Heterotrimeric Kinesin-II Is Required for the Assembly of Motile 9 (cid:49) 2 Ciliary Axonemes on Sea Urchin Embryos

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 (cid:49) 0 ciliary axonemes that lack central pair MTs. Control embryos show no such defect and form swimming blastulae with normal, motile, 9 (cid:49) 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 inter-mediate and for formation of stable central pair MTs. Thus, kinesin-II SDS-PAGE was performed in duplicate on 7.5% resolving gels, and one gel was stained with Coomassie blue while the second gel was transferred to nitrocellulose and probed with K2.4 anti–kinesin-II primary antibody (1.0 or 1.5 (cid:109) g/ml) overnight at 4 (cid:56) C and then with horseradish peroxidase– conjugated goat anti–mouse secondary antibody (1:2,000) for 1 h at room Blots were developed using the enhanced chemilumines-cence detection system (Amersham Corp., Arlington Heights, IL) ex- posed for 2 min ( S. purpuratus MT or 4–20 min ( L. pictus blastulae protein). For densitometry, 100 (cid:109) g of blastulae protein, deciliated blastulae protein, and cilia protein were loaded in adjacent lanes and, as-suming equivalent protein transfer occurred in adjacent lanes, K2.4- labeled bands were analyzed on Western blots using a Stratagene (La Jolla, CA) Eagle Eye II gel documentation system. and focal single focal in en face were onto transparency. K2.4-injected embryo blastomeres reveal a normal arrangement of centrioles ( B ), radiating intracellular array of MTs ( C ), and striated rootlets ( D ). However, oblique sections across an axoneme on K2.4-injected embryos suggest an absence of central pair MTs ( B ) that are present in control axoneme oblique sections ( A ). Axonemal cross sections at higher magnification reveal that, while control axonemes present a typical arrangement of nine outer doublet MTs around a central pair of singlet MTs ( E ), axonemes from the paralyzed, K2.4-injected embryos completely lack central pair MTs and possess only the nine outer doublets ( F–I ). A pair of long oblique sections also reveals the clear difference between central pair presence in a control axoneme ( J ) and absence in an injected embryo axoneme ( K ). The panel of axoneme cross sections from K2.4-injected embryos ( F–I ) displays the variety of patterns of axonemal content exhibited in the absence of the central pair, including axonemes that appear devoid of either central pair or centralized amorphous material ( F ), axonemes with some centralized amorphous material ( G and H ), as well as axonemes where apparently all nine outer doublets can be counted as they spiral into the central cavity ( I ). way as in K2.4-injected embryos, initially forming a 7- (cid:109) m-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- (cid:109) m 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 (cid:109) m) 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.