Ancient Leishmania coronin (CRN12) is involved in microtubule remodeling during cytokinesis

Amogh A. Sahasrabuddhe 0 Ramesh C. Nayak 0 Chhitar M. Gupta 0 0 Division of Molecular and Structural Biology, Central Drug Research Institute , M.G. Marg, Lucknow 226001 , India - Summary In general, coronins play an important role in actin-based processes, and are expressed in a variety of eukaryotic cells, including Leishmania. Here, we show that Leishmania coronin preferentially distributes to the distal tip during cytokinesis, and interacts with microtubules through a microtubule-based motor, kinesin K39. We further show that reduction in coronin levels by 40-50% in heterozygous coronin mutants results in generation of bipolar cells (25-30%), specifically in the log phase, owing to unregulated growth of the corset microtubules. Further analysis of bipolar cells revealed that the main cause of generation of bipolar cell morphology is the intrusion of the persistently growing corset microtubules into the other daughter cell corset from the opposite direction. This defect in cytokinesis, e c n e i c S l l e C fo Introduction l Coronins represent an evolutionarily conserved family of WDa run reeupkeaartyoatcetsinf-rboimndyienagstptroohteuimnsanasn(dXaarveiewreidteally., 2e0x0p8re;sMseodrgaamnoanndg Jo Fernandez, 2008). Whereas higher eukaryotes express variable number of coronin isotypes (Uetrecht and Bear, 2006), only unique coronins are expressed in the lower eukaryotic organisms (Xavier et al., 2008). These proteins localize mainly at the sites where active actin-network remodeling takes place, such as leading edge, phagocytic-cup and immunological synapse (Gandhi and Goode, 2008; de Hostos, 2008; Nal et al., 2004). Using various genetic approaches, coronins have been shown to affect several actin-based cellular functions, such as cell locomotion, phagocytosis, macropinocytosis, cytokinesis and phagosome formation (Nagasaki et al., 2001; Rappleye et al., 1999; Bharathi et al., 2004; Yan et al., 2005). Apart from actin, coronins have also been reported to associate with microtubules. The purified coronin proteins from Saccharomyces cerevisiae (CRN11) and Drosophila melanogaster (CRN7) have been reported to bind microtubules and to crosslink microtubules and actin filaments (Goode et al., 1999). Genetic analyses have revealed that these proteins regulate microtubule-based cellular functions, such as nuclear migration and axonal guidance (Heil-Chapdelaine et al., 1998; Goode et al., 1999). These studies taken together indicate that coronins play an important role in actin and microtubule-based processes. However, despite the presence of these proteins in a number of lower eukaryotes, including Plasmodium falciparum (Tardieux et al., 1998), Trichomonas vaginalis (Bricheux et al., 2000), Babesia species (Figueroa et al., 2004), Acanthamoeba healyi (Baldo et al., 2005) and Leishmania donovani (Nayak et al., 2005), little is known about their functions in these organisms. however, disappears upon episomal gene complementation. Additionally, our attempts to prepare homozygous mutants were unsuccessful, as only the aneuploid cells survive the selection process. These results indicate that coronin regulates microtubule remodeling during Leishmania cytokinesis and is essentially required for survival of these parasites in culture. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/10/1691/DC1 Leishmania are an important group of flagellated kinetoplastid parasites that are transmitted to humans by the bite of sand fly. They cause a wide spectrum of human diseases, including Kalaazar (Desjeux, 2004). Unlike other eukaryotes, the cytoskeleton of these pathogens is marked by a dense microtubular corset that surrounds the entire cell body and defines their shape. However, the flagellar pocket and distal tip regions are devoid of the microtubular framework. The flagellar pocket is the only site that facilitates endocytosis, recycling of cell surface molecules and accumulation of several membrane-bound receptors (Bonhivers et al., 2008; Krishnamurthy et al., 2005; Hung et al., 2004). Although various cytoskeletal proteins become redistributed to the distal tip during cell division (Kratzerova et al., 2001; Gerald et al., 2007), no specialized functions have so far been attributed to this site. Previously, we identified a novel homolog of coronin in Leishmania species (Nayak et al., 2005), which belongs to the phylogenetically oldest clade of coronin proteins, and has recently been renamed into CRN12 (Morgan and Fernandez, 2008). We also reported that Leishmania CRN12 associates with filamentous actin in Leishmania promastigotes (Nayak et al., 2005; Kapoor et al., 2008), and is retained in the flagellar pocket region and in a few cells at the distal tip (Nayak et al., 2005). We have now further explored the intracellular distribution of Leishmania CRN12 in both the resting and dividing Leishmania cells. In addition, we have generated Leishmania CRN12 deletion mutants, and studied the effects of CRN12 depletion on the growth and cell division of the mutant cells. The results reported here indicate that Leishmania CRN12 plays a crucial role in corset-microtubule remodeling during cytokinesis. Results Leishmania CRN12 accumulates at the posterior ends in dividing cells and co-localizes with tubulin and kinesin K39 Our earlier studies have shown that CRN12 in detergent-treated Leishmania promastigotes is retained in the flagellar pocket region and also in a few cells at the posterior pole (Nayak et al., 2005). To explore this further, we analyzed the intracellular CRN12 distribution in both the resting and dividing Leishmania promastigotes. The analyses revealed that CRN12 was invariably concentrated at the posterior ends throughout cytokinesis until the final stage of separation of the daughter cells (Fig. 1B). However, in resting cells, CRN12 accumulation at the posterior end was not a regular feature as only about 27% (26.95.6%, n=1780) of these cells showed posterior accumulation (Fig. 1A). Interestingly, in the CRN12 accumulation zones, actin was only faintly stained. As posterior ends are rich in dynamic microtubules, we analyzed whether Leishmania CRN12 was present together with the Fig. 1. Intracellular distribution of CRN12 and actin in resting and dividing Leishmania promastigotes. (A) Immunofluorescence images showing colocalization of CRN12 and actin at most of the places and a predominant localization of CRN12 in the flagellar pocket region in the resting cells. (B) Immunofluorescence images showing localization of both CRN12 and actin in the dividing cells. All these cells show a characteristic pattern of CRN12 accumulation at both the flagellar end and posterior pole. Arrowheads indicate posterior pole accumulation of CRN12 in the dividing cells. Ph, phase; Trans, transmission; Coro, CRN12; Act, actin. Scale bars: 5 m. dynamic microtubules at these sites. The dynamic microtubules can be stained with tyrosinated -tubulin an (...truncated)