Guangshuo Ou

Functional coordination of intraflagellar transport motors

Cilia have diverse roles in motility and sensory reception, and defects in cilia function contribute to ciliary diseases such as Bardet–Biedl syndrome (BBS). Intraflagellar transport (IFT) motors assemble and maintain cilia by transporting ciliary precursors, bound to protein complexes called IFT particles, from the base of the cilium to their site of incorporation at the distal tip. In Caenorhabditis elegans, this is accomplished by two IFT motors, kinesin-II and osmotic avoidance defective (OSM)-3 kinesin, which cooperate to form two sequential anterograde IFT pathways that build distinct parts of cilia. By observing the movement of fluorescent IFT motors and IFT particles along the cilia of numerous ciliary mutants, we identified three genes whose protein products mediate the functional coordination of these motors. The BBS proteins BBS-7 and BBS-8 are required to stabilize complexes of IFT particles containing both of the IFT motors, because IFT particles in bbs-7 and bbs-8 mutants break down into two subcomplexes, IFT-A and IFT-B, which are moved separately by kinesin-II and OSM-3 kinesin, respectively. A conserved ciliary protein, DYF-1, is specifically required for OSM-3 kinesin to dock onto and move IFT particles, because OSM-3 kinesin is inactive and intact IFT particles are moved by kinesin-II alone in dyf-1 mutants. These findings implicate BBS ciliary disease proteins and an OSM-3 kinesin activator in the formation of two IFT pathways that build functional cilia.

Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons

Cilia have diverse roles in motility and sensory reception and their dysfunction contributes to cilia-related diseases. Assembly and maintenance of cilia depends on the intraflagellar transport (IFT) of axoneme, membrane, matrix and signalling proteins to appropriate destinations within the organelle. In the current model, these diverse cargo proteins bind to multiple sites on macromolecular IFT particles, which are moved by a single anterograde IFT motor, kinesin-II, from the ciliary base to its distal tip, where cargo-unloading occurs. Here, we describe the observation of fluorescent IFT motors and IFT particles moving along distinct domains within sensory cilia of wild-type and IFT-motor-mutant Caenorhabditis elegans. We show that two anterograde IFT motor holoenzymes, kinesin-II and Osm-3–kinesin, cooperate in a surprising way to control two pathways of IFT that build distinct parts of cilia. Instead of each motor independently moving its own specific cargo to a distinct destination, the two motors function redundantly to transport IFT particles along doublet microtubules adjacent to the transition zone to form the axoneme middle segment. Next, Osm-3–kinesin alone transports IFT particles along the distal singlet microtubules to stabilize the distal segment. Thus, the subtle coordinate activity of these IFT motors creates two sequential transport pathways.

Functional genomics of the cilium, a sensory organelle

Cilia and flagella play important roles in many physiological processes, including cell and fluid movement, sensory perception, and development. The biogenesis and maintenance of cilia depend on intraflagellar transport (IFT), a motility process that operates bidirectionally along the ciliary axoneme. Disruption in IFT and cilia function causes several human disorders, including polycystic kidneys, retinal dystrophy, neurosensory impairment, and Bardet-Biedl syndrome (BBS). To uncover new ciliary components, including IFT proteins, we compared C. elegans ciliated neuronal and nonciliated cells through serial analysis of gene expression (SAGE) and screened for genes potentially regulated by the ciliogenic transcription factor, DAF-19. Using these complementary approaches, we identified numerous candidate ciliary genes and confirmed the ciliated-cell-specific expression of 14 novel genes. One of these, C27H5.7a, encodes a ciliary protein that undergoes IFT. As with other IFT proteins, its ciliary localization and transport is disrupted by mutations in IFT and bbs genes. Furthermore, we demonstrate that the ciliary structural defect of C. elegans dyf-13(mn396) mutants is caused by a mutation in C27H5.7a. Together, our findings help define a ciliary transcriptome and suggest that DYF-13, an evolutionarily conserved protein, is a novel core IFT component required for cilia function.

Mechanism of transport of IFT particles in C. elegans cilia by the concerted action of kinesin-II and OSM-3 motors.

The assembly and function of cilia on Caenorhabditis elegans neurons depends on the action of two kinesin-2 motors, heterotrimeric kinesin-II and homodimeric OSM-3–kinesin, which cooperate to move the same intraflagellar transport (IFT) particles along microtubule (MT) doublets. Using competitive in vitro MT gliding assays, we show that purified kinesin-II and OSM-3 cooperate to generate movement similar to that seen along the cilium in the absence of any additional regulatory factors. Quantitative modeling suggests that this could reflect an alternating action mechanism, in which the motors take turns to move along MTs, or a mechanical competition, in which the motors function in a concerted fashion to move along MTs with the slow motor exerting drag on the fast motor and vice versa. In vivo transport assays performed in Bardet-Biedl syndrome (BBS) protein and IFT motor mutants favor a mechanical competition model for motor coordination in which the IFT motors exert a BBS protein–dependent tension on IFT particles, which controls the IFT pathway that builds the cilium foundation.

Polarized myosin produces unequal-size daughters during asymmetric cell division.

Daughter Diversity Asymmetric cell division generates cell diversity and maintains tissue homeostasis. In early Caenorhabditis elegans embryos, the mitotic spindle is pulled toward one side of the cell by the molecular motor, dynein, and the cell divides into two unequally sized daughters. However, other types of asymmetric cell divisions (for example, in Drosophila neuroblasts) start with a centrally localized spindle. In this latter case, the mechanism by which two differently sized daughters are created is not understood. Ou et al. (p. 677, published online 30 September; see the Perspective by Grill) studied asymmetric cell divisions in the Q neuroblast lineage during C. elegans development and found that when the spindle was centred, myosin II accumulated at higher levels on the side of what will become the smaller daughter cell, giving rise to asymmetric myosin-based contractile forces acting on the membrane. Motor proteins help to produce developmentally distinct daughter cells during development Asymmetric positioning of the mitotic spindle before cytokinesis can produce different-sized daughter cells that have distinct fates. Here, we found an asymmetric division in the Caenorhabditis elegans Q neuroblast lineage that began with a centered spindle but generated different-sized daughters, the smaller (anterior) of which underwent apoptosis. During this division, more myosin II accumulated anteriorly, suggesting that asymmetric contractile forces might produce different-sized daughters. Indeed, partial inactivation of anterior myosin by chromophore-assisted laser inactivation created a more symmetric division and allowed the survival and differentiation of the anterior daughter. Thus, the balance of myosin activity on the two sides of a dividing cell can govern the size and fate of the daughters.

Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure–frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a “canonical” IFT motor, whereas OSM-3 is an “accessory” IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.

Proteomic study and marker protein identification of caenorhabditis elegans lipid droplets

Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved across almost all species. Many metabolic syndromes are directly linked to the over-storage of neutral lipids in LDs. The study of LDs in Caenorhabditis elegans (C. elegans) has been difficult because of the lack of specific LD marker proteins. Here we report the purification and proteomic analysis of C. elegans lipid droplets for the first time. We identified 306 proteins, 63% of these proteins were previously known to be LD-proteins, suggesting a similarity between mammalian and C. elegans LDs. Using morphological and biochemical analyses, we show that short-chain dehydrogenase, DHS-3 is almost exclusively localized on C. elegans LDs, indicating that it can be used as a LD marker protein in C. elegans. These results will facilitate further mechanistic studies of LDs in this powerful genetic system, C. elegans.

Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development.

Conditional gene knockout animals are valuable tools for studying the mechanisms underlying cell and developmental biology. We developed a conditional knockout strategy by spatiotemporally manipulating the expression of an RNA-guided DNA endonuclease, CRISPR-Cas9, in Caenorhabditis elegans somatic cell lineages. We showed that this somatic CRISPR-Cas9 technology provides a quick and efficient approach to generate conditional knockouts in various cell types at different developmental stages. Furthermore, we demonstrated that this method outperforms our recently developed somatic TALEN technique and enables the one-step generation of multiple conditional knockouts. By combining these techniques with live-cell imaging, we showed that an essential embryonic gene, Coronin, which is associated with human neurobehavioral dysfunction, regulates actin organization and cell morphology during C. elegans postembryonic neuroblast migration and neuritogenesis. We propose that the somatic CRISPR-Cas9 platform is uniquely suited for conditional gene editing-based biomedical research.

The PKD protein qilin undergoes intraflagellar transport

Cilia play diverse roles in motility and sensory reception, and defects in their formation and function underlie cilia-related human diseases [1]. One such disease is polycystic kidney disease (PKD), a heritable nephropathy associated with defects in the formation and function of sensory (also known as primary) cilia within renal tubules of the kidney [2]. Because the assembly and maintenance of these sensory cilia depends upon the intraflagellar transport (IFT) of axoneme and ciliary membrane components, such as polycystins [3], defective IFT is one of the factors that can contribute to PKD.

Dynamic phosphorylation of CENP-A at Ser68 orchestrates its cell-cycle-dependent deposition at centromeres.

The H3 histone variant CENP-A is an epigenetic marker critical for the centromere identity and function. However, the precise regulation of the spatiotemporal deposition and propagation of CENP-A at centromeres during the cell cycle is still poorly understood. Here, we show that CENP-A is phosphorylated at Ser68 during early mitosis by Cdk1. Our results demonstrate that phosphorylation of Ser68 eliminates the binding of CENP-A to the assembly factor HJURP, thus preventing the premature loading of CENP-A to the centromere prior to mitotic exit. Because Cdk1 activity is at its minimum at the mitotic exit, the ratio of Cdk1/PP1α activity changes in favor of Ser68 dephosphorylation, thus making CENP-A available for centromeric deposition by HJURP. Thus, we reveal that dynamic phosphorylation of CENP-A Ser68 orchestrates the spatiotemporal assembly of newly synthesized CENP-A at active centromeres during the cell cycle.