Wu, Jui-Ching et al. (2011) International Worm Meeting "PAR proteins regulate the localization of LET-99 during asymmetric division."

Rose, Lesilee S., Espiritu, Eugenel B., Wu, Jui-Ching

[International Worm Meeting, 2011]

Positioning of the mitotic spindle is essential for a number of developmental processes, including asymmetric divisions. During such divisions, the spindle must be aligned with the axis of cell polarity and the spindle is often displaced so that division gives daughters of unequal size. In many systems, PAR polarity proteins establish polarization of the cell and regulate spindle movements via a complex including Ga, GPR and LIN-5. In C. elegans one-cell embryos, GPR and LIN-5 are asymmetrically localized and activate cortical forces that pull on astral microtubules to position the spindle. We previously showed that LET-99 is a key regulator of GPR asymmetry. LET-99 is asymmetrically localized at the cortex in a lateral-posterior band pattern, where it inhibits GPR localization. Quantitative analysis of LET-99 localization in mutant backgrounds shows that PAR-3 inhibits cortical LET-99 localization at the anterior cortex, while a gradient of PAR-1 inhibits LET-99 at the posterior-most cortex. Additionally, cytoplasmic polarity mediators are not required. PAR-1 is a Ser/Thr kinase, which associates with LET-99 in immunoprecipitations from embryo extracts and in pull-down assays. In other systems, phosphorylation of targets by PAR-1 is known to generate binding sites for 14-3-3 proteins, which causes dissociation of the targets from the cortex. We therefore tested LET-99 for association with the C. elegans 14-3-3 protein, PAR-5, using bacterially expressed His-tagged LET-99 incubated with embryo extracts. PAR-5 bound to His-LET-99 in wild-type extracts, but PAR-5 binding was greatly diminished in extracts from par-1(RNAi) embryos. We used computer predictions for 14-3-3 binding sites followed by yeast-two hybrid (Y2H) assays to identify two LET-99 serine residues essential for PAR-5 binding. To determine the in vivo relevance of these sites, we introduced S to A mutations into an otherwise full-length rescuing LET-99 transgene (LET-99-AA). When transferred into a let-99 deletion mutant background, the transgene encoded LET-99-AA protein was mislocalized. Instead of being present in a band pattern, LET-99-AA localized to the entire posterior cortex of the one-cell embryo, similar to what was observed for LET-99 in par-1 mutant embryos. These and other results strongly support the model that PAR-5 binds to LET-99 to dissociate it from the cortex, and that this interaction is regulated by phosphorylation of LET-99 by PAR-1 at the posterior. In the future, we will test the hypothesis that PAR-1 directly phosphorylates LET-99 using in vitro kinase assays. We will also test LET-99 interacting proteins identified in Y2H screens for a role in anchoring LET-99 at the cortex.

Wu, Jui-ching et al. (2011) International Worm Meeting "Independent evolution of PP1 phosphatases for regulation of diverse sperm motility structures."

Chu, Diana, Mirosoian, Susan, Cintra, Thais, Wu, Tammy, Wu, Jui-Ching, Estrada, Rodrigo, Go, Aiza, Routman, Eric, Samson, Mark, Jow, Margaret

[International Worm Meeting, 2011]

The rapid evolution of male reproductive proteins has generated distinctive sperm motility structures, including tubulin-based flagella in mammals and Major Sperm Protein (MSP)-based pseudopods in nematodes. Despite such molecular divergence, we have found that sperm-specific PP1 phosphatases, which function in mouse sperm motility, are required for male fertility in C. elegans. We were thus interested to determine how GSP-3/4 can function in motility in species with morphologically distinct sperm. First, using genetic, molecular, and cell biological approaches in C. elegans, we find the sperm-specific PP1 phosphatases GSP-3 and GSP-4 (GSP-3/4) are functionally redundant and required for male fertility in worms. During meiosis, GSP-3/4 are required for proper sperm meiotic chromosome segregation and localize to sperm chromatin. Post-meiosis, GSP-3/4 shift localization from chromatin to pseudopods, suggesting GSP-3/4 are also required for motility. Consistent with this, gsp-3/4 mutant spermatids develop shorter pseudopods with greatly reduced pseudopod treadmilling activity and are thus immotile. GSP-3/4 spatially regulate MSP: they concentrate at the pseuodpodial base and regulate MSP disassembly during morphogenesis. Thus, GSP-3/4 regulate sperm motility through modulating MSP cytoskeleton dynamics during sperm activation. How have sperm-specific PP1 phosphatases come to play a central role in sperm motility function in mouse and nematodes given that GSP-3/4 regulate aspects of motility specific to nematode sperm? To determine if sperm-specific PP1 phosphatases in nematodes and mammals arose from a common ancestor that adapted sperm-specificity, we conducted a phylogenetic analysis of PP1 proteins in species from yeast to humans. We determined that sperm-specific PP1 phosphatases evolved in separate lineages and not from a common ancestor. Therefore, animals from a broad range of species, regardless of their sperm morphology, have separately adapted PP1 phosphatases as critical regulators of male reproduction.

Jui-ching Wu et al. (2003) International Worm Meeting "Genetic Analysis of Asymmetric LET-99 localization"

Lesilee S Rose, Meng-Fu B Tsou, Jui-ching Wu

[International Worm Meeting, 2003]

For a cell to divide asymmetrically, spindle position has to be coordinated with the cell polarity axis, so that different cell fate determinants can be segregated into different daughter cells after the cleavage. In C. elegans, a symmetric cortical PAR protein localization is required for generating polarity in the early embryo; however, how spindle orientation is related to this asymmetric cue remains unclear. Our lab has identified a maternal lethal effect gene, let-99, which plays a crucial role in spindle orientation. let-99 mutant embryos have defects in nuclear rotation and anaphase spindle positioning, although PAR protein localization and cell polarity are normal. We have found that the LET-99 protein is enriched as a posterior band at the cell cortex in cells undergoing asymmetric cell division. Furthermore, this localization pattern is dependent on asymmetric PAR-3 and PAR-2 localization. Finally, the mislocalization of LET-99 correlates with defects in nuclear rotation in both par-3 and par-2 mutant embryos (see abstract by Tsou and Rose). These observations lead to the model that the PAR-dependent asymmetry of LET-99 is essential for nuclear rotation. Although LET-99 is asymmetrically localized to the cortex in a PAR-3/PAR-2 dependent manner, how this asymmetry is generated remains to be elucidated. We are currently examining the genetic and molecular requirements for generating asymmetric LET-99 localization. Our data show that LET-99 localization is also dependent on PAR-1, a Ser/Thr kinase that is localized to the posterior cortex in response to PAR-3/PAR-2 asymmetry cues. In par-1 mutant embryos, LET-99 is localized to the entire posterior cortex instead of a discrete post erior band. This result suggests that posteriorly localized PAR-1 plays a role in restricting high levels of LET-99 from the very posterior cortex. Moreover, LET-99 is similarly mislocalized to the entire posterior cortex in a par-1 mutant allele that bears a point mutation in the kinase domain, indicating PAR-1 kinase activity is required for regulating LET-99 localization. To further test which aspects of LET-99 localization are important for nuclear rotation, we are reexamining nuclear rotation in par-1 mutant embryos. Moreover, we are beginning structure-function studies of the LET-99 protein to further elucidate the mechanisms that generate LET-99 asymmetry.

Jui-ching Wu et al. (2002) West Coast Worm Meeting "Genetic Analysis of Asymmetric LET-99 Localization"

Meng-Fu Bryan Tsou, Lesilee S Rose, Jui-ching Wu

[West Coast Worm Meeting, 2002]

Asymmetric cell division, which generates two daughter cells with different cell fates, is important for normal development. Two events are necessary for cells to divide asymmetrically. First, polarity has to be established to position cell fate determinants asymmetrically. Second, the mitotic spindle has to be properly oriented to correctly segregate cell fate determinants into different daughter cells.

Jui-ching Wu et al. (2005) International Worm Meeting "PAR-dependent localization of cortical LET-99 asymmetry"

Jui-ching Wu, Lesilee Rose

[International Worm Meeting, 2005]

Asymmetric cell division, which generates daughter cells with different cell fates, is important for the development of the C. elegans embryo. To achieve an asymmetric cell division, the spindle has to be oriented to the cell polarity axis. The PAR proteins are crucial for the establishment of cell polarity in P lineage cells that divide asymmetrically, as well as for proper spindle orientation. PAR proteins are localized to either anterior or posterior cortical domains and studies by others have revealed a localization pathway that leads to the asymmetric distribution of downstream factors: Anterior PAR-3 and posterior PAR-2 mutually exclude each other, which allows PAR-1 to localize to the posterior cortex. PAR-1 asymmetry then results in anterior localization of cytoplasmic MEX-5/6, which leads to the posterior localization of certain cell fate determinants. How asymmetric PAR domains control spindle positioning is less understood. Our lab previously showed that the LET-99 protein plays an important role in spindle positioning downstream of PAR polarity cues. LET-99 is required for both the nuclear rotation event that orients the spindle on the polarized axis, as well as for asymmetric anaphase movements that contribute to unequal division. LET-99 is localized at the cortex, with the highest levels being present in a posterior cortical band in asymmetrically dividing P lineage cells; this band does not overlap with the anterior PAR domain. Previous work showed that LET-99 localization is dependent on both PAR-3 and PAR-2, in a manner that indicates PAR-3 inhibits LET-99 localization at the anterior cortex. We are examining other components of the polarity pathway to determine which are required for normal LET-99 localization. We have found that in par-1 mutants, high levels of LET-99 are localized to the entire posterior cortex instead of a discrete band, but the anterior levels of LET-99 are still low. We have also found that LET-99 is mislocalized to the entire posterior cortex in par-4 mutant embryos. Preliminary data suggest that mex-5/6 do not have a direct role in generating the LET-99 pattern. We hypothesize that PAR-3 and the associated PAR-6 and PKC-3 proteins inhibit LET-99 localization to the anterior cortex, while PAR-1 and/or PAR-4 directly inhibits LET-99 localization at the very posterior. To test this model, we are currently quantifying PAR-1 levels in wild type to determine if there is a gradient of PAR-1: more PAR-1 at the posterior could result in formation of the LET-99 band. We are also examining LET-99 localization in double mutant combinations. Models for the molecular mechanism of LET-99 localization will be discussed.

Jui-ching Wu et al. (2004) West Coast Worm Meeting "Regulation of Asymmetric LET-99 Localization in C. elegans Embryos"

Jui-ching Wu, Lesilee S Rose

[West Coast Worm Meeting, 2004]

During cell division, the position of spindle determines the cleavage plane, which bisects the mother cell and separates cytoplasmic materials into the daughter cells. Accurate spindle positioning is of great importance during asymmetric divisions, because the cleavage plane has to be coordinated with the cell polarity axis. In C. elegans , embryonic development begins with a series of asymmetric divisions, during which the cells are polarized along the anterior-posterior axis, and the mitotic spindle is oriented on to the same axis. PAR proteins have been shown to play a crucial role in establishment of cell polarity, and previous studies in our lab show that LET-99 plays a role in connecting the PAR polarity cues to spindle orientation. LET-99 is localized to the entire cell cortex, but the highest levels are present in a posterior cortical band in cells that divide asymmetrically. This asymmetric LET-99 localization pattern is PAR-2 and PAR-3-dependent, and the mislocalization of LET-99 correlates with nuclear rotation defects observed in par-2 and par-3 mutants. This suggests that LET-99 asymmetry is important for spindle orientation. We are currently investigating the basis by which LET-99 asymmetry is generated. To determine which other aspects of the canonical PAR pathway are required for LET-99 localization, we have examined LET-99 localization in mutants for genes that act downstream of PAR-2 and PAR-3. Our data show that LET-99 is mislocalized to the entire posterior cortex in par-1 mutant embryos. However, the pattern is not affected in the mex-5;mex-6 mutant background. Quantification of staining intensities shows that more LET-99 is recruited to the posterior cortex in par-1 mutant embryos than in wild type. Together with previous studies, these results lead to a model that PAR-3 and PAR-1 inhibit cortical LET-99 localization at the anterior and posterior cortex respectively to generate the band pattern. We are currently testing other polarity proteins for a role in LET-99 localization, as well as creating LET-99::GFP constructs for use in determining the regions of LET-99 required for asymmetric localization.

Jui-ching Wu et al. (2006) Development & Evolution Meeting "PAR-3 and PAR-1 cooperate to generate the LET-99 band pattern during asymmetric division"

Jui-ching Wu, Lesilee Rose

[Development & Evolution Meeting, 2006]

During asymmetric cell divisions in early worm embryos, cell polarity is regulated by PAR proteins. PAR proteins also specify spindle positioning via a G protein signaling pathway that is required for the forces that move the spindle. Our lab previously showed that the LET-99 protein acts downstream of the PAR proteins and negatively regulates G protein signaling. Unlike several PAR proteins that localize to either the anterior or posterior cortex, LET-99 localizes in a distinct pattern as a posterior cortical band. We previously showed that cortical LET-99 asymmetry is dependent on PAR-3, a member of the anterior PAR-3/PAR-6/PKC-3 complex. To further elucidate how the PAR polarity pathway determines the LET-99 band pattern, we examined LET-99 localization in other polarity and spindle positioning mutants. We found that LET-99 localization is dependent on the cortical part of the polarity pathway, but not cytoplasmic proteins such as MEX-5/6. In the absence of the posteriorly localized PAR-1 kinase, LET-99 is recruited to the entire posterior cortex at high levels, which indicates that PAR-1 inhibits LET-99 localization at the posterior in wild type. The maintenance of low LET-99 levels at the anterior cortex in par-1 mutants shows that PAR-3 inhibits LET-99 localization independently of PAR-1. Further, quantification of staining intensities revealed a gap between the anterior PAR-3 and the posterior PAR-1 domains that correlates with the region of highest cortical LET-99 intensity. We conclude that the cortical LET-99 band is generated due to inhibition by PAR-3 and PAR-1. In a par-1 kinase mutant allele, LET-99 is also mislocalized to the entire posterior cortex. This result suggests that LET-99 localization is regulated by phosphorylation. Moreover, we found that nuclear rotation is less robust in par-1 mutant embryos when the extrinsic asymmetry of cell shape is removed. We propose that the anterior PAR-3/PAR-6/PKC-3 complex and posterior PAR-1 both inhibit LET-99 localization through their kinase activities, and the resulting cortical LET-99 band is required for efficient nuclear rotation during asymmetric cell division. We have generated a rescuing HA-tagged let-99 transgene with the goal of determining which LET-99 sequences respond to PAR-3 and PAR-1.

Chen, Shang-Yang et al. (2019) International Worm Meeting "Male meiotic chromosome segregation is not subjected to rigorous checkpoint regulation."

Chen, Shang-Yang, Wu, Jui-Ching

[International Worm Meeting, 2019]

During male meiosis, the duplicated chromosomes undergo two consecutive separation events. Because chromosome segregation is strictly regulated by the spindle assembly checkpoint (SAC), we asked whether both male meiotic divisions are also regulated by the same principle. Although outer kinetochore BUB-1, acting as SAC signaling platform, is disassembled and reassembled between two divisions, we found the SAC target securin is degraded during first division and remained undetectable through the second division. Interestingly, though administration of proteasome inhibitor arrested primary spermatocytes at anaphase I, division of secondary spermatocytes was not affected by inhibition of proteasome. These results show that SAC-dependent protein degradation does not play role in the second male meiotic chromosome segregation. To test whether SAC is indeed dispensable from second male meiotic division, we examined the division of primary and secondary spermatocytes with monopolar spindle. Surprisingly, monopolar spindle only minimally delayed both divisions, with timely disassembly of the outer kinetochore. Additionally, SAC-defective mutant mdf-1 did not show significant effect in monopolar primary and secondary spermatocytes. Furthermore, spermatocytes with defective spindle would skip division and proceed to final stage that generates abnormal spermatids. Taken together, our results show that the male meiotic chromosome segregation is not subjected to rigorous checkpoint control.

Lin, Yi-hsiu et al. (2015) International Worm Meeting "GSP-2 PP1 phosphatase regulates chromosome segregation events during sperm meiotic divisions."

Lin, Yi-hsiu, Wu, Jui-Ching

[International Worm Meeting, 2015]

Faithful chromosome segregation is crucial for development and cellular function. It has been shown that, during mitosis and oocyte meiosis, PP1 phosphatases prevents premature separation of sister chromatids by antagonizing Aurora B kinase activities. In sperm meiosis, however, it is unclear whether the chromosome segregation events are also under the control of PP1 phosphatases. We set out to examine the role of PP1 phosphatases GSP-1 and GSP-2 in sperm meiotic divisions. By fertility assessment, gsp-1 males were capable of siring viable progeny. Contrarily, females mated with gsp-2 mutant males generated both dead embryos and viable progeny. When examined by immunofluorescence staining, gsp-1 mutant male germline showed normal chromosome segregation. However, typical anaphase I meiotic spindles were never detected in gsp-2 mutant spermatocytes. Live imaging of gsp-2 mutant spermatocytes showed that during the first meiotic division the chromosomes were often not aligned properly, resulting in failure of resolution. Immunofluorescence staining of the Aurora B kinase AIR-2 showed that, instead of localize to the center of the bivalents, AIR-2 is spread through the surface of chromosomes in both gsp-1 and gsp-2 mutant spermatocytes. Our results show that at least GSP-2 PP1 phosphatase is required for sperm meiotic chromosome segregation. To further define whether GSP-1 plays a supportive role with GSP-2, the progression of sperm meiotic divisions in gsp-1;gsp-2 double mutant males are currently under examination.

Espiritu, Eugenel et al. (2015) International Worm Meeting "Polarity proteins regulate the localization of the spindle-positioning mediator, LET-99 ."

Wu, Jui-Ching, Rose, Lesilee, Espiritu, Eugenel, Richarson, Ella

[International Worm Meeting, 2015]

To produce cellular diversity during development, cells will undergo asymmetric divisions to produce daughters with different cellular fates. Spindle positioning is essential for these divisions, where the mitotic spindle must align with the axis of cell polarity so that cell fate determinants are distributed unequally to the daughter cells. In many systems, the conserved PAR proteins establish polarization of the cell. PAR proteins also regulate spindle movements via a complex including Ga, GPR and LIN-5, which recruit the microtubule motor dynein. In the C. elegans embryo, our lab previously identified LET-99 as a key regulator of GPR asymmetry in embryos. LET-99 is asymmetrically localized at the cortex in a posterior-lateral band pattern, where it inhibits GPR localization. Our analysis of LET-99 in mutant backgrounds showed that PAR-3 and the kinase PAR-1 inhibit cortical LET-99 localization at the anterior and posterior-most cortex respectively. In other systems, phosphorylation of targets by PAR-1 generates binding sites for 14-3-3 proteins, which alters the targets' localization. We found that the 14-3-3 protein PAR-5 bound to His::LET-99 in wild-type embryo extracts, but PAR-5 binding was greatly diminished after PAR-1 depletion. Computer predictions and yeast-two hybrid data identified two LET-99 serine residues essential for PAR-5 binding. We introduced S-to-A mutations at these residues in an otherwise full-length rescuing LET-99 transgene (LET-99-AA). In a let-99 deletion background, the LET-99-AA protein mislocalized to the entire posterior cortex of the one-cell embryo, similar to LET-99 in par-1 mutant embryos. These and other results support a model that PAR-1 phosphorylates LET-99 to create binding sites for PAR-5, which prevents LET-99 association with the posterior-most cortex. To begin to determine how LET-99 localization is restricted from the anterior, we analyzed LET-99 after depletion of anterior PAR components. We found that the PAR-3 associated proteins, PAR-6 and PKC-3, restrict LET-99 localization from the anterior. We are now testing if PAR-1 or PKC-3 can directly phosphorylate LET-99 in vitro. Additionally, we are determining how phosphorylation may regulate LET-99 affinity for lipids as we have determined that LET-99 can associate to negatively charged lipids in vitro. Funding for this work was provided by the NIH T32 MCB Training Program, Schwall Medical Research Fellowship, and the Schwall Dissertation Year Fellowship to EE and R01GM068744 to LR.