Kinesin-13 Regulates Flagellar, Interphase, and Mitotic Microtubule Dynamics in Giardia intestinalis

Abstract

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.Giardia intestinalis is the most frequently identified protozoan cause of intestinal morbidity worldwide (59) and has a two-stage life cycle: a "trophozoite," or flagellate, form that attaches to the intestinal microvilli and a cyst form that can persist in the environment (1, 21). As in other eukaryotes, the giardial microtubule cytoskeleton creates a stable scaffold for intracellular trafficking, for organelle attachment, and for cell polarization (21). However, other important functions of the microtubule cytoskeleton are dynamic and rely upon both intrinsic dynamic instability-stochastic switches between microtubule growth and shrinkage phases (43)-and active regulation of microtubule assembly and/or disassembly. Microtubule dynamics, for example, are critical during cell division in Giardia where the two nuclei (30) undergo mitosis with extranuclear spindles that penetrate at polar nuclear openings (58), followed by the duplication and repositioning of eight flagella into the daughter cells (75). Beyond descriptions of cytoskeletal movements, we currently have little understanding of the molecular mechanism of active regulation of interphase and mitotic microtubule dynamics in Giardia.One class of conserved regulators of microtubule dynamics that mediate interactions between microtubule plus ends and other organelles are t...

EB1 and EB3 promote cilia biogenesis by several centrosome-related mechanisms

“…EB3 interacts with the MT depolymerizing kinesin-13 MCAK (Lee et al, 2008). Kinesin-13 family proteins have been implicated in cilium disassembly in protists (Blaineau et al, 2007;Dawson et al, 2007;Piao et al, 2009), and EB3 might, therefore, stabilize the axoneme by counteracting such proteins.…”

Flagellar regeneration requires cytoplasmic microtubule depolymerization and kinesin-13

“…In mammalian cells, treatment with taxol reduces cilia length whereas nocodazole treatment results in longer cilia, and changes of cilia length correlate with increase of cytoplasmic free tubulins (Sharma et al, 2011). In contrast, opposite phenotypes of flagella in Giardia are observed with these treatments (Dawson et al, 2007). To learn whether these drugs affect flagellar length in Chlamydomonas, steady state cells were treated for 4 h with taxol or nocodazole.…”

“…Overexpression of a kinesin-13 homologue in Leishmania, which is localized along the length of the flagellum, results in shorter flagella whereas its depletion by RNA interference (RNAi) in Trypanosome leads to longer flagella (Blaineau et al, 2007). In Giardia, overexpression of a kinesin-13 rigor mutant S280N causes longer flagella and disrupts cytoplasmic microtubule dynamics (Dawson et al, 2007).…”

Centrosomal protein CEP104/ChlamydomonasFAP256 moves to the ciliary tip during cilia assembly

“…Among these are the MT plus-end-binding proteins EB1 and EB3 and the nonprocessive kinesin motor kinesin-13. The latter has been shown to be involved in MT depolymerization at the plus and minus ends of MTs (Hunter et al, 2003;Wordeman, 2005) and homologs of Kinesin-13 have been linked to axonemal assembly and disassembly through overexpression and knockout studies in a variety of protistans (Blaineau et al, 2007;Chan and Ersfeld, 2010;Dawson et al, 2007). Another member of the kinesin superfamily, Kif19A, which depolymerizes MTs from the plus ends, also localizes at the tips of motile cilia.…”

“…Similarly, depletion of Kif24, a kinesin-13 family protein that specifically depolymerizes centriolar MTs, resulted in aberrant cilia (Kobayashi et al, 2011). Another class of MT binding proteins, the EB family of MT plus-end-binding proteins, which help in recruiting other proteins to the plus ends of MTs, were reported to be localized to the flagellar tips in Chlamydomonas, Giardia and mammalian motile cilia (Pedersen et al, 2003;Dawson et al, 2007;Schrøder et al, 2011 andBrooks andWallingford, 2012). The Chlamydomonas homolog of an EB family protein, CrEB1, localizes to the tip of the flagellar axoneme and interacts with IFT protein IFT172, indicating its possible role in regulating IFT events at the flagellar tip (Pedersen et al, 2003;Pedersen et al, 2005).…”