α-tubulin regulation by 5' introns in Saccharomyces cerevisiae.

Across eukaryotic genomes, multiple α- and ß-tubulin genes require regulation to ensure sufficient production of tubulin heterodimers. Features within these gene families that regulate expression remain underexplored. Here, we investigate the role of the 5' intron in regulating α-tubulin expression in Saccharomyces cerevisiae. We find that the intron in the α-tubulin, TUB1, promotes α-tubulin expression and cell fitness during microtubule stress. The role of the TUB1 intron depends on proximity to the TUB1 promoter and sequence features that are distinct from the intron in the alternative α-tubulin isotype, TUB3. These results lead us to perform a screen to identify genes that act with the TUB1 intron. We identified several genes involved in chromatin remodeling, α/ß-tubulin heterodimer assembly, and the spindle assembly checkpoint. We propose a model where the TUB1 intron promotes expression from the chromosomal locus and that this may represent a conserved mechanism for tubulin regulation under conditions that require high levels of tubulin production.

Tubulin isotype regulation maintains asymmetric requirement for α-tubulin over ß-tubulin.

How cells regulate α- and ß-tubulin to meet the demand for αß-heterodimers and avoid consequences of monomer imbalance is not understood. We investigate the role of gene copy number and how shifting expression of α- or ß-tubulin genes impacts tubulin proteostasis and microtubule function in Saccharomyces cerevisiae. We find that α-tubulin gene copy number is important for maintaining excess α-tubulin protein compared to ß-tubulin protein. Excess α-tubulin prevents accumulation of super-stoichiometric ß-tubulin, which leads to loss of microtubules, formation of non-microtubule assemblies of tubulin, and disrupts cell proliferation. In contrast, sub-stoichiometric ß-tubulin or overexpression of α-tubulin has minor effects. We provide evidence that yeast cells equilibrate α-tubulin protein concentration when α-tubulin isotype expression is increased. We propose an asymmetric relationship between α- and ß-tubulins, in which α-tubulins are maintained in excess to supply αß-heterodimers and limit the accumulation of ß-tubulin monomers.

Microtubule cytoskeleton: Revealing new readers of the tubulin code.

Microtubule networks are thought to be controlled by an elaborate program of tubulin posttranslational modifications and proteins that selectively bind to modified states. A new study identifies proteins that bind tyrosinated tubulin, revealing a novel recognition mechanism.

Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype-Specific Brain Malformations.

Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different ß-tubulin genes, TUBB2A or TUBB3. RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in ß-tubulins and located in the exchangeable GTP-binding pocket of ß-tubulin. To determine the impact of T178M on ß-tubulin function we created an analogous mutation in the ß-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with purified mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of ß-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our findings demonstrate the importance of tubulin's GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development.

Specialist α-tubulins for pluralist microtubules.

α- and ß-tubulins are encoded by multigene families, but the role of tubulin diversity for microtubule function has been a longstanding mystery. A new study (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202010155) shows that the two budding yeast α-tubulins have distinct roles during mitotic spindle positioning.

Bone marrow derived cells facilitate urinary bladder regeneration by attenuating tissue inflammatory responses.

INTRODUCTION: Inflammatory responses following tissue injury are essential for proper tissue regeneration. However, dysfunctional or repetitive inflammatory tissue assaults can lead to poor tissue regeneration and ultimate tissue failure via fibrosis. Previous attempts at urinary bladder tissue regeneration utilizing polymeric and biologic scaffolding materials tended to elicit these responses leading to poor tissue regeneration. Recent advances in bladder regeneration utilizing bone marrow derived mesenchymal stem cells (MSCs) and CD34(+) hematopoietic stem/progenitor cells (HSPCs) with biocompatible citric acid based scaffolds have provided an environment that not only promotes the growth of architecturally germane and physiologically functional tissue, but also modulates aspects of the innate immune response. MATERIAL AND METHODS: Within this study MSCs, CD34(+) HSPCs, or MSC/CD34(+) HSPC seeded POC [poly (1,8-octanediol-co-citrate)] scaffolds were utilized in an established rodent bladder augmentation model to evaluate inflammation as it pertains to bladder tissue regeneration. RESULTS: Quantified data from post-augmentation regenerated tissue samples at the 4 week time-point demonstrated that POC/MSC and POC/MSC + CD34(+) HSPC grafts markedly reduced the presence of pro-inflammatory CD68(+) macrophages and MPO(+) neutrophils compared to unseeded POC or POC/CD34(+) HSPC-only seeded grafts. Pro-inflammatory cytokines TNFα and IL-1b were also significantly down-regulated with a concomitant increase in the anti-inflammatory cytokines IL-10 and IL-13 in the aforementioned POC/MSC and POC/MSC + CD34(+) HSPC composites. Furthermore, this led to fewer instances of bladder tissue granuloma formation combined with greater muscle content and tissue angiogenic events as previous data has demonstrated. CONCLUSIONS: Data indicates that POC/MSC and POC/MSC + CD34(+) HSPC grafts attenuate the innate inflammatory response and promote bladder tissue regeneration.