Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport.

Daniel J Barbosa,Bram Prevo,Dhanya K Cheerambathur,Joana Duro,Reto Gassmann,Ana X Carvalho,Julie Ahringer

doi:10.1371/journal.pgen.1006941

Abstract

The microtubule-based motor dynein generates pulling forces for centrosome centration and mitotic spindle positioning in animal cells. How the essential dynein activator dynactin regulates these functions of the motor is incompletely understood. Here, we dissect the role of dynactin's microtubule binding activity, located in the p150 CAP-Gly domain and an adjacent basic patch, in the C. elegans zygote. Analysis of p150 mutants engineered by genome editing suggests that microtubule tip tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex and for generating robust cortical pulling forces. Instead, mutations in p150's CAP-Gly domain inhibit cytoplasmic pulling forces responsible for centration of centrosomes and attached pronuclei. The centration defects are mimicked by mutations of α-tubulin's C-terminal tyrosine, and both p150 CAP-Gly and tubulin tyrosine mutants decrease the frequency of early endosome transport from the cell periphery towards centrosomes during centration. Our results suggest that p150 GAP-Gly domain binding to tyrosinated microtubules promotes initiation of dynein-mediated organelle transport in the dividing one-cell embryo, and that this function of p150 is critical for generating cytoplasmic pulling forces for centrosome centration.

Highlights

Cytoplasmic dynein 1 is the major microtubule (MT) minus-end directed motor in animals and transports various cargo from the cell periphery to the cell interior
The motor cytoplasmic dynein 1 uses microtubule filaments as tracks to transport cargo from the cell periphery to the cell center, where the microtubule minus ends are embedded at the centrosome
In C. elegans hermaphrodite adults, 302 out of 959 somatic cells are neurons, yet we did not find evidence for a p135dnc-1 isoform at the mRNA level (S3D Fig), nor Dynactin p150 helps dynein pull on microtubules conserved cytoskeleton-associated protein glycine-rich (CAP-Gly) domain predicted to bind the end-binding homology (EBH) domain of EB and the EEY/F motifs present at the C-termini of EB and α-tubulin

Summary

Introduction

Cytoplasmic dynein 1 (dynein) is the major microtubule (MT) minus-end directed motor in animals and transports various cargo from the cell periphery to the cell interior. To generate pulling force, dynein is either attached to anchor proteins fixed at the cell cortex (cortical pulling) [1,2,3,4], or dynein is anchored on organelles in the cytoplasm (cytoplasmic pulling) [5,6]. In the latter instance, dynein generates MT length-dependent pulling forces by working against viscous drag as it transports organelles along MTs toward centrosomes. In animal cells, +TIP binding of dynactin recruits dynein to growing MT ends [9,15,16,17,18]

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