Construction of an Inducible TBCE Overexpression System to Probe Tubulin Cofactor-Mediated Microtubule Homeostasis
We are studying a new regulator of microtubule homeostasis, wdA, in the fungus Aspergillus nidulans. Mutations in wdA confer cold-sensitive lethal mitotic catastrophe, suggesting an important role in microtubule-based processes. Previous genetic studies showed that deletion of TBCA causes a near-lethal phenotype that is suppressed by deletion of wdA, suggesting opposing roles in regulating tubulin homeostasis. To investigate this relationship, an inducible TBCE overexpression construct was designed using the alcA alcohol dehydrogenase promoter system. The construct was generated through PCR amplification, restriction digestion, ligation into the pSDW194 shuttle vector, transformation into Escherichia coli, and diagnostic restriction analysis. Candidate plasmids produced restriction patterns consistent with the predicted alcA::TBCE construct. Although transformation into A. nidulans and phenotypic characterization remain future work, the completed construct provides a tool to examine how altered TBCE dosage influences tubulin balance and microtubule homeostasis in vivo. These studies will help clarify the role of tubulin cofactors in maintaining a dynamic and reversible tubulin homeostasis network.