Modeling disease-correlated TUBA1A mutation in budding yeast reveals a molecular basis for tubulin dysfunction

Abstract

Malformations of cortical development (MCD) of the human brain are a likely consequence of defective neuronal migration, and/or proliferation of neuronal progenitor cells, both of which are dictated in part by microtubule-dependent transport of various cargoes, including the mitotic spindle. Throughout the evolutionary spectrum, proper spindle positioning depends on cortically anchored dynein motors that exert forces on astral microtubules emanating from spindle poles. A single heterozygous amino acid change, G436R, in the conserved TUBA1A α-tubulin gene was reported to account for MCD in patients. The mechanism by which this mutation disrupts microtubule function in the developing cerebral cortex is not understood.Studying the consequence of tubulin mutations in mammalian cells is challenging partly because of the large number of α-tubulin isotypes expressed. To overcome this challenge, we have generated a budding yeast strain expressing the mutated tubulin (Tub1 G437R in yeast) as one of the main sources of α-tubulin (in addition to Tub3, another α-tubulin isotype in this organism). Although viability of the yeast was unimpaired by this mutation, they became reliant on Tub3, as was apparent by the synthetic lethality of this mutant in combination with tub3∆. We find that Tub1 G437R assembles into microtubules that support normal G1 activity, but lead to enhanced dynein-dependent nuclear migration phenotypes during G2/M, and a consequential disruption of spindle positioning. We find that this mutation impairs the interaction between She1 -a negative regulator of dynein -and microtubules, as was apparent from a yeast two-hybrid assay, a co-sedimentation assay, and from live cell imaging.We conclude that a weaker interaction between She1 and Tub1 G437R -containing microtubules results in enhanced dynein activity, ultimately leading to the spindle positioning defect. Our results provide the first evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation, and sheds light on a mechanism that may be causative of neurodevelopmental diseases.

Tubulin mutations in neurodevelopmental disorders as a tool to decipher microtubule function

“…To what extent are specific pathways and cross‐talking pathways altered? Using this elegant system, we demonstrated that one MCD β‐tubulin mutation impairs binding of the yeast EB1 +TIPS [154]. If our observations in yeast apply to mammalian tubulin, MT +TIPS complexes might be reduced in neurons, thereby impairing neuron migration or neural mitotic spindle orientation [86,201].…”

“…More recently, we discovered that an α‐tubulin mutation dampened MT association with a negative dynein regulator. Therefore, not only might impaired dynein motor activity be implicated, in some cases dynein motor overactivity might also be implicated [154].…”

“…In such a case, the isoform’s specific function is lost and the mutation behaves like a typical ‘loss‐of‐function’ mutation. On the other hand, however, a mutated tubulin isoform that is effectively incorporated into MTs may impair the intrinsic MT dynamics, or MT interaction with partners, which may in turn disrupt MT or dynein function [153,154]. When this happens, the mutant tubulin is expected to have an effect, even if it is expressed over and above a wild‐type tubulin pool (in other words, the mutant tubulin is transdominant).…”

“…More recently, we used yeast to model the TUBA1A G436R mutation that maps onto the external surface of the MT. This is a mutation that has been found in a patient with pachygyria (reduced cortex gyration) and severe microcephaly [154]. We have created a yeast cell in which the essential α‐tubulin gene harbors this specific mutation.…”

“…This is the first evidence of impaired interaction between MTs and a dynein regulator caused by tubulin mutation. Combined with the fact that dynein motor domains have been found mutated in a number of MCD patients (see § Modulating microtubule dynamics and motor functions: MAPs and +TIPS ), this evidence sheds light on a MAP‐dependent dynein deregulation that may be causative of MCD diseases [154].…”