The Cofactor-Dependent Pathways for α- and β-Tubulins in Microtubule Biogenesis Are Functionally Different in Fission Yeast

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 93-103
Author(s):  
Pippa A Radcliffe ◽  
Miguel Angel Garcia ◽  
Takashi Toda
Keyword(s):  
Protein Folding ◽  
Cell Growth ◽  
Genetic Analysis ◽  
Cell Viability ◽  
Detailed Analysis ◽  
Fission Yeast ◽  
Gene Disruption ◽  
Genetic Interactions ◽  
Evolutionarily Conserved ◽  
Β Tubulin

Abstract The biogenesis of microtubules in the cell comprises a series of complex steps, including protein-folding reactions catalyzed by chaperonins. In addition a group of evolutionarily conserved proteins, called cofactors (A to E), is required for the production of assembly-competent α-/β-tubulin heterodimers. Using fission yeast, in which alp11+, alp1+, and alp21+, encoding the homologs for cofactors B, D, and E, respectively, are essential for cell viability, we have undertaken the genetic analysis of alp31+, the homolog of cofactor A. Gene disruption analysis shows that, unlike the three genes mentioned above, alp31+ is dispensable for cell growth and division. Nonetheless, detailed analysis of alp31-deleted cells demonstrates that Alp31A is required for the maintenance of microtubule structures and, consequently, the proper control of growth polarity. alp31-deleted cells show genetic interactions with mutations in β-tubulin, but not in α-tubulin. Budding yeast cofactor A homolog RBL2 is capable of suppressing the polarity defects of alp31-deleted cells. We conclude that the cofactor-dependent biogenesis of microtubules comprises an essential and a nonessential pathway, both of which are required for microtubule integrity.

scholarly journals Fission Yeast Pob1p, Which Is Homologous to Budding Yeast Boi Proteins and Exhibits Subcellular Localization Close to Actin Patches, Is Essential for Cell Elongation and Separation

1999 ◽  
Vol 10 (8) ◽  
pp. 2745-2757 ◽  
Author(s):  
Mika Toya ◽  
Yuichi Iino ◽  
Masayuki Yamamoto
Keyword(s):  
Cell Growth ◽  
Fission Yeast ◽  
Cell Separation ◽  
Gene Disruption ◽  
Host Cells ◽  
Ph Domain ◽  
Division Plane ◽  
Sam Domain ◽  
Growing Cell ◽  
Growth Loss

The fission yeast pob1 gene encodes a protein of 871 amino acids carrying an SH3 domain, a SAM domain, and a PH domain. Gene disruption and construction of a temperature-sensitivepob1 mutant indicated that pob1 is essential for cell growth. Loss of its function leads to quick cessation of cellular elongation. Pob1p is homologous to two functionally redundant Saccharomyces cerevisiaeproteins, Boi1p and Boi2p, which are necessary for cell growth and relevant to bud formation. Overexpression of pob1inhibits cell growth, causing the host cells to become round and swollen. In growing cells, Pob1p locates at cell tips during interphase and translocates near the division plane at cytokinesis. Thus, this protein exhibits intracellular dynamics similar to F-actin patches. However, Pob1p constitutes a layer, rather than patches, at growing cell tips. It generates two split discs flanking the septum at cytokinesis. The pob1-defective cells no longer elongate but swell gradually at the middle, eventually assuming a lemon-like morphology. Analysis using the pob1-ts allele revealed that Pob1p is also essential for cell separation. We speculate that Pob1p is located on growing plasma membrane, possibly through the function of actin patches, and may recruit proteins required for the synthesis of cell wall.

scholarly journals Evolutionarily conserved genetic interactions with budding and fission yeast MutS identify orthologous relationships in mismatch repair-deficient cancer cells

2014 ◽  
Vol 6 (9) ◽  
Author(s):  
Elena Tosti ◽  
Joseph A Katakowski ◽  
Sonja Schaetzlein ◽  
Hyun-Soo Kim ◽  
Colm J Ryan ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Cancer Cells ◽  
Mismatch Repair ◽  
Genetic Interactions ◽  
Evolutionarily Conserved

scholarly journals Adenine Addition Restores Cell Viability and Butanol Production in Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) Cultivated at 37°C

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Keiji Kiyoshi ◽  
Sohei Kawashima ◽  
Kosuke Nobuki ◽  
Toshimori Kadokura ◽  
Atsumi Nakazato ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Viability ◽  
Spore Formation ◽  
Butanol Production ◽  
Metabolomic Analysis ◽  
Primary Metabolites ◽  
Content Type ◽  
Atp Levels ◽  
Wide Range ◽  
Multiple Primary

ABSTRACT We have developed butanol-producing consolidated bioprocessing from cellulosic substrates through coculture of cellulolytic clostridia and butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4. However, the butanol fermentation by strain N1-4 (which has an optimal growth temperature of 30°C) is sensitive to the higher cultivation temperature of 37°C; the nature of this deleterious effect remains unclear. Comparison of the intracellular metabolites of strain N1-4 cultivated at 30°C and 37°C revealed decreased levels of multiple primary metabolites (notably including nucleic acids and cofactors) during growth at the higher temperature. Supplementation of the culture medium with 250 mg/liter adenine enhanced both cell growth (with the optical density at 600 nm increasing from 4.3 to 10.2) and butanol production (increasing from 3.9 g/liter to 9.6 g/liter) at 37°C, compared to those obtained without adenine supplementation, such that the supplemented 37°C culture exhibited growth and butanol production approaching those observed at 30°C in the absence of adenine supplementation. These improved properties were based on the maintenance of cell viability. We further showed that adenine supplementation enhanced cell viability during growth at 37°C by maintaining ATP levels and inhibiting spore formation. This work represents the first demonstration (to our knowledge) of the importance of adenine-related metabolism for clostridial butanol production, suggesting a new means of enhancing target pathways based on metabolite levels. IMPORTANCE Metabolomic analysis revealed decreased levels of multiple primary metabolites during growth at 37°C, compared to 30°C, in C. saccharoperbutylacetonicum strain N1-4. We found that adenine supplementation restored the cell growth and butanol production of strain N1-4 at 37°C. The effects of adenine supplementation reflected the maintenance of cell viability originating from the maintenance of ATP levels and the inhibition of spore formation. Thus, our metabolomic analysis identified the depleted metabolites that were required to maintain cell viability. Our strategy, which is expected to be applicable to a wide range of organisms, permits the identification of the limiting metabolic pathway, which can serve as a new target for molecular breeding. The other novel finding of this work is that adenine supplementation inhibits clostridial spore formation. The mechanism linking spore formation and metabolomic status in butanol-producing clostridia is expected to be the focus of further research.

scholarly journals GLIS Family Zinc Finger 1 was First Linked With Preaxial Polydactyly I in Humans by Stepwise Genetic Analysis

2022 ◽  
Vol 9 ◽  
Author(s):  
Jie-Yuan Jin ◽  
Pan-Feng Wu ◽  
Fang-Mei Luo ◽  
Bing-Bing Guo ◽  
Lei Zeng ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Cell Viability ◽  
Rna Sequencing ◽  
Zinc Finger ◽  
Nuclear Translocation ◽  
Genetic Etiology ◽  
Preaxial Polydactyly ◽  
Genetic Mechanisms ◽  
And Migration

Background: Preaxial polydactyly (PPD) is one of the most common developmental malformations, with a prevalence of 0.8–1.4% in Asians. PPD is divided into four types, PPD I–IV, and PPD I is the most frequent type. Only six loci (GLI1, GLI3, STKLD1, ZRS, pre-ZRS, and a deletion located 240 kb from SHH) have been identified in non-syndromic PPD cases. However, pathogenesis of most PPD patients has never been investigated. This study aimed to understand the genetic mechanisms involved in the etiology of PPD I in a family with multiple affected members.Methods: We recruited a PPD I family (PPD001) and used stepwise genetic analysis to determine the genetic etiology. In addition, for functional validation of the identified GLIS1 variant, in vitro studies were conducted. GLIS1 variants were further screened in additional 155 PPD cases.Results: We identified a GLIS1 variant (NM_147193: c.1061G > A, p.R354H) in the PPD001 family. In vitro studies showed that this variant decreased the nuclear translocation of GLIS1 and resulted in increased cell viability and migration. RNA sequencing revealed abnormal TBX4 and SFRP2 expression in 293T cells transfected with mutant GLIS1. Additionally, we identified a GLIS1 variant (c.664G > A, p.D222N) in another PPD case.Conclusion: We identified two GLIS1 variants in PPD I patients and first linked GLIS1 with PPD I. Our findings contributed to future molecular and clinical diagnosis of PPD and deepened our knowledge of this disease.

scholarly journals Genome-wide screen for cell growth regulators in fission yeast

2017 ◽  
Vol 130 (12) ◽  
pp. 2049-2055 ◽  
Author(s):  
Louise Weston ◽  
Jessica Greenwood ◽  
Paul Nurse
Keyword(s):  
Cell Growth ◽  
Fission Yeast ◽  
Growth Regulators ◽  
Genome Wide

scholarly journals Water Uptake in PHBV/Wollastonite Scaffolds: A Kinetics Study

2019 ◽  
Vol 3 (3) ◽  
pp. 74 ◽  
Author(s):  
Ribas ◽  
Montanheiro ◽  
Montagna ◽  
Prado ◽  
Campos ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Phase Separation ◽  
Cell Growth ◽  
Cell Viability ◽  
Water Uptake ◽  
Kinetic Mechanism ◽  
Kinetics Study ◽  
Biological Applications ◽  
Thermally Induced Phase Separation ◽  
Thermally Induced

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a widely studied polymer and it has been found that porous PHBV materials are suitable for substrates for cell cultures. A crucial factor for scaffolds designed for tissue engineering is the water uptake. This property influences the transport of water and nutrients into the scaffold, which promotes cell growth. PHBV has significant hydrophobicity, which can harm the production of cells. Thus, the addition of α-wollastonite (WOL) can modify the PHBV scaffold’s water uptake. To our knowledge, a kinetics study of water uptake of α-wollastonite phase powder and the PHBV matrix has not been reported. In this work, PHBV and WOL, (PHBV/WOL) films were produced with 0, 5, 10, and 20 wt % of WOL. Films were characterized, and the best concentrations were chosen to produce PHBV/WOL scaffolds. The addition of WOL in concentrations up to 10 wt % increased the cell viability of the films. MTT analysis showed that PHBV/5%WOL and PHBV/10%WOL obtained cell viability of 80% and 98%, respectively. Therefore, scaffolds with 0, 5 and 10 wt % of WOL were fabricated by thermally induced phase separation (TIPS). Scaffolds were characterized with respect to morphology and water uptake in assay for 65 days. The scaffold with 10 wt % of WOL absorbed 44.1% more water than neat PHBV scaffold, and also presented a different kinetic mechanism when compared to other samples. Accordingly, PHBV/WOL scaffolds were shown to be potential candidates for biological applications.

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