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.

LET-711, the Caenorhabditis elegans NOT1 ortholog, is required for spindle positioning and regulation of microtubule length in embryos.

Spindle positioning is essential for the segregation of cell fate determinants during asymmetric division, as well as for proper cellular arrangements during development. In Caenorhabditis elegans embryos, spindle positioning depends on interactions between the astral microtubules and the cell cortex. Here we show that let-711 is required for spindle positioning in the early embryo. Strong loss of let-711 function leads to sterility, whereas partial loss of function results in embryos with defects in the centration and rotation movements that position the first mitotic spindle. let-711 mutant embryos have longer microtubules that are more cold-stable than in wild type, a phenotype opposite to the short microtubule phenotype caused by mutations in the C. elegans XMAP215 homolog ZYG-9. Simultaneous reduction of both ZYG-9 and LET-711 can rescue the centration and rotation defects of both single mutants. let-711 mutant embryos also have larger than wild-type centrosomes at which higher levels of ZYG-9 accumulate compared with wild type. Molecular identification of LET-711 shows it to be an ortholog of NOT1, the core component of the CCR4/NOT complex, which plays roles in the negative regulation of gene expression at transcriptional and post-transcriptional levels in yeast, flies, and mammals. We therefore propose that LET-711 inhibits the expression of ZYG-9 and potentially other centrosome-associated proteins, in order to maintain normal centrosome size and microtubule dynamics during early embryonic divisions.

The torsin-family AAA+ protein OOC-5 contains a critical disulfide adjacent to Sensor-II that couples redox state to nucleotide binding.

A subgroup of the AAA+ proteins that reside in the endoplasmic reticulum and the nuclear envelope including human torsinA, a protein mutated in hereditary dystonia, is called the torsin family of AAA+ proteins. A multiple-sequence alignment of this family with Hsp100 proteins of known structure reveals a conserved cysteine in the C-terminus of torsin proteins within the Sensor-II motif. A structural model predicts this cysteine to be a part of an intramolecular disulfide bond, suggesting that it may function as a redox sensor to regulate ATPase activity. In vitro experiments with OOC-5, a torsinA homolog from Caenorhabditis elegans, demonstrate that redox changes that reduce this disulfide bond affect the binding of ATP and ADP and cause an attendant local conformational change detected by limited proteolysis. Transgenic worms expressing an ooc-5 gene with cysteine-to-serine mutations that disrupt the disulfide bond have a very low embryo hatch rate compared with wild-type controls, indicating these two cysteines are essential for OOC-5 function. We propose that the Sensor-II in torsin family proteins is a redox-regulated sensor. This regulatory mechanism may be central to the function of OOC-5 and human torsinA.

LET-99 opposes Galpha/GPR signaling to generate asymmetry for spindle positioning in response to PAR and MES-1/SRC-1 signaling.

G-protein signaling plays important roles in asymmetric cell division. In C. elegans embryos, homologs of receptor-independent G protein activators, GPR-1 and GPR-2 (GPR-1/2), function together with Galpha (GOA-1 and GPA-16) to generate asymmetric spindle pole elongation during divisions in the P lineage. Although Galpha is uniformly localized at the cell cortex, the cortical localization of GPR-1/2 is asymmetric in dividing P cells. In this report, we show that the asymmetry of GPR-1/2 localization depends on PAR-3 and its downstream intermediate LET-99. Furthermore, in addition to its involvement in spindle elongation, Galpha is required for the intrinsically programmed nuclear rotation event that orients the spindle in the one-cell. LET-99 functions antagonistically to the Galpha/GPR-1/2 signaling pathway, providing an explanation for how Galpha-dependent force is regulated asymmetrically by PAR polarity cues during both nuclear rotation and anaphase spindle elongation. In addition, Galpha and LET-99 are required for spindle orientation during the extrinsically polarized division of EMS cells. In this cell, both GPR-1/2 and LET-99 are asymmetrically localized in response to the MES-1/SRC-1 signaling pathway. Their localization patterns at the EMS/P2 cell boundary are complementary, suggesting that LET-99 and Galpha/GPR-1/2 signaling function in opposite ways during this cell division as well. These results provide insight into how polarity cues are transmitted into specific spindle positions in both extrinsic and intrinsic pathways of asymmetric cell division.

LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues in C. elegans embryos.

Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of cellular polarity. We provide evidence that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99 is a DEP domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, the data indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle.