphase, produces faster elongation rates after early anaphase, and exhibits frequent spindle collapses. We find that the carboxy-terminal domain interacts with the plus-end tracking protein EB1/Bim1 and recruits Bim1 to the midzone to maintain midzone length. Overall, our results suggest that the Ase1 domains provide cells with a modular system to tune midzone activity and control elongation rates.

scholarly journals Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker.

1993 ◽  
Vol 120 (1) ◽  
pp. 129-139 ◽  
Author(s):  
M Algrain ◽  
O Turunen ◽  
A Vaheri ◽  
D Louvard ◽  
M Arpin
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Actin Filaments ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Binding Domains ◽  
Carboxy Terminal Domain ◽  
Cell Surface Structures ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Ezrin, a widespread protein present in actin-containing cell-surface structures, is a substrate of some protein tyrosine kinases. Based on its primary and secondary structure similarities with talin and band 4.1 it has been suggested that this protein could play a role in linking the cytoskeleton to the plasma membrane (Gould, K.L., A. Bretscher, F.S. Esch, and T. Hunter. 1989. EMBO (Eur. Mol. Biol. Organ.), J. 8:4133-4142; Turunen, O., R. Winqvist, R. Pakkanen, K.-H. Grzeschik, T. Wahlström, and A. Vaheri. 1989. J. Biol. Chem. 264:16727-16732). To test this hypothesis, we transiently expressed the complete human ezrin cDNA, or truncated cDNAs encoding the amino- and carboxy-terminal domains of the protein, in CV-1 cells. Protein epitope tagging was used to unambiguously determine the subcellular distribution of the protein encoded by the transfected cDNA. We show that this protein is concentrated underneath the dorsal plasma membrane in all actin-containing structures and is partially detergent insoluble. The amino-terminal domain displays the same localization but is readily extractable by nonionic detergent. The carboxy-terminal domain colocalizes with microvillar actin filaments as well as with stress fibers and remains associated with actin filaments after detergent extraction, and with disorganized actin structures after cytochalasin D treatment. Our results clearly demonstrate that ezrin interacts with membrane-associated components via its amino-terminal domain, and with the cytoskeleton via its carboxy-terminal domain. The amino-terminal domain could include the main determinant that restricts the entire protein to the cortical cytoskeleton in contact with the dorsal plasma membrane and its specialized microdomains such as microvilli, microspikes and lamellipodia.

scholarly journals Organization of spindle microtubules in Ochromonas danica.

1980 ◽  
Vol 87 (3) ◽  
pp. 531-545 ◽  
Author(s):  
D H Tippit ◽  
L Pillus ◽  
J Pickett-Heaps
Keyword(s):  
Average Length ◽  
Numerical Data ◽  
Tracking Errors ◽  
Mitotic Apparatus ◽  
Ochromonas Danica ◽  
Spindle Poles ◽  
Late Anaphase ◽  
Early Anaphase ◽  
Spindle Microtubules ◽  
Spindle Elongation

The entire framework of microtubules (MTs) in the mitotic apparatus of Ochromonas danica is reconstructed (except at the spindle poles) from transverse serial sections. Eleven spindles were sectioned and used for numerical data, but only four were reconstructed: a metaphase, an early anaphase, a late anaphase, and telophase. Four major classes of MTs are observed: (a) free MTs (MTs not attached to either pole); (b) interdigitated MTs (MTs attached to one pole which laterally associate with MTs from the opposite pole); (c) polar MTs (MTs attached to one pole); (d) kinetochore MTs (kMTs). Pole-to-pole MTs are rare and may be caused by tracking errors. During anaphase, the kMTs, free MTs, and polar MTs shorten until most disappear, while interdigitated MTs lengthen. In the four reconstructed spindles, the number of MTs decreases between early anaphase and telophase from 881 to 285, while their average length increases from 1.66 to 4.98 micron. The total length of all the MTs in the spindle (placed end to end) remains at 1.42 +/- 0.04 mm between these stages. At late anaphase and telophase the spindle is comprised mainly of groups of interdigitated MTs. Such MTs from opposite poles form a region of overlap in the middle of the spindle. During spindle elongation (separation of the poles), the length of the overlap region does not decrease. These results are compatible with theories that suggest that MTs directly provide the force that elongates the spindle, either by MT polymerization alone or by MT sliding with concomitant MT polymerization.

scholarly journals Kinetochore microtubule dynamics and the metaphase-anaphase transition.

1995 ◽  
Vol 131 (3) ◽  
pp. 721-734 ◽  
Author(s):  
Y Zhai ◽  
P J Kronebusch ◽  
G G Borisy
Keyword(s):  
Dynamic Behavior ◽  
Mammalian Cells ◽  
Chromosome Movement ◽  
Relative Contribution ◽  
Anaphase Onset ◽  
Early Anaphase ◽  
Order Of Magnitude ◽  
Spindle Elongation ◽  
Dramatic Shift ◽  
The Stability

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.

scholarly journals Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 674
Author(s):  
Francesco Capriglia ◽  
Francesca Rizzo ◽  
Giuseppe Petrosillo ◽  
Veronica Morea ◽  
Giulia d’Amati ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Molecular Mechanisms ◽  
Mitochondrial Protein ◽  
Trna Synthetase ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Mitochondrial Functions ◽  
Carboxy Terminal

The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

scholarly journals What’s all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry

2021 ◽  
Author(s):  
Blase Matthew LeBlanc ◽  
Rosamaria Yvette Moreno ◽  
Edwin Escobar ◽  
Mukesh Kumar Venkat Ramani ◽  
Jennifer S Brodbelt ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phosphorylation State ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Protein Encoding ◽  
Small Nuclear Rnas ◽  
Rnap Ii ◽  
Carboxy Terminal ◽  
Encoding Genes

RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and...

scholarly journals The signal transducer gp130: Solution structure of the carboxy-terminal domain of the cytokine receptor homology region

2008 ◽  
Vol 8 (1) ◽  
pp. 5-12 ◽  
Author(s):  
Thomas Kernebeck ◽  
Stefan Pflanz ◽  
Peter C. Heinrich ◽  
Axel Wollmer ◽  
Joachim Grötzinger ◽  
...  
Keyword(s):  
Solution Structure ◽  
Cytokine Receptor ◽  
Signal Transducer ◽  
Homology Region ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

Study on the association between CTD peptides and zinc(ii)-dipicolylamine appended beta-cyclodextrin

RSC Advances ◽  
2015 ◽  
Vol 5 (98) ◽  
pp. 80434-80440 ◽  
Author(s):  
Saihui Zhang ◽  
Yantao Shi ◽  
Wei Wang ◽  
Zhi Yuan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Beta Cyclodextrin ◽  
Carboxy Terminal

Association between zinc(ii)-dipicolylamine appended beta-cyclodextrin and CTD (carboxy-terminal domain of RNA polymerase II) peptides with different phosphorylation patterns was studied by ITC and NMR.

scholarly journals A Functional Interaction between the Carboxy-Terminal Domain of RNA Polymerase II and Pre-mRNA Splicing

1997 ◽  
Vol 136 (1) ◽  
pp. 5-18 ◽  
Author(s):  
Lei Du ◽  
Stephen L. Warren
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Functional Interaction ◽  
Carboxy Terminal Domain ◽  
Localization Signal ◽  
Nuclear Domains ◽  
Carboxy Terminal

In the preceding study we found that Sm snRNPs and SerArg (SR) family proteins co-immunoprecipitate with Pol II molecules containing a hyperphosphorylated CTD (Kim et al., 1997). The association between Pol IIo and splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged (Kim et al., 1997). The latter findings led us to hypothesize that a phosphorylated form of the CTD interacts with pre-mRNA splicing components in vivo. To test this idea, a nested set of CTD-derived proteins was assayed for the ability to alter the nuclear distribution of splicing factors, and to interfere with splicing in vivo. Proteins containing heptapeptides 1-52 (CTD52), 1-32 (CTD32), 1-26 (CTD26), 1-13 (CTD13), 1-6 (CTD6), 1-3 (CTD3), or 1 (CTD1) were expressed in mammalian cells. The CTD-derived proteins become phosphorylated in vivo, and accumulate in the nucleus even though they lack a conventional nuclear localization signal. CTD52 induces a selective reorganization of splicing factors from discrete nuclear domains to the diffuse nucleoplasm, and significantly, it blocks the accumulation of spliced, but not unspliced, human β-globin transcripts. The extent of splicing factor disruption, and the degree of inhibition of splicing, are proportional to the number of heptapeptides added to the protein. The above results indicate a functional interaction between Pol II's CTD and pre-mRNA splicing.

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