Epistatic, synthetic, and balancing interactions among tubulin missense mutations affecting neurite growth inCaenorhabditis elegans

Abstract

Mutations in tubulins affect microtubule (MT) dynamics and functions during neuronal differentiation and their genetic interaction provides insights into the regulation of MT functions. We previously used C. elegans touch receptor neurons to analyze the cellular impact of tubulin mutations and reported the phenotypes of 67 tubulin missense mutations, categorized into three classes: loss-of-function ( lf), antimorphic ( anti), and neomorphic ( neo) alleles. In this study, we isolated 54 additional tubulin alleles through suppressor screens in sensitized backgrounds that caused excessive neurite growth. These alleles included 32 missense mutations not analyzed before, bringing the total number of mutations in our collection to 99. Phenotypic characterization of these newly isolated mutations identified three new types of alleles: partial lf and weak neo alleles of mec-7/β-tubulin that had subtle effects and strong anti alleles of mec-12/α-tubulin. We also discovered complex genetic interactions among the tubulin mutations, including the suppression of neo mutations by intragenic lf and anti alleles, additive and synthetic effects between mec-7 neo alleles, and unexpected epistasis, in which weaker neo alleles masked the effects of stronger neo alleles in inducing ectopic neurite growth. We also observed balancing between neo and anti alleles, whose respective MT-hyperstablizing and -destabilizing effects neutralized each other.

MBL-1/Muscleblind regulates neuronal differentiation and controls the splicing of a terminal selector inCaenorhabditis elegans

“…To study the effects of MT dynamics, we tracked EBP-2::GFP comets in the TRNs using the juIs338[mec-4p::ebp-2::GPF + ttx-3p::RFP] transgene based on a previous protocol [22]. Day-one adults were mounted with 100 nm polystyrene beads (Polysciences #00876-15; 5-fold dilution) on an agarose pad, and their PLMs were imaged for 1 min with 150-ms exposure time and 350-ms time intervals.…”

The Expression and Function of Tubulin Isotypes in Caenorhabditis elegans

“…Through extensive genetic screens, a large collection of mec-12 and mec-7 missense mutations (99 alleles in total) had been isolated and characterized for their effects on neurite growth ( Zheng et al, 2017 ; Lee et al, 2021 ). In addition to many loss-of-function mutants, two distinct types of gain-of-function alleles were found among the missense alleles, and they had much stronger effects on neurite growth than the deletion alleles.…”

“…Lastly, studies of the mec-12 and mec-7 alleles also found complex genetic interactions between the tubulin mutations, including epistatic, additive, synthetic, and balancing effects ( Lee et al, 2021 ). For example, the effects of neomorphic gain-of-function mutants can be suppressed by loss-of-function and antimorphic gain-of-function mutations.…”

“…Neomorphic alleles often show additive effects as expected, but sometimes a weaker neomorphic allele could also mask the effects of a stronger neomorphic allele in inducing ectopic neurite growth. Moreover, the microtubule-stabilizing and -destabilizing effects of neomorphic and antimorphic alleles were found in some cases to neutralize each other to allow normal neurite growth pattern in the double mutants ( Lee et al, 2021 ). These results are in line with earlier molecular genetics studies on tubulins in other organisms ( Morris et al, 1979 ) and can help understand how tubulin isotypes work together.…”

Musleblind-1 regulates microtubule cytoskeleton in C. elegans mechanosensory neuron through tubulin mRNAs

“…Mechanosensory neurons of C. elegans, responsible for gentle touch sensation, have been used to study microtubule regulation and neuronal polarization in vivo (39)(40)(41). In this study, we have identified a mutation in the muscleblind-1 gene, as a suppressor of the touch neuron developmental defect in loss of kinesin-13 family microtubule depolymerase, klp-7.…”