Morris RL and Scholey JM (1997), Heterotrimeric kinesin-II is required for the a...

ECB-ART-36725

J Cell Biol 1997 Sep 08;1385:1009-22. doi: 10.1083/jcb.138.5.1009.

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Heterotrimeric kinesin-II is required for the assembly of motile 9+2 ciliary axonemes on sea urchin embryos.

Morris RL , Scholey JM .

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Heterotrimeric kinesin-II is a plus end- directed microtubule (MT) motor protein consisting of distinct heterodimerized motor subunits associated with an accessory subunit. To probe the intracellular transport functions of kinesin-II, we microinjected fertilized sea urchin eggs with an anti-kinesin-II monoclonal antibody, and we observed a dramatic inhibition of ciliogenesis at the blastula stage characterized by the assembly of short, paralyzed, 9+0 ciliary axonemes that lack central pair MTs. Control embryos show no such defect and form swimming blastulae with normal, motile, 9+2 cilia that contain kinesin-II as detected by Western blotting. Injection of anti-kinesin-II into one blastomere of a two-cell embryo leads to the development of chimeric blastulae covered on one side with short, paralyzed cilia, and on the other with normal, beating cilia. We observed a unimodal length distribution of short cilia on anti-kinesin-II-injected embryos corresponding to the first mode of the trimodal distribution of ciliary lengths observed for control embryos. This short mode may represent a default ciliary assembly intermediate. We hypothesize that kinesin-II functions during ciliogenesis to deliver ciliary components that are required for elongation of the assembly intermediate and for formation of stable central pair MTs. Thus, kinesin-II plays a critical role in embryonic development by supporting the maturation of nascent cilia to generate long motile organelles capable of producing the propulsive forces required for swimming and feeding.

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Species referenced: Echinodermata
Genes referenced: dnah3 KRP85 LOC100887844 LOC100893907 LOC115919910 LOC578392 pole
???displayArticle.antibodies??? KRP85 Ab3 LOC373178 Ab4

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	Figure 1. Specificity of K2.4 anti–kinesin-II antibody. (A) Coomassie blue–stained gel of MTs polymerized and pelleted in the presence of AMP-PNP from S. purpuratus cytosol (lane 1) or in the presence of ATP (lane 2). An identical gel was transferred to nitrocellulose and then probed with K2.4 to show that the antibody recognizes the 85-kD SpKRP85 subunit of kinesin-II and follows the kinesin-II as it pellets with microtubules in the presence of AMP-PNP (lane 1′) but not appreciably in the presence of ATP (lane 2′). (B) Coomassie blue–stained gel of whole L. pictus ciliated blastulae (lane 1), deciliated blastulae (lane 2), and isolated cilia (lane 3) shown loaded at 100 μg per lane. An identical gel was transferred to nitrocellulose and then probed with K2.4 to show that the antibody strongly and specifically recognizes the 85-kD subunit of kinesin-II in L. pictus whole blastulae (lane 1′) and deciliated blastulae (lane 2′). K2.4 also detects KRP85 in isolated cilia (lane 3′) but at a much lower level (5–10% by weight total protein) than in embryos. Increasing the exposure time for the enhanced chemiluminescence–detected blot from 4 min (lanes 1′, 2′, and 3′) to 20 min (lane 3′′) clearly reveals the KRP85 band in cilia during the embryonic stage at which K2.4 exerted its inhibitory effect.
	Figure 2. Effect of K2.4 anti–kinesin-II antibody microinjection on mitosis and cell division. This series of video frames illustrates the development of two L. pictus embryos, one microinjected with K2.4 (left embryo) and the other with SUK-4 (right embryo), through the swimming blastula stage. These two embryos were fertilized at 19:40, injected at 20:12, and as revealed by the recorded time/date stamps, are pictured here at 0:52, 1:55, 5:26, 8:05, and 13:12 h after fertilization. In the final frame, the SUK-4–injected embryo on the right starts to swim out of the injection chamber and into the sea water, leaving the stationary K2.4-injected embryo behind. The intracellular oil droplets confirm that these embryos were successfully injected. The dark vertical lines are the edges of the microinjection chamber. Bar, 40 μm.
	Figure 3. Effect of K2.4 anti–kinesin-II antibody microinjection on ciliogenesis during sea urchin development. Images of live embryos (A and B) show that an embryo injected with K2.4 at the one-cell stage (A) develops to the swimming blastula stage, but only forms short, paralyzed cilia, while an uninjected embryo (B) undergoes normal ciliogenesis and forms long, rapidly beating cilia over its entire surface. Differences in ciliary length are more easily seen in embryos fixed in 3% glutaraldehyde in calcium-free sea water (C and D), where the short paralyzed cilia with knoblike structures at the tips (C, inset) are seen on a K2.4-injected embryo (C), while an uninjected control embryo (D) possesses long cilia without terminal knobs. High-magnification images of the embryo surface juxtaposed to the coverslip on K2.4-injected and control embryos (E and F) show the different lengths, but similar density per unit area, of cilia on K2.4-injected embryos (E) and on uninjected (F) embryos. Four of the short cilia on the K2.4-injected embryo pictured (E) are indicated with arrowheads. Image pairs (A and B, C and D, and E and F) are shown at the same scale. The cilium in C, inset was 11 μm long. Bars, 20 μm.
	Figure 4. Two views of a half-paralyzed chimeric blastula resulting from the microinjection of one cell of a two-cell embryo with the K2.4 anti– kinesin-II antibody. The oil droplet (O) marks the injected half of the embryo on the left side of these video-enhanced contrast images, where many short, immotile cilia are clearly visible (arrowheads). In contrast, many long, motile cilia (arrows) are visible on the uninjected, control, right half of the blastula. The embryo has spun clockwise between these two images taken ∼5 s apart, driven by its long motile cilia, which are most clear when their movements are impeded by the adjacent unciliated embryo (lower right of each image) or by the wall of the injection chamber (bottom). The close proximity of short paralyzed cilia to long motile cilia is apparent at the bottom of these images. Bar, 20 μm.
	Figure 5. Relative frequency of occurrence of different lengths of cilia in K2.4-injected and control SUK4-injected, nonspecific Ig– injected, and uninjected embryos. Relative frequencies were determined by pooling individual cilia length measurements from all cells at each condition and dividing the number of cilia in each length bin by the total number of cilia measured for length. Cilia were binned such that the 2-μm bin contains cilia of length 1.00– 2.99 μm, the 4-μm bin contains cilia of length 3.00 to 4.99 μm, etc. Data are derived from 214 cilia on seven K2.4-injected embryos, 73 cilia on four SUK-4–injected embryos, 76 cilia from two nonspecific IgG–injected embryos, and 246 cilia on seven uninjected embryos. Open arrowheads indicate the mode of short cilia present on all embryos, whereas closed arrowheads indicate the major mode of normal length cilia that are present on control but not on K2.4-injected embryos.
	Figure 7. Effect of anti–kinesin-II antibody microinjection at one-cell stage on subsequent gastrulation and spiculogenesis. Gastrulation to form an archenteron (left) and formation of spicules (middle) occurred at similar times in uninjected (UN) and K2.4-injected (K2.4) embryos. Archenteron images are oriented with animal pole to the left; the blastopore of the K2.4-injected embryo faces toward the objective lens and lies out of the image plane. While control embryos continued to swim with their normal cilia (right), K2.4-injected embryos never grew normal length or motile cilia (arrowheads) during even the longest observation periods of >5 d. Archenteron is shown 4.5 d after fertilization and injection. In a separate batch of embryos, spicules and cilia are shown 1.5 d after fertilization and injection. Archenteron panels are same scale; spicule and cilia panels are same scale. Bars, 20 μm.
	Figure 8. Model of role of kinesin-II during ciliogenesis in early sea urchin embryos. In the K2.4-injected embryos described in this study (A), basal bodies (bb) localize properly, and ciliary growth begins in the absence of kinesin-II function. An axoneme (long gray rectangles labeled ax) has formed, but it lacks components that confer motility and lacks a central pair. (1) Cytoplasmic dynein transports membrane vesicles (gray circles) and associated axonemal proteins (black squares) towards the minus ends of cytoplasmic MTs (black lines) located near the basal body. Upon arrival at the base of the cilium, cytoplasmic dynein may dissociate from the vesicles (2) as vesicles fuse with the plasma membrane at the ciliary base (3) delivering their membrane and associated proteins. But while vesicle delivery proceeds at a normal rate and required membrane can progress up the axoneme (4, wavy gray arrow), axonemal cargoes and their associated axonemal proteins can not proceed up into the axoneme toward the plus ends of the axonemal MTs without the activity of kinesin-II, and therefore they remain at the base of the cilium (5). The “procilium” that grows from this process is short (averaging 7 μm), perhaps because of the reduced delivery of axonemal structural proteins, immotile due to the absence of central pair MTs, and has a swelling at its distal tip perhaps because of disorganized or insufficient capping structures. In the control embryos (B), axonemal growth begins in much the same way as in K2.4-injected embryos, initially forming a 7-μm-long procilium assembly intermediate. Throughout axonemal growth, cytoplasmic dynein transports membrane vesicles and associated axonemal proteins including kinesin-II (1) to the base of the cilium. Upon arrival near the basal body, cytoplasmic dynein may dissociate from the vesicles (2) as the vesicles fuse with the plasma membrane (3) delivering membrane that can progress up the axoneme as needed (4). After rapid establishment of the 7-μm procilium (gray) by a mechanism not requiring kinesin-II function, elongation of the axoneme and assembly of central pair microtubules (black) is now supported by the activity of kinesin-II as it transports axonemal structural proteins or MT-stabilizing factors up the axoneme (5) toward the plus ends of the axonemal microtubules at the cilium tip. Ciliary growth (wavy black arrow), supported by kinesin-II–driven transport along the axoneme, produces a cilium that is both long (most often 18 μm) because of sufficient delivery of axonemal structural proteins (6) and motile because of the presence of an organized central pair. The bulbous swelling at the tip of the cilium is absent, perhaps because of sufficient delivery of ciliary cap structures and subsequent proper organization and stabilization of the axonemal tip.

References [+] :

Aizawa, Kinesin family in murine central nervous system. 1992, Pubmed