LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring.

The anaphase spindle determines the position of the cytokinesis furrow, such that the contractile ring assembles in an equatorial zone between the two spindle poles. Contractile ring formation is mediated by RhoA activation at the equator by the centralspindlin complex and midzone microtubules. Astral microtubules also inhibit RhoA accumulation at the poles. In the Caenorhabditis elegans one-cell embryo, the astral microtubule-dependent pathway requires anillin, NOP-1, and LET-99. LET-99 is well characterized for generating the asymmetric cortical localization of the Gα-dependent force-generating complex that positions the spindle during asymmetric division. However, whether the role of LET-99 in cytokinesis is specific to asymmetric division and whether it acts through Gα to promote furrowing are unclear. Here we show that LET-99 contributes to furrowing in both asymmetrically and symmetrically dividing cells, independent of its function in spindle positioning and Gα regulation. LET-99 acts in a pathway parallel to anillin and is required for myosin enrichment into the contractile ring. These and other results suggest a positive feedback model in which LET-99 localizes to the presumptive cleavage furrow in response to the spindle and myosin. Once positioned there, LET-99 enhances myosin accumulation to promote furrowing in both symmetrically and asymmetrically dividing cells

PAR-dependent and geometry-dependent mechanisms of spindle positioning.

During intrinsically asymmetric division, the spindle is oriented onto a polarized axis specified by a group of conserved PAR proteins. Extrinsic geometric asymmetry generated by cell shape also affects spindle orientation in some systems, but how intrinsic and extrinsic mechanisms coexist without interfering with each other is unknown. In some asymmetrically dividing cells of the wild-type Caenorhabditis elegans embryo, nuclear rotation directed toward the anterior cortex orients the forming spindle. We find that in such cells, a PAR-dependent mechanism dominates and causes rotation onto the polarized axis, regardless of cell shape. However, when geometric asymmetry is removed, free nuclear rotation in the center of the cell is observed, indicating that the anterior-directed nature of rotation in unaltered embryos is an effect of cell shape. This free rotation is inconsistent with the prevailing model for nuclear rotation, the specialized cortical site model. In contrast, in par-3 mutant embryos, a geometry-dependent mechanism becomes active and causes directed nuclear rotation. These results lead to the model that in wild-type embryos both PAR-3 and PAR-2 are essential for nuclear rotation in asymmetrically dividing cells, but that PAR-3 inhibits geometry-dependent rotation in nonpolarized cells, thus preventing cell shape from interfering with spindle orientation

A novel function for the Caenorhabditis elegans torsin OOC-5 in nucleoporin localization and nuclear import.

Torsin proteins are AAA+ ATPases that localize to the endoplasmic reticular/nuclear envelope (ER/NE) lumen. A mutation that markedly impairs torsinA function causes the CNS disorder DYT1 dystonia. Abnormalities of NE membranes have been linked to torsinA loss of function and the pathogenesis of DYT1 dystonia, leading us to investigate the role of the Caenorhabditis elegans torsinA homologue OOC-5 at the NE. We report a novel role for torsin in nuclear pore biology. In ooc-5-mutant germ cell nuclei, nucleoporins (Nups) were mislocalized in large plaques beginning at meiotic entry and persisted throughout meiosis. Moreover, the KASH protein ZYG-12 was mislocalized in ooc-5 gonads. Nups were mislocalized in adult intestinal nuclei and in embryos from mutant mothers. EM analysis revealed vesicle-like structures in the perinuclear space of intestinal and germ cell nuclei, similar to defects reported in torsin-mutant flies and mice. Consistent with a functional disruption of Nups, ooc-5-mutant embryos displayed impaired nuclear import kinetics, although the nuclear pore-size exclusion barrier was maintained. Our data are the first to demonstrate a requirement for a torsin for normal Nup localization and function and suggest that these functions are likely conserved

LET-99 inhibits lateral posterior pulling forces during asymmetric spindle elongation in C. elegans embryos

GPR-1/2 (regulators of Gα signaling necessary for asymmetric cell division) receives a positional cue from Let-99, resulting in its appropriate distribution around the posterior cortex

TorsinA regulates the LINC to moving nuclei.

How LINC complexes are regulated to connect nuclei to the cytoskeleton during nuclear migration is unknown. Saunders et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201507113) show that the AAA+ ATPase torsinA and its partner LAP1 are required for nuclear migration during fibroblast polarization by mediating the dynamics of LINC complexes

LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring.

The anaphase spindle determines the position of the cytokinesis furrow, such that the contractile ring assembles in an equatorial zone between the two spindle poles. Contractile ring formation is mediated by RhoA activation at the equator by the centralspindlin complex and midzone microtubules. Astral microtubules also inhibit RhoA accumulation at the poles. In the Caenorhabditis elegans one-cell embryo, the astral microtubule-dependent pathway requires anillin, NOP-1, and LET-99. LET-99 is well characterized for generating the asymmetric cortical localization of the Gα-dependent force-generating complex that positions the spindle during asymmetric division. However, whether the role of LET-99 in cytokinesis is specific to asymmetric division and whether it acts through Gα to promote furrowing are unclear. Here we show that LET-99 contributes to furrowing in both asymmetrically and symmetrically dividing cells, independent of its function in spindle positioning and Gα regulation. LET-99 acts in a pathway parallel to anillin and is required for myosin enrichment into the contractile ring. These and other results suggest a positive feedback model in which LET-99 localizes to the presumptive cleavage furrow in response to the spindle and myosin. Once positioned there, LET-99 enhances myosin accumulation to promote furrowing in both symmetrically and asymmetrically dividing cells

Mitotic Spindle Positioning in the EMS Cell of Caenorhabditis elegans Requires LET-99 and LIN-5/NuMA

Asymmetric divisions produce daughter cells with different fates, and thus are critical for animal development. During asymmetric divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types, a cortically localized complex consisting of Gα, GPR-1/2, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynactin/dynein, which exerts pulling forces on astral microtubules to physically position the spindle. The conserved PAR polarity proteins are known to regulate both cytoplasmic asymmetry and spindle positioning in many cases. However, spindle positioning also occurs in response to cell signaling cues that appear to be PAR-independent. In the four-cell Caenorhabditis elegans embryo, Wnt and Mes-1/Src-1 signaling pathways act partially redundantly to align the spindle on the anterior/posterior axis of the endomesodermal (EMS) precursor cell. It is unclear how those extrinsic signals individually contribute to spindle positioning and whether either pathway acts via conserved spindle positioning regulators. Here, we genetically test the involvement of Gα, LIN-5, and their negative regulator LET-99, in transducing EMS spindle positioning polarity cues. We also examined whether the C. elegans ortholog of another spindle positioning regulator, DLG-1, is required. We show that LET-99 acts in the Mes-1/Src-1 pathway for spindle positioning. LIN-5 is also required for EMS spindle positioning, possibly through a Gα- and DLG-1-independent mechanism

The kinases PIG-1 and PAR-1 act in redundant pathways to regulate asymmetric division in the EMS blastomere of C. elegans

The PAR-1 kinase of C. elegans is localized to the posterior of the one-cell embryo and its mutations affect asymmetric spindle placement and partitioning of cytoplasmic components in the first cell cycle. However, par-1 mutations do not cause failure to restrict the anterior PAR polarity complex to the same extent as mutations in the posteriorly localized PAR-2 protein. Further, it has been difficult to examine the role of PAR-1 in subsequent divisions due to the early defects in par-1 mutant embryos. Here we show that the PIG-1 kinase acts redundantly with PAR-1 to restrict the anterior PAR-3 protein for normal polarity in the one-cell embryo. By using a temperature sensitive allele of par-1, which exhibits enhanced lethality when combined with a pig-1 mutation, we have further explored roles for these genes in subsequent divisions. We find that both PIG-1 and PAR-1 regulate spindle orientation in the EMS blastomere of the four-cell stage embryo to ensure that it undergoes an asymmetric division. In this cell, PIG-1 and PAR-1 act in parallel pathways for spindle positioning, PIG-1 in the MES-1/SRC-1 pathway and PAR-1 in the Wnt pathway