Analyses of microtubule arrays in Giardia lamblia and fission yeast: Implications for the evolution of the cytoskeleton

Comparative analyses of evolutionarily diverse eukaryotes are essential for understanding cytoskeletal evolution. I used fluorescence microscopy and kymographic analysis to describe and quantify microtubule dynamics in living fission yeast cells expressing GFP:alpha-tubulin. I determined that the cytoplasmic microtubule bundles are composed of several individual microtubules that behave independently, with only one microtubule present at the cell tip at a given time. Cytoplasmic arrays are cell cycle regulated and microtubule growth is inhibited upon the transition to mitosis. During mitosis I detected a novel class of short, dynamic microtubules during spindle assembly, and documented the dynamic behavior of microtubule plus ends in mitotic spindles during anaphase. The cytoplasmic astral microtubules in anaphase have similar dynamics to interphase arrays. Finally spindle collapse occurs by depolymerization of individual microtubules alternating between each half spindle.; Although it is an evolutionarily basal eukaryote, the microtubule cytoskeleton of the binucleate parasite Giardia lamblia is complex. Because mitosis in Giardia had not been previously described, I used indirect immunofluorescence with antibodies against alpha-tubulin to visualize the microtubule arrays in Giardia and specifically to identify the mitotic spindle. Two independent spindles form a basket around each nucleus and elongate along the left-right axis of the cell. Transmission electron microscopy (TEM) was used to confirm that mitosis is semi-open, the spindle is extra-nuclear and microtubules enter the nucleus through openings in the nuclear membrane adjacent to the spindle poles. I also confirmed the presence of a single centromere locus on each chromosome through the localization of CENP-A:GFP. The essential kinesin BimC shows a conserved localization in Giardia spindles. Centrin, a conserved protein associated with basal bodies and centrosomes, localized to the flagellar basal bodies in interphase and the spindle poles during mitosis, linking segregation of chromosomes and flagella during mitosis.; Unlike Chlamydomonas, flagellar length in Giardia is affected by the anti-microtubule drugs taxol and nocodazole. The microtubule binding proteins EB1 and kinesin-13 also localize to flagellar tips and exit points from the cell body. These data support the balance point model for regulation of flagellar length and a different mechanism of regulation for the internal versus external axonemes.