Despite being essential for spatial cell division control, the mechanisms governing spindle positioning remain incompletely understood. In the Caenorhabditis elegans one-cell stage embryo, the spindle becomes asymmetrically positioned during anaphase through the action of as-yet unidentified cortical force generators that pull on astral microtubules and that depend on two G alpha proteins and associated proteins. We performed spindle-severing experiments following temporally restricted gene inactivation and drug exposure, and established that microtubule dynamics and dynein are both required for generating efficient pulling forces. We found that the G alpha-associated proteins GPR-1/2 and LIN-5 interact in vivo with LIS-1, a component of the dynein complex. Moreover, we discovered that the LIN-5, GPR-1/2 and the G alpha proteins promote the presence of the dynein complex at the cell cortex. Our findings suggest a mechanism by which the G alpha proteins enable GPR-1/2 and LIN-5 recruitment to the cortex, thus ensuring the presence of cortical dynein. Together with microtubule dynamics, this allows pulling forces to be exerted and proper cell division to be achieved.

Heterotrimeric G proteins are crucial for asymmetric cell division, but the mechanisms of signal activation remain poorly understood. Here, we establish that the evolutionarily conserved protein RIC-8 is required for proper asymmetric division of one-cell stage C. elegans embryos. Spindle severing experiments demonstrate that RIC-8 is required for generation of substantial pulling forces on astral microtubules. RIC-8 physically interacts with GOA-1 and GPA-16, two Galpha subunits that act in a partially redundant manner in one-cell stage embryos. RIC-8 preferentially binds to GDP bound GOA-1 and is a guanine nucleotide exchange factor (GEF) for GOA-1. Our analysis suggests that RIC-8 acts before the GoLoco protein GPR-1/2 in the sequence of events leading to Galpha activation. Furthermore, coimmunoprecipitation and in vivo epistasis demonstrate that inactivation of the Gbeta subunit GPB-1 alleviates the need for RIC-8 in one-cell stage embryos. Our findings suggest a mechanism in which RIC-8 favors generation of Galpha free from Gbetagamma and enables GPR-1/2 to mediate asymmetric cell division.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Non-pulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and ten deletions, we have identified an additional thirty eight novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, twenty missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic whereas four familial cases with three showing maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA binding domain, indicating its plausible role in gene regulation. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

The Drosophila no distributive disjunction (nod) gene encodes a kinesin-like protein that has been proposed to push chromosomes toward the metaphase plate during female meiosis. We report that the nonmotor domain of the nod protein can mediate direct binding to DNA. Using an antiserum prepared against bacterially expressed nod protein, we show that during prometaphase nod protein is localized on oocyte chromosomes and is not restricted to either specific chromosomal regions or to the kinetochore. Thus, motor-based chromosome-microtubule interactions are not limited to the centromere, but extend along the chromosome arms, providing a molecular explanation for the polar ejection force.

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the G␣ ␣ proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using coimmunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the G␤ ␤ protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two G␣ ␣ proteins can activate the same pathway and that their dual presence is normally needed to counter G␤ ␤␥ ␥. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA-16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between G␣ ␣ proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.

There was an error published in Development 137, 237-247.In Fig. 2D, owing to the PPH-6 film being inadvertently flipped during scanning, the two lanes labelled as input instead showed flowthrough. A revised Fig. 2 with the correct input samples in D is shown below. In addition, a note has been added to the end of the legend to clarify the lack of GFP-SAPS-1 signal in these input lanes.This error does not affect the conclusions of this experiment or of the paper. The authors apologise to readers for this mistake. Western blot analysis using PPH-6 antibodies on wild-type or pph-6(RNAi) embryonic extracts. The blot was reprobed with α-tubulin antibodies as a loading control (bottom). Note the presence of two species, with the lower one exhibiting the predicted molecular weight of PPH-6 (∼37 kDa). Note also that the ratio between these two species varies among extracts (compare A with inputs in C). Similar variability is observed for SAPS-1 (B,C). The variation might be due to differences in the developmental stages of the embryos in the different preparations. (B) Western blot analysis of wild-type or saps-1(RNAi) embryonic extracts probed with SAPS-1 antibodies. Note presence of two major specific species, exhibiting the predicted molecular weight of the splice variants of SAPS-1 (∼80 kDa and 87 kDa). A non-specific band (NS) served as a loading control. (C) Coimmunoprecipitation from wild-type, pph-6(RNAi) or saps-1(RNAi) embryonic extracts using PPH-6 antibodies. Western blots were probed with antibodies against PPH-6, SAPS-1 or α-tubulin, as indicated. In the second row, the input is exposed 10 times longer than the IP. Input/IP=1:50. In three independent experiments, we observed that PPH-6 antibodies retrieved more PPH-6 from the saps-1(RNAi) extract than from the pph-6(RNAi) extract, despite similar depletion levels of PPH-6. Perhaps PPH-6 not bound to SAPS-1 is more accessible to PPH-6 antibodies. (D) Extract from embryos expressing GFP-SAPS-1 or from wild-type embryos immunoprecipitated with GFP antibodies and analyzed by western blot with GFP or PPH-6 antibodies, as indicated. Note that only the PPH-6 species with the lower molecular weight co-immunoprecipitates with GFP-SAPS-1. Input/IP=1:50 (overall levels of GFP-SAPS-1 protein in the embryonic lysates are low, hence the lack of detection of GFP-SAPS-1 in the input lanes). 2689

SUMMARYAsymmetric cell division is an evolutionarily conserved process that gives rise to daughter cells with different fates. In one-cell stage C. elegans embryos, this process is accompanied by asymmetric spindle positioning, which is regulated by anterior-posterior (A-P) polarity cues and driven by force generators located at the cell membrane. These force generators comprise two G proteins, the coiled-coil protein LIN-5 and the GoLoco protein GPR-1/2. The distribution of GPR-1/2 at the cell membrane is asymmetric during mitosis, with more protein present on the posterior side, an asymmetry that is thought to be crucial for asymmetric spindle positioning. The mechanisms by which the distribution of components such as GPR-1/2 is regulated in time and space are incompletely understood. Here, we report that the distribution of the Gb subunit GPB-1, a negative regulator of force generators, varies across the cell cycle, with levels at the cell membrane being lowest during mitosis. Furthermore, we uncover that GPB-1 trafficks through the endosomal network in a dynamin-and RAB-5-dependent manner, which is most apparent during mitosis. We find that GPB-1 trafficking is more pronounced on the anterior side and that this asymmetry is regulated by A-P polarity cues. In addition, we demonstrate that GPB-1 depletion results in the loss of GPR-1/2 asymmetry during mitosis. Overall, our results lead us to propose that modulation of Gb trafficking plays a crucial role during the asymmetric division of one-cell stage C. elegans embryos.

Abstract. The nod kinesin-like protein is localized along the arms of meiotic chromosomes and is required to maintain the position of achiasmate chromosomes on the developing meiotic spindle. Here we show that the localization of ectopically expressed nod protein on mitotic chromosomes precisely parallels that observed for wild-type nod protein on meiotic chromosomes. Moreover, the carboxyl-terminal half of the nod protein also binds to chromosomes when overexpressed in mitotic cells, whereas the overexpressed amino-terminal motor domain binds only to microtubules. Chromosome localization of the carboxyl-terminal domain of nod depends upon an 82-amino acid region comprised of three copies of a sequence homologous to the DNAbinding domain of HMG 14/17 proteins. These data map the two primary functional domains of the nod protein in vivo and provide a molecular explanation for the directing of the nod protein to a specific subcellular component, the chromosome.