A neuron has a polarized structure with a long axon and several dendrites which is established and maintained by microtubule cytoskeleton. Microtubules are heteropolymers of alpha and beta-tubulin dimers and have intrinsic polarity with a fast growing plus end and a slow growing minus end. We are using Touch receptor neurons (TRN) of C. elegans, to understand the basis of this organization and regulation of neuronal microtubules. One of the major factors that depolymerize microtubules is the kinesin-13 family protein KLP-7. The loss of
klp-7 causes excess stabilization of microtubule cytoskeleton leading to ectopic neurite extension in touch neurons and other neurons as well (Ghosh-Roy et al 2012; Puri et al 2019). We found that this phenotype in
klp-7(0) can be reversed by a microtubule-destabilizing drug colchicine or in backgrounds lacking either alpha or beta tubulin. We hypothesized that the suppressors of
klp-7(0) neuronal phenotype might encode for novel regulators of the neuronal microtubule cytoskeleton. With this idea, we did a forward genetic screen in
klp-7(0) mutant using Ethyl Methane Sulfonate(EMS). We isolated 26 mutants that suppress the ectopic posterior extension phenotype of ALM neuron in
klp-7(0), out of 12,422 F1's from a clonal screening. To identify the exact causal mutant, the suppressors were crossed four times with (N2) Bristol to collect 10 individual recombinants. We sequenced the pooled lysates of these recombinants and the FASTQ sequences of Whole Genome Sequencing (WGS) data were analyzed on the Galaxy user interface using MimodD tools. The list of SNP variants was used to generate mapping plots, which gave putative homozygous EMS SNPs for a given suppressor. These SNPs affect genes that encode for tubulin subunits, RNA-binding protein, Ca2+ dependent secretion activators, kinases and ECM components. We will present a comprehensive analysis of the characterization of a few mutants to unravel their roles in regulating microtubule cytoskeleton. References: Ghosh-Roy, A., Goncharov, A., Jin, Y., Chisholm A.D. (2012). Kinesin-13 and tubulin posttranslational modifications regulate microtubule growth in axon regeneration. Developmental Cell 23(4), 716-728 Puri D, Ponniah K, Biswas K, Basu A, Lundquist EA, Ghosh-Roy A. (2019) WNT Signaling Establishes Microtubule Polarity in Neuron Through the Regulation of Kinesin 13 Family Microtubule Depolymerizing Factor. (Sneak peek in Cell Press).
http://dx.doi.org/10.2139/ssrn.3456296