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Interrogation of ?-tubulin alleles using high-resolution fitness measurements reveals a distinct cytoplasmic function in spindle alignment.

ABSTRACT: ?-Tubulin has a well-established role in nucleating the assembly of microtubules, yet how phosphorylation regulates its activity remains unclear. Here, we use a time-resolved, fitness-based SGA approach to compare two ?-tubulin alleles, and find that the genetic interaction profile of ?tub-Y362E is enriched in spindle positioning and cell polarity genes relative to that of ?tub-Y445D, which is enriched in genes involved in spindle assembly and stability. In ?tub-Y362E cells, we find a defect in spindle alignment and an increase in the number of astral microtubules at both spindle poles. Our results suggest that the ?tub-Y362E allele is a separation-of-function mutation that reveals a role for ?-tubulin phospho-regulation in spindle alignment. We propose that phosphorylation of the evolutionarily conserved Y362 residue of budding yeast ?-tubulin contributes to regulating the number of astral microtubules associated with spindle poles, and promoting efficient pre-anaphase spindle alignment.

SUBMITTER: Shulist K

PROVIDER: S-EPMC5595808 | biostudies-other | 2017 Sep

REPOSITORIES: biostudies-other

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Interrogation of γ-tubulin alleles using high-resolution fitness measurements reveals a distinct cytoplasmic function in spindle alignment.

Shulist Kristian K Yen Eric E Kaitna Susanne S Leary Allen A Decterov Alexandra A Gupta Debarun D Vogel Jackie J

Scientific reports 20170912 1

γ-Tubulin has a well-established role in nucleating the assembly of microtubules, yet how phosphorylation regulates its activity remains unclear. Here, we use a time-resolved, fitness-based SGA approach to compare two γ-tubulin alleles, and find that the genetic interaction profile of γtub-Y362E is enriched in spindle positioning and cell polarity genes relative to that of γtub-Y445D, which is enriched in genes involved in spindle assembly and stability. In γtub-Y362E cells, we find a defect in ...[more]

PMID: 28900268

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Project description:Infections by parasitic nematodes inflict a huge burden on the health of humans and livestock throughout the world. Anthelmintic drugs are the first line of defense against these infections. Unfortunately, resistance to these drugs is rampant and continues to spread. To improve treatment strategies, we must understand the genetics and molecular mechanisms that underlie resistance. Studies of the fungus Aspergillus nidulans and the free-living nematode Caenorhabditis elegans discovered that a beta-tubulin gene is mutated in benzimidazole (BZ) resistant strains. In parasitic nematode populations, three beta-tubulin alleles, F167Y, E198A, and F200Y, have long been correlated with resistance. Additionally, improvements in sequencing technologies have identified new alleles - E198V, E198L, E198K, E198I, and E198Stop - also correlated with BZ resistance. However, none of these alleles have been proven to cause resistance. To empirically demonstrate this point, we independently introduced the F167Y, E198A, and F200Y alleles as well as two of the newly identified alleles, E198V and E198L, into the BZ susceptible C. elegans N2 genetic background using the CRISPR-Cas9 system. These genome-edited strains were exposed to both albendazole and fenbendazole to quantitatively measure animal responses to BZs. We used a range of concentrations for each BZ compound to define response curves and found that all five of the alleles conferred resistance to BZ compounds equal to a loss of the entire beta-tubulin gene. These results prove that the parasite beta-tubulin alleles cause resistance. The E198V allele is found at low frequencies along with the E198L allele in natural parasite populations, suggesting that it could affect fitness. We performed competitive fitness assays and demonstrated that the E198V allele reduces animal health, supporting the hypothesis that this allele might be less fit in field populations. Overall, we present a powerful platform to quantitatively assess anthelmintic resistance and effects of specific resistance alleles on organismal fitness in the presence or absence of the drug.

Dataset Information

Interrogation of ?-tubulin alleles using high-resolution fitness measurements reveals a distinct cytoplasmic function in spindle alignment.

Publications

Interrogation of γ-tubulin alleles using high-resolution fitness measurements reveals a distinct cytoplasmic function in spindle alignment.

Similar Datasets

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