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Wnt5a-Ror signaling is a conserved pathway that regulates morphogenetic processes during vertebrate development [R. T. Moon et al., Development 119, 97–111 (1993); I. Oishi et al., Genes Cells 8, 645–654 (2003)], but its downstream signaling events remain poorly understood. Through a large-scale proteomic screen in mouse embryonic fibroblasts, we identified the E3 ubiquitin ligase Pdzrn3 as a regulatory target of the Wnt5a-Ror pathway. Upon pathway activation, Pdzrn3 is degraded in a β-catenin–independent, ubiquitin-proteasome system–dependent manner. We developed a flow cytometry-based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 stability. Epistatically, Pdzrn3 is regulated independently of Kif26b, another Wnt5a-Ror effector. Wnt5a-dependent degradation of Pdzrn3 requires phosphorylation of three conserved amino acids within its C-terminal LNX3H domain [M. Flynn, O. Saha, P. Young, BMC Evol. Biol. 11, 235 (2011)], which acts as a bona fide Wnt5a-responsive element. Importantly, this phospho-dependent degradation is essential for Wnt5a-Ror modulation of cell migration. Collectively, this work establishes a Wnt5a-Ror cell morphogenetic cascade involving Pdzrn3 phosphorylation and degradation.

In multicellular organisms, asymmetric cell division generates cell type diversity during both development and tissue homeostasis. For successful asymmetric division to proceed, a cellular axis of polarity, or asymmetry, must be established via any of several molecular mechanisms. This step is followed by two sequential processes requiring precise coordination of the various components of the cytoskeleton. First, the mitotic spindle must be positioned in alignment with the axis of polarity; second, cytokinesis must occur at the right position and time, as instructed by the spindle, to segregate chromosomes and bisect the axis of polarity. In this dissertation I present my contributions to two research projects related to cytoskeletal regulation during asymmetric cell division.Chapter I reviews the state of the field prior to my studies and introduces the two research projects in context. Chapter II is a manuscript in revision for Journal of Cell Science that relates our findings from the first project, which aimed to understand the role of the DEPDC1 homolog LET-99 during cytokinesis. LET-99 was previously characterized as a negative regulator of the dynein-containing force generating complex during spindle positioning, but had also been shown to play a separable role in cytokinesis. With others, I demonstrated that during the first mitotic division LET-99 and the Rac1 homolog CED-10 have antagonistic roles in regulating the balance of branched vs linear actin during cytokinesis to achieve robust and timely furrowing. These roles appear to be global rather than spatially restricted to the contractile ring. However, the findings suggest that CED-10 does not interact with LET-99 to promote spindle positioning in the first mitotic division, further confirming that LET-99’s role as a regulator of the actomyosin cytoskeleton and cytokinesis is separable from its role in spindle positioning.Chapter III is a manuscript in preparation that documents our investigations into the role of CED-10 in spindle orientation of the asymmetrically dividing EMS cell. Two signaling pathways that regulate this spindle orientation were previously identified: a Wnt/Frizzled pathway and a MES-1/SRC-1 pathway. I determined that CED-10 works upstream or at the level of SRC-1 in the MES-1/SRC-1 signaling pathway and furthermore that CED-10 contributes to cortical localization of the same dynein-containing force generating complex involved in spindle positioning at the one-cell stage. I also found that CED-10’s molecular function in this context may be to promote branched actin formation at the EMS/P2 contact, but this evidence is partially contradicted by our findings that loss of the branched-actin regulator GEX-3 does not enhance the rate of EMS spindle rotation defects in Wnt/MOM-2-depleted embryos. An additional curious finding is that CED-10 has a previously unreported role in P1 spindle rotation, as do the branched actin nucleator Arp2/ARX-2 (Arp2) and the Frizzled ortholog MOM-5.Although this research was performed using nematodes as a model organism, the results have broad implications for all animals because most of the molecular components involved are evolutionarily conserved. By understanding in detail the mechanisms of different types of asymmetric divisions, we can understand both normal development and diseases such as cancer, which involves the dysregulation of asymmetric division.

Asymmetric cell division is essential for the creation of cell types with different identities and functions. The EMS blastomere of the four-cell embryo undergoes an asymmetric division in response to partially redundant signaling pathways. One pathway involves a Wnt signal emanating from the neighboring P2 cell, while the other pathway is defined by the receptor-like MES-1 protein localized at the EMS/P2 cell contact, and the cytoplasmic kinase SRC-1. In response to these pathways, the EMS nuclear-centrosome complex rotates so that the spindle forms on the anterior-posterior axis; after division, the daughter cell contacting P2 becomes the endodermal precursor cell. Here we identify the Rac1 homolog, CED-10, as a new component of the MES-1/SRC-1 pathway. Loss of CED-10 affects both spindle positioning and endoderm specification. Although MES-1 is still present at the EMS/P2 contact in embryos, SRC-1 dependent phosphorylation is reduced. These and other results suggest that CED-10 acts downstream of MES-1 and upstream of, or at the level of, SRC-1 activity. In addition, we find that the branched actin regulator ARX-2 is enriched at the EMS/P2 cell contact site, in a CED-10 dependent manner. Loss of ARX-2 results in spindle positioning defects, suggesting that CED-10 acts through branched actin to promote the asymmetric division of the EMS cell.

Metazoan eggs have a specialized coat of extracellular matrix that aids in sperm-egg recognition. The coat is rapidly remodeled after fertilization to prevent polyspermy and establish a more permanent barrier to protect the developing embryo. In nematodes, this coat is called the vitelline layer, which is remodeled into the outermost layer of a rigid and impermeable eggshell. We have identified three key components of the vitelline layer structural scaffold - PERM-2, PERM-4 and CBD-1, the first such proteins to be described in the nematode C. elegant. CBD-1 recruited PERM-2 and PERM-4 to the nascent vitelline layer via two N- terminal chitin-binding domains. After fertilization, all three proteins redistributed from the zygote surface to the outer eggshell. Depletion of PERM-2 and PERM-4 from the scaffold led to a porous vitelline layer that permitted soluble factors to leak through the eggshell and resulted in embryonic death. In addition to its role in vitelline layer assembly, CBD-1 is also known to scaffold a protein complex required for fertilization and egg activation (EGG-1-5/CHS-1/MBK-2). We found the PERM complex and EGG complex to be structurally and functionally independent, and regulated through distinct domains of CBD-1. CBD-1 is thus a multifaceted regulator that promotes distinct aspects of vitelline layer assembly and egg activation. In sum, our findings identify the first vitelline layer components in nematodes, and provide a foundation through which to explore both conserved and species-specific strategies used by animals to build protective barriers following fertilization.

Holmes et al 1 presented interesting data suggesting that the presence of an apolipoprotein E (ApoE) [epsilon]4 allele did not effect the rate of progression of cognitive impairment. A detailed description of the methodology is given elsewhere. 2 ApoE genotyping was performed on genomic DNA extracted from whole blood samples according to established procedures. 3 The mean age of the patients was 79; 38 (75%) were women and they had mean CAMCOG scores of 48.

During cytokinesis, signals from the central spindle stimulate the accumulation of active RhoA-GTPase and thus contractile ring components at the equator, while the astral microtubules inhibit such components at the polar cortex. The DEPDC1 family protein LET-99 is required for furrow ingression in the absence of the central spindle signal, and for timely onset of furrowing even in the presence of the central spindle signal. Here we show that LET-99 works downstream or independently of RhoA-GTP and antagonizes branched F-actin and the Rac protein CED-10 to promote furrow initiation. This interaction with CED-10 is separable from LET-99’s function in spindle positioning. We also characterize a new role for LET-99 in regulating cortical stability, where LET-99 acts in parallel with the actomyosin scaffolding protein anillin, but LET-99 does not antagonize CED-10 in this case. We propose that LET-99 acts in a pathway that inhibits the Rac CED-10 to promote the proper balance of branched versus linear F-actin for cytokinesis, and that LET-99 also regulates another factor that contributes to cortical stability.