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Rose Lab - Research

Spindle positioning during asymmetric division

The C. elegans embryo has emerged as an excellent model in which to study spindle positioning during asymmetric division, in part due its invariant cell lineage and the ability to follow movements of nuclei and spindles in living embryos using DIC microscopy. These attributes, coupled with the current capabilities for live-imaging of microtubule behavior using GFP-tagged proteins, and depletion of microtubule associated proteins through both mutation and RNA interference (RNAi), allow a dissection of the molecular mechanisms of spindle positioning.

Control
A transgenic embryo expressing GFP::alpha-tubulin & mCherry::Histone H2B undergoing its first mitotic division. The asymmetric movements of centration, nuclear rotation and spindle displacement are shown.
Imaged by spinning disc confocal. Espiritu et. al, Dev Bio, 2012.

The C. elegans embryo undergoes a series of asymmetric divisions in which the spindle is aligned on the axis of polarity specified by the conserved PAR polarity proteins. In the 1-cell embryo, the pronuclei meet in the posterior, but then move towards the center of the embryo (centration). The entire pronuclear-centrosome complex rotates 90° (nuclear rotation) to align the centrosomes on the anterior/posterior axis (A/P) of the embryo. At the end of metaphase, the entire spindle shifts towards the posterior and during anaphase the spindle elongates asymmetrically, with the posterior spindle pole moving at a faster rate compared to the anterior spindle pole. Together these metaphase/anaphase movements, referred to hereafter as posterior spindle displacement, result in an unequal cleavage such that the anterior daughter AB cell is slightly larger than P1. The posterior pole movements are also accompanied by vigorous transverse oscillations. Similar nuclear rotation and spindle displacement movements occur in the P1 cell. The daughters of P1, EMS and P2 also undergo asymmetric unequal divisions, although EMS division is controlled by cell signaling pathways rather than the PAR proteins. Our research program focuses on identifying the molecular mechanisms by which the PAR proteins and other signals regulate spindle positioning.


LET-99: a novel regulator of G protein activity during asymmetric division

The PAR proteins, heterotrimeric Gα subunits and their activators (GPR and LIN-5 in C. elegans) act to regulate spindle positioning in several species. In C. elegans one-cell embryos, GPR and LIN-5 are required to generate forces acting from the cortex that pull on astral microtubules to move the spindle, potentially via regulation of the microtubule motor dynein. Our studies identified LET-99, a DEPDC1 family protein, as another intermediate that is required for both the nuclear rotation events that orient the spindle and for spindle displacement. The highest levels of cortical LET-99 protein are restricted to a posterior lateral band through inhibition by both PAR-3 at the anterior and PAR-1 at the posterior (a, b in diagram below). LET-99, along with other PAR polarity cues, then inhibits the cortical localization of GPR/LIN-5 in the band region, resulting in the asymmetric cortical accumulation of GPR/LIN-5 at the posterior-most cortex during spindle displacement. Significantly, we also showed that GPR-1/2 and LIN-5 are asymmetrically localized by LET-99 and the PARs to the opposite anterior cortex during the time of anteriorly directed nuclear rotation (a). These results provide the basis for our working model that the inhibition of G protein signaling in the LET-99 band region leads to three cortical force generation domains in the embryo (anterior, posterior lateral band and posterior); laser ablation studies that map the cortical forces support this model.

We are currently dissecting the molecular mechanisms by which the PAR proteins regulate LET-99, and how LET-99 in turn regulates GPR localization. In addition, we are examining the role of LET-99 and G protein signaling in divisions that are not regulated by PAR polarity.

Control Model for PAR-dependent spindle positioning. Gα/GPR signaling is required for forces that pull on microtubules from the cortex (+), whereas LET-99 inhibits force. Note that LET-99 and GPR/LIN-5 localize to the entire cortex, but only the regions with highest levels are indicated for simplicity. (a) Nuclear rotation & centration. (b) Spindle displacement during metaphase/anaphase. (c) Zoom of dashed area in (b). Espiritu & Rose, eLS, 2013


C. elegans OOC-5 as a model for torsin function at nuclear pores.



Schematic of the C. elegans germline. VanGompel et al., MBoC, 2015

In the course of our studies on genes involved in asymmetric division, we identified a gene, ooc-5, as being required for the re-establishment of PAR polarity during the second asymmetric division in the embryo, as well as for normal oogenesis in adult C. elegans. OOC-5 is one of three C. elegans homologs of human torsinA, which is defective in individuals with the neuromuscular disease early onset dystonia. Like torsin A, OOC-5 is localized to the lumen of the endoplasmic reticulum and nuclear envelope. Torsins are part of the AAA+ family of proteins and may use ATP hydrolysis to dissociate or unfold target proteins. However, their basic cellular function remains to be elucidated. Interestingly, other C. elegans researchers showed that depletion of the NPP-1 nucleoporin, a component of the nuclear pore complex (NPC), leads to an asymmetric division phenotype highly similar to that of ooc-5 mutants. We have found that multiple nucleoporins are mislocalized in ooc-5 mutants. We are further characterizing nuclear envelope phenotypes in ooc-5, using C. elegans as a model system to decipher the role of torsins at the nuclear envelope and nuclear pores. Additionally, we are beginning to examine how ooc-5 and the NPC are involved in polarity regulation in embryos.