The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 μm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope–based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at ∼1.1 μm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end–directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.

Polarity establishment, asymmetric division, and acquisition of cell fates are critical steps during early development. In this review, we discuss processes that set up the embryonic axes, with an emphasis on polarity establishment and asymmetric division. We begin with the first asymmetric division in the C. elegans embryo, where symmetry is broken by the local inactivation of actomyosin cortical contractility. This contributes to establishing a polarized distribution of PAR proteins and associated components on the cell cortex along the longitudinal embryonic axis, which becomes the anterior-posterior (AP) axis. Thereafter, AP polarity is maintained through reciprocal negative interactions between the anterior and posterior cortical domains. We then review the mechanisms that ensure proper positioning of the centrosomes and the mitotic spindle in the one-cell embryo by exerting pulling forces on astral microtubules. We explain how a ternary complex comprised of Gα (GOA-1/GPA-16), GPR-1/GPR-2, and LIN-5 is essential for anchoring the motor protein dynein to the cell cortex, where it is thought to exert pulling forces on depolymerizing astral microtubules. We proceed by providing an overview of cell cycle asynchrony in two-cell embryos, as well as the cell signaling and spindle positioning events that underly the subsequent asymmetric divisions, which establish the dorsal-ventral and left-right axes. We then discuss how AP polarity ensures the unequal segregation of cell fate regulators via the cytoplasmic proteins MEX-5/MEX-6 and other polarity mediators, before ending with an overview of how the fates of the early blastomeres are specified by these processes.

cell division. In C. elegans embryos, homologs of receptorindependent G protein activators, GPR-1 and GPR-2 (GPR-1/2), function together with Gα (GOA-1 and GPA-16) to generate asymmetric spindle pole elongation during divisions in the P lineage. Although Gα 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, Gα is required for the intrinsically programmed nuclear rotation event that orients the spindle in the one-cell. LET-99 functions antagonistically to the Gα/GPR-1/2 signaling pathway, providing an explanation for how Gα-dependent force is regulated asymmetrically by PAR polarity cues during both nuclear rotation and anaphase spindle elongation. In addition, Gα 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/P 2 cell boundary are complementary, suggesting that LET-99 and Gα/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.

G protein signaling pathways regulate mitotic spindle positioning during cell division in many systems. In Caenorhabditis elegans embryos, G alpha subunits act with the positive regulators GPR-1/2 and LIN-5 to generate cortical pulling forces for posterior spindle displacement during the first asymmetric division. GPR-1/2 are asymmetrically localized at the posterior cortex by PAR polarity cues at this time. Here we show that LIN-5 colocalizes with GPR-1/2 in one-cell embryos during spindle displacement. Significantly, we also find that LIN-5 and GPR-1/2 are localized to the opposite, anterior cortex in a polarity-dependent manner during the nuclear centration and rotation movements that orient the forming spindle onto the polarity axis. The depletion of LIN-5 or GPR-1/2 results in decreased centration and rotation rates, indicating a role in force generation at this stage. The localization of LIN-5 and GPR-1/2 is largely interdependent and requires G alpha. Further, LIN-5 immunoprecipitates with G alpha in vivo, and this association is GPR-1/2 dependent. These results suggest that a complex of G alpha/GPR-1/2/LIN-5 is asymmetrically localized in response to polarity cues, and this may be the active signaling complex that transmits asymmetries to the force generation machinery during both nuclear rotation and spindle displacement.

Asymmetric cell divisions play an important role in generating diversity during metazoan development. In the early C. elegans embryo, a series of asymmetric divisions are crucial for establishing the three principal axes of the body plan (AP, DV, LR) and for segregating determinants that specify cell fates. In this review, we focus on events in the one-cell embryo that result in the establishment of the AP axis and the first asymmetric division. We first describe how the sperm-derived centrosome initiates movements of the cortical actomyosin network that result in the polarized distribution of PAR proteins. We then briefly discuss how components acting downstream of the PAR proteins mediate unequal segregation of cell fate determinants to the anterior blastomere AB and the posterior blastomere P1. We also review how a heterotrimeric G protein pathway generates cortically based pulling forces acting on astral microtubules, thus mediating centrosome and spindle positioning in response to AP polarity cues. In addition, we briefly highlight events involved in establishing the DV and LR axes. The DV axis is established at the four-cell stage, following specific cell-cell interactions that occur between P2 and EMS, the two daughters of P1, as well as between P2 and ABp, a daughter of AB. The LR axis is established shortly thereafter by the division pattern of ABa and ABp. We conclude by mentioning how findings made in early C. elegans embryos are relevant to understanding asymmetric cell division and pattern formation across metazoan evolution.

Chemosensation in the nervous system of the nematode Caenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.

No abstract

Studies of about 20 maternally expressed genes are providing an understanding of mechanisms of patterning and cell-fate determination in the early Caenorhabditis elegans embryo. The analyses have revealed that fates of the early blastomeres are specified by a combination of intrinsically asymmetric cell divisions and two types of cell-cell interactions: inductions and polarizing interactions. In this review we summarize the current level of understanding of the molecular mechanisms underlying these processes in the specification of cell fates in the pregastrulation embryo.