PMC

Ribbon presentation of the GiKIN2a monomer. Red, α-helices; blue, β-sheets; green, neck-linker. ADP is displayed in the active site as ball-and-stick model, and the magnesium ion is shown in purple. The area of the mutated residue on helix α2a is indicated in yellow. Strands and helices are numbered according to the convention. The figure was made using PyMOL ().

Structural comparison of the human kinesin 1MKJ with the giardial kinesin-2 homolog GiKIN2a. (a) Superposition of the C^α atoms of human kinesin structure PDB:1MKJ (blue) and GiKIN2a (green; RMS deviation = 1.0 Å over 294 residues. The ADP nucleotide in the active site is shown as red sticks. (b) Superposition of the docked neck linker from PDB:1MKJ (blue) with the docked neck linker of GiKIN2a (green). The figure was made using PyMOL ().

The GiKIN2aT104N mutant kinesin acts as a rigor mutant in vitro. (a) Superose 12 gel filtration profile of purified pig brain αβ-tubulin and kinesin motor mutant GiKIN2aT104N. Key to elution profile line colors: blue, 280-nm absorbance spectrum of unbound αβ-tubulin; green, 280 nm absorbance spectrum αβ-tubulin in complex with kinesin motor mutant GiKIN2aT105N; and red, the corresponding 260-nm absorbance spectra of both and (b) 12.5% SDS-PAGE showing the elution fractions from 12 to 15 ml in 0.5-ml steps of the Superose 12 gel filtration chromatography of αβ-tubulin and GiKIN2aT104N. The leftmost lane contains the molecular weight marker.

Comparison of the active site of (a) GiKIN2a and (b) GiKIN2aT104N. The nucleotide ADP is displayed as ball and stick, the Mg²⁺ is shown as a purple sphere, and the coordinating water molecules are shown as blue spheres. Several of the important residues are displayed in ball-and-stick. (a) The Mg²⁺ is coordinated in an octahedral cage by one of the oxygen atoms of the β-phosphate of ADP, the hydroxyl oxygen of T104, and four waters. D241 forms hydrogen bonds with one of the water molecules coordinating Mg²⁺. The distance between Gly 97 of the P-loop and the Gly244 is 5.86 Å. (b) The mutated residue N104 has moved away from the Mg²⁺ and is no longer involved in its octahedral coordination. The oxygen atoms of D241 are directly stabilizing the Mg²⁺. The distance between Gly 97 of the P-loop and the Gly244 decreased to 5.75 Å. Dashed lines indicate hydrogen bonds. The figure were made using PyMOL ().

Kinesin-2 and IFT raft complex homologues localize to cytoplasmic regions of axonemes, as well as to external regions including flagellar tips and flagellar pores. In fixed Giardia trophozoites, both kinesin-2 GFP fusions (GiKIN2a and GiKIN2b) localized to cytoplasmic and membrane-bound regions of each of the eight axonemes, concentrating at distal flagellar tips (a–h; see live images in Supplementary Figure S3). In addition, both IFT complex A and B raft homologues (IFT140 and IFT81) localized to similar regions of the axonemes, though both show a pronounced localization to cytoplasmic regions of the posteriolateral axonemes, and the flagellar pores, where the transition from cytoplasmic to membrane-bound regions of the axonemes occurs (i–p). Note localization foci at the distal flagellar tips (arrowheads). The inset (panel l) depicts GFP foci on membrane-bound regions of the right caudal flagella. Red, anti-α-tubulin; green, GFP fusion; blue, DAPI. Scale bar, 2 μm.

Ultrastructure of cytoplasmic and membrane-bound regions of giardial axonemes. The giardial microtubule cytoskeleton is comprised of four main structures (panel a, schematic; panel b, anti-tubulin immunostaining). The eight flagellar axonemes are caudal (cfl), anterior (afl), posteriolateral (pfl), and ventral (vfl), the ventral adhesive disk (vd), the “funis” (fn), and the “median body” (mb; ). Basal bodies localize between the two nuclei, with the exception of the two anterior (afl) axoneme basal bodies (a and b). In panel c, transmission electron micrographs demonstrate the conserved ultrastructure of cytoplasmic portions of the axonemes. OD, outer doublet; CP, central pair; BB, basal body; and TZ, transition zone. Scale bar, 200 nm. In panel d, the basal body ultrastructure is presented in further detail. (e) The transition from the cytoplasmic to the membrane-bound regions of the caudal axonemes is shown and illustrates electron-dense material at the plasma membrane (pm). Scanning electron microscopy shows a “collar” region around the exit point of the axoneme (fp, flagellar pore) from the cell body in panel f.

The ectopic expression of the rigor GiKIN2aT104N mutant results in significantly shortened axonemes. The induced expression of the rigor kinesin-2 transgene (GiKIN2aT280N) acts as a dominant negative allele and results in significantly shorter flagella (d–f), than the uninduced control (a–c, and see quantitation of flagellar length in panel g). Axonemes were traced and labeled for ease of interpretation, and these schematics indicate the positions of the eight flagella arranged in four pairs: afl, anterior flagella; pfl, posteriolateral flagella; vfl, ventral flagella; and cfl, caudal flagella. Also, note the increased α-tubulin immunostaining that localizes at the distal tips of axonemes in these representative images of over 200 flagella measured (g). In panel g, various lengths of membrane-bound regions of flagella were quantified before and after induction of the dominant negative GiKIN2aT104N mutant for 24 h, using both anti-α-tubulin and anti-α14-giardin immunostaining (which stains external regions of axonemes). Statistically significant differences based on two-tailed Student t tests of induced and uninduced flagellar length measurements (from distal tips to the cell body; *p < 0.05. Error bars, SD). Numbers above axoneme type (afl, pfl, vfl, and cfl) denote the total number of axonemes measured for each treatment. Red, anti-α-tubulin; green, anti-α14-giardin; blue, DAPI. Scale bar, 2 μm.