Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis

Meredith S. Sagolla 0 Scott C. Dawson 0 1 Joel J. Mancuso 0 W. Zacheus Cande ) 0 0 345 LSA, Department of Molecular and Cell Biology, University of California Berkeley , Berkeley, CA 94720 , USA 1 Present address: Section of Microbiology, University of California Davis , Davis, CA 95616 , USA - In the binucleate parasite Giardia intestinalis, two diploid nuclei and essential cytoskeletal structures including eight flagella are duplicated and partitioned into two daughter cells during cell division. The mechanisms of mitosis and cytokinesis in the binucleate parasite Giardia are poorly resolved, yet have important implications for the maintenance of genetic heterozygosity. To articulate the mechanism of mitosis and the plane of cell division, we used three-dimensional deconvolution microscopy of each stage e of mitosis to monitor the spatial relationships of conserved icen cayntdoltohgeicsaplimndalrekeprosletso. tUhesimngitobtoicthspliignhdtl-esa,nthdetcreanntsrmo misseiroens c electron microscopy, we determined that Giardia has a llS semi-open mitosis with two extranuclear spindles that e access chromatin through polar openings in the nuclear fC membranes. In prophase, the nuclei migrate to the cell o midline, followed by lateral chromosome segregation in l a n r u o Introduction J Giardia intestinalis, a widespread zoonotic intestinal parasite, has two life-cycle stages: a binucleate, double diploid flagellate or trophozoite form that attaches to the intestinal microvilli, and an infectious cyst form that persists in the environment (Adam, 2001; Gillin et al., 1996). Over several million cases of malabsorptive diarrhea by giardiasis are estimated to occur annually worldwide, and waterborne outbreaks of giardiasis are frequent in areas where unfiltered waters are routinely contaminated. (Savioli et al., 2006). Although Giardia is well known in terms of disease (Adam, 2001; Savioli et al., 2006) and has received much recent attention due to evolutionary controversies (Baldauf, 2003; Baldauf et al., 2000; Best et al., 2004; Ciccarelli et al., 2006; Dacks et al., 2002; Graczyk, 2005; Knight, 2004; Sogin et al., 1989), there remains little knowledge of the assembly and division of the complex giardial cytoskeletal systems required for the life cycle of the parasite, including the mechanism of mitosis. Like all diplomonads, Giardia has two diploid nuclei (2n=10), which are both transcriptionally active and identical in DNA content (Adam et al., 1988; Kabnick and Peattie, 1990; Wiesehahn et al., 1984; Yu et al., 2002). In addition, Giardia is bilaterally symmetrical and possesses a complex and distinctive microtubule cytoskeleton that establishes anteriorposterior, left-right and dorsal-ventral polarity and, importantly, plays a major role in its virulence (Elmendorf et al., 2003). The microtubule cytoskeleton of the trophozoite, or anaphase. Taxol treatment results in lagging chromosomes and half-spindles. Our analysis supports a nuclear migration model of mitosis with lateral chromosome segregation in the left-right axis and cytokinesis along the longitudinal plane (perpendicular to the spindles), ensuring that each daughter inherits one copy of each parental nucleus with mirror image symmetry. Fluorescence in situ hybridization (FISH) to an episomal plasmid confirms that the nuclei remain separate and are inherited with mirror image symmetry. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/23/4889/DC1 intestinal form, is characterized by four main elements: eight flagellar axonemes and basal bodies, the ventral disc, the funis and the median body (Fig. 1A,B). Giardia is unique among diplomonads in that it possesses the ventral disc, a novel organelle composed of an overlapping spiral lamella of microtubules that mediates attachment to the intestinal microvilli (or a laboratory substrate), most likely by a suctionbased mechanism (Hansen et al., 2006). To complete cell division, the complex giardial cell must duplicate and partition both diploid nuclei as well as the multiple cytoskeletal structures. Thus, a detailed analysis of mitosis in Giardia at both the ultrastructural and molecular level is needed to resolve two intriguing questions. First, how does Giardia coordinate the division of two equivalent nuclei and a complex microtubule cytoskeleton? Second, how conserved is the mechanism of mitosis in a highly divergent and putatively early branching eukaryote? Understanding chromosome segregation in Giardia, however, has been hampered principally by the lack of cytological descriptions of intermediate stages of mitosis, including the inability to identify the mitotic spindle (Solari et al., 2003). Several prior studies have sought to identify the stages of mitosis using primarily light microscopy and chromatin staining, yet have not described a mitotic spindle (Crva and Nohynkova, 1992; Filice, 1952). Furthermore, recent debate concerning the mechanism of giardial cell division has lead to proposals of unconventional mechanisms of chromosome segregation, Fig. 1. Giardia has two equivalent nuclei and a complex microtubule cytoskeleton. (A) The microtubule cytoskeleton of the trophozoite is characterized by eight flagellar axonemes and basal bodies (afl, anterior; c, caudal; pfl, posterior; vfl, ventral), the ventral adhesive disc (vd), the funis (fn) and the median body (mb) (diagramed in A). All basal bodies are between the nuclei. (B) Anti-tubulin labeling reveals the microtubule arrays, including the eight flagella, and median body. The ventral disk is more weakly stained than the other structures. Although images of Giardia are frequently presented as two-dimensional projections in dorsal perspective as in A, describing cell division in Giardia requires analysis of cell morphology in three dimensions. (C) The cell is shown in 3D from a side angle. The spatial positions of the four nuclei following nuclear division are shown arrayed along the leftright (L-R) and dorsal-ventral (D-V) axes. Three possible planes of cytokinesis are represented in C; longitudinal [anterior-posterior (A-P)], transverse (L-R) and frontal (D-V). The dorsal-posterior nuclei are marked with asterisks in C,D. including the role of non-spindle microtubular organelles in cell division (Benchimol, 2004b; Solari et al., 2003) or of ce the presence of multiple or incongruent planes of division n (Benchimol, 2004a; Ghosh et al., 2001; Yu et al., 2002). e ic Owing to the cytoskeletal complexity of the binucleate lS Giardia cell, understanding the mechanism of mitosis and cell le division requires the use of three-dimensional (3D) imaging C and monitoring of crucial aspects of the spindle such as fo the spatial orientation of spindle poles and kinetochore la attachments of chromosomes (at the centromere) to the spindle. rn Centrin, a ubiquitous protein found in association with basal u bodies and centrioles, is located with the centrioles at the Jo spindl (...truncated)