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[Cell Cycle 8:5, 697-699; 1 March 2009]; ©2009 Landes Bioscience Extra View Trypanosome TOR complex 2 functions in cytokinesis Antonio Barquilla and Miguel Navarro* IB UT E. Instituto de Parasitología y Biomedicina “López-Neyra”; Consejo Superior de Investigaciones Científicas; CSIC; (Spanish National Research Council); Avda. del Conocimiento s/n; Granada, Spain NO T DI other hand, the remodeling of cell morphology and extensive reorganization of cell components that occurs during the developmental differentiation processes requires a quiescent cell cycle in order to redistribute organelles efficiently. Growth and differentiation seem to have opposite effects on cell cycle progression and, therefore, both processes must be tightly coordinated and interconnected. In sum, regulation of cell growth, cell cycle progression and cytokinesis are highly controlled processes in trypanosomatids, although it is poorly understood.1 Target of rapamycin (TOR) is a serine/threonine kinase that couples nutrient availability to activation of processes that lead to cell growth.2,3 Basically, TOR responds positively to nutrients (especially amino acids) and activates and orchestrates ribosome production, translation initiation, and transcription to generate macromolecules, thus leading to cell mass accumulation. Metazoans seemed to be subjected to more complex regulation through the involvement of the insulin/insulin-like growth factor system, which couples TOR activation to energy levels, growth factors or variation in oxygen concentration in order to orchestrate overall body cell growth. In sum, TOR participates in the maintenance of energy and amino acid homeostasis. Upon nutrient starvation or energy depletion, TOR activity is inhibited, triggering processes aimed to maintain cellular viability and overcome unfavorable conditions, such as autophagy, the process of obtaining molecules and energy from recycling cellular components by direct degradation. We have recently shown that control of cell growth in Trypanosoma brucei is achieved by two functionally distinct TOR kinases through signaling by two distinct TOR complexes (TORC).4 TbTOR1, which localizes to the nucleus, regulates temporal aspects of cell growth by signaling through TORC1. In contrast, cytosolic TbTOR2 binds exclusively to TORC2 and regulates actin cytoskeleton remodeling, cell polarization and cytokinesis. We reported the potent trypanocidal effect of the specific TOR inhibitor rapamycin, which impairs cell proliferation. Rapamycin inhibition of T. brucei proliferation is exclusively mediated by prevention of TORC2 complex formation, while TORC1 is unaffected, which is the opposite of what occurs in other eukaryotes. Taking advantage of this unique feature, we have studied the rapamycin-mediated inhibition of TORC2 signaling in a context where TORC1 signaling is unaltered. TORC1 is not inhibited by rapamycin, thus protein synthesis is unaffected, which indicates that the dramatic morphological defects in cell polarization, endocytosis and cytokinesis are the results of selective TORC2 inhibition. Furthermore, we found other TOR-like proteins, TbTOR-like 1 OS CI EN CE .D O TOR (target of rapamycin) is a kinase of the phosphatidylinositol kinase-related kinase (PIKK) family that controls cell growth in eukaryotes in response to nutrients, energy conditions and growth factors. We have recently identified two trypanosome TOR orthologs, named TbTOR1 and TbTOR2, and two other proteins with significant homology to yeast or mammalian TORs, named TbTOR-like 1 and TbTOR-like 2. TbTOR1 depletion results in arrest of bloodstream trypanosomes in G1, concomitant to protein synthesis inhibition; however, TbTOR2 depletion leads to dramatic morphological defects in cell polarization, endocytosis and cytokinesis. Rapamycin inhibits T. brucei cell growth by prevention of TORC2 complex formation, without any effect on TORC1 contrary to what generally occurs in other eukaryotes. Based on the unique features of T. brucei and its distal position in the eukaryotic cell lineage, we describe our views on the function of the TOR protein as a major regulator of cell growth and cytokinesis and discuss a possible role in the developmental differentiation processes. ST R Key words: Trypanosoma brucei, TOR, cytokinesis, TORC2, developmental differentiation © 20 09 LA ND ES BI Cell cycle progression and cell polarization are intertwined processes in unicellular protozoan parasites. On one hand, highly polarized cell morphology and cell growth is a key aspect of trypanosome biology. Trypanosomes contain a number of single copy organelles that must be duplicated and correctly segregated to the progeny. The entire cell body is surrounded by a tight array of microtubules associated with the plasma membrane, except in the so-called flagellar pocket, an evagination of the plasma membrane where the flagellum emerges from the cell body. This is the only area where endo- and exocytosis can take place, thus determining the high degree of polarization required for the regulation of the trafficking network. Cytokinesis differs from the classical processes observed in mammals and yeast as a cleavage furrow forms along the longitudinal/helical axis of the vermiform parasite. Therefore, any defect in cell polarization impairs both correct cytokinesis and/ or endo/exocytosis processes, leading to non-viable progeny. On the *Correspondence to: Miguel Navarro; Consejo Superior de Investigaciones Cientificas CSIC; Molecular Biology; Av del Conocimiento s/n; PTCS; Armilla, Granada 18100 Spain; Tel.: 34.958181651; Fax: 34.958181633; Email: [email protected]. es Submitted: 12/22/08; Accepted: 01/09/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/7808 www.landesbioscience.com Cell Cycle 697 IB UT E. Trypanosome TORC2 in cytokinesis has been observed.4 Interestingly, by scanning electron microscopy, we detected cells containing two flagella pairs upon TbTOR2 depletion, suggesting that cytokinesis was not completed successfully when cells re-entered the next round of the cell cycle (Fig. 1B). Furthermore, an incomplete cleavage-furrow during this abnormal cytokinesis process and successive rounds of duplication of organelles results in aberrant cells containing various nuclei and flagella (Fig. 1C). Depletion of TbTOR2 also results in cells with severely inhibited cytokinesis (Fig. 1D). Little is known about the molecules that are directly involved in the regulation of the cytokinesis process in T. brucei.1 Cytokinesis does not occur via the constriction of an actomyosin ring, like in mammals and yeast cells. Actin polarization via TORC2 may play an important role in the movement and segregation of organelles or/and in the ingression of the cleavage furrow in bloodstream trypanosomes. However, actin depletion did not significantly alter cytokinesis in the procyclic form, while bloodstream trypanosomes were dramatically affected.8 Thus, an additional direct role for TbTORC2 in cytokinesis, independent of actin cytoskeleton remodeling, cannot be ruled out. Supporting this view, TOR proteins have been found in close association with the basal bodies in Chlamydomonas reindhardtii9 and the kinetoplast in T. brucei.4 Since actin polarization does not seem to be required in procyclic trypanosomes,8 functional characterization of TbTOR2 in the procyclic form would be informative and address whether TbTORC2 functions in cytokinesis by an additional mechanism independent of actin. TbTOR2 Regulates Endocytosis and Cytokinesis © 20 09 LA ND ES BI OS CI EN CE .D O A major conserved readout of TORC2 signaling in eukaryotes is the polarization of the actin cytoskeleton.5,6 Although little is known regarding the functional relevance of TOR-mediated actin cytoskeleton remodeling in mammals, it is essential for cell cycle-dependent actin cytoskeleton organization in yeast. Actin is highly polarized towards the endocytic pathway in bloodstream T. brucei and is essential for endocytosis. Actin-depleted cells showed reduced endocytosis, leading to the formation of an enlarged flagellar pocket, the so-called Big Eye phenotype. TORC2 loss-of-function experiments in T. brucei also produced depolarization of the actin cytoskeleton, which led to impaired endocytosis, showing that actin polarization by TORC2 is conserved in this ancient eukaryote and suggesting that this function was acquired very early in eukaryote evolution.4 Moreover, reduced TbTORC2 signaling produced defects in cytokinesis, resulting in trypanosome cells that were unable to segregate their organelles properly, increasing the number of cells containing multiple nuclei and kinetoplasts.4 The trypanosome cell cycle, which consists of the G1, S, G2 and M phases, possesses a well-coordinated kinetoplast (mitochondrial DNA) cycle in which segregation occurs prior to the onset of nuclear mitosis.7 Thus, cell cycle phases can be easily distinguished by a DNA-staining dye, such as DAPI, because mitochondrial DNA segregates earlier than daughter nuclei. Concomitant with mitochondrial DNA division, basal bodies and flagellum duplication occurs,7 providing excellent cell cycle markers for nuclear S phase. Because flagellum duplication occurs during the S phase, cells analyzed by scanning electron microscopy can be subdivided into cells with one flagella, which are cells in G1 phase (Fig. 1A, lower cell), and cells with two flagella, which are in the S/G2 phase of the cell cycle (Fig. 1A, upper cell). Later, cells with two flagella in G2 undergo nuclear mitosis, segregation of organelles, and finally cytokinesis leading to two daughter cells with a single flagellum. The cytokinesis process is triggered when the trypanosome cell is about to duplicate its organelles, which is followed by the ingression of a cleavage furrow that bisects the cell and the segregation of organelles, especially the basal bodies and the flagella. Finally, the abscission takes place, resulting in the separation of the two daughter cells. Importantly, kinetoplast and flagella separation are affected upon TbTOR2 RNAi, since an increased number of cells containing one kinetoplast with two nuclei NO T DI and TbTOR-like 2, with similar domain structure to that found in other TOR proteins, although our results suggest that they are not components of TORC1 or TORC2 signaling.4 ST R Figure 1. Scanning electron micrographs of Trypanosoma brucei upon TbTOR2 depletion results in cytokinesis defects. (A) Cells in G1 (lower cell) and in S-phase (upper cell). (B–D) Cells showing defects of the cytokinesis process upon TbTOR2 RNA interference, which leads to aberrant cells containing several co-joined cell bodies. Scale bar: 3 μm. 698 Rapamycin Inhibits TbTORC2 Formation Rapamycin is a potent trypanocidal agent that prevents cell proliferation by interfering with cytokinesis. Classical rapamycin action occurs through binding to TORC1, which affects TORC1 kinase activity and, in turn, inhibits cell growth, protein synthesis and cell cycle progression. In contrast, rapamycin treatment of the bloodstream form trypanosomes led to non-viable multinucleated multiflagellated cells. The effect of rapamycin on trypanosomes correlated with the effects of TbTOR2 and TbAVO3 depletion, which are both members of TORC2. Pulldown experiments confirmed that TORC1 was rapamycin insensitive because the non-conserved rapamycin-binding domain of TbTOR1, which is in not capable of binding to the inhibitory complex FKBP12rapamycin. This result led to the unexpected finding that rapamycin specifically inhibited TORC2 signaling in T. brucei, in contrast to what occurs in other eukaryotes. Furthermore, we demonstrated that TbFKBP12-rapamycin binds exclusively to free TbTOR2, Cell Cycle 2009; Vol. 8 Issue 5 Trypanosome TORC2 in cytokinesis IB UT E. ST R © 20 09 LA ND ES BI OS CI EN CE .D O TORC1 activity and cell growth are both prerequisites for proliferation in the long slender bloodstream form and are likely to occur in other proliferative stages found in the Tsetse as well. However, as important as proliferation, it is essential the adaptation of the parasite to drastic environmental changes when traverses through the Tsetse fly and transits from the insect to the mammalian host, and vice versa.11 Autophagic processes, regulated by TORC1 function in mammals and yeast, occur during differentiation processes in Leishmania, T. cruzi and T. brucei, and play an essential role during progression through the life cycle.12-14 Cell growth is an energetically expensive process and must be tightly regulated in order to maintain cell homeostasis. We have recently found that reduction in TbTORC1 signaling promotes cell resistance to unfavorable situations (Barquilla A and Navarro M, unpublished results). Therefore, situations that compromise cell viability might be accompanied by downregulation of TORC1 signaling, thus promoting temporal resistance to overcome critical conditions. The results of our study support this view as the quiescent state induced by TORC1 loss-offunction promotes cell survival in unfavorable conditions. Another feature of the inhibition of TORC1 signaling would be its role in the cell cycle regulation during differentiation processes carried out in distinct stages during the trypanosome life cycle. T. cruzi differentiation from the proliferative epimastigote to the infective metacyclic promastigote, a process called metacyclogenesis, is triggered by nutrient deprivation, a situation that compromises TORC1 signaling in mammals and yeast. Differentiation involves extensive morphological and biochemical remodeling, which is accompanied by a G1 cell cycle arrest. As mentioned before, cytokinesis is an exquisitely regulated process and cytoskeleton remodeling by TbTORC2 during differentiation processes may be crucial for the correct timing and completion. Thus, inhibition of cell cycle progression during differentiation processes may be essential in order to avoid abnormal cell division. DI Speculations on TOR Signaling During the Trypanosome Life Cycle References 1. Hammarton TC, Monnerat S, Mottram JC. Cytokinesis in trypanosomatids. Curr Opin Microbiol 2007; 10:520-7. 2. Sarbassov D, Ali SM, Sabatini DM. Growing roles for the mTOR pathway. Curr Opin Cell Biol 2005; 17:596-603. 3. Martin DE, Hall MN. The expanding TOR signaling network. Curr Opin Cell Biol 2005; 17:158-66. 4. Barquilla A, Crespo JL, Navarro M. Rapamycin inhibits trypanosome cell growth by preventing TOR complex 2 formation. Proc Natl Acad Sci USA 2008; 105:14579-84. 5. Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 2004; 6:1122-8. 6. Schmidt A, Kunz J, Hall MN. TOR2 is required for organization of the actin cytoskeleton in yeast. Proc Natl Acad Sci USA 1996; 93:13780-5. 7. Woodward R, Gull K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. JCS 1990; 95:49-57. 8. Garcia-Salcedo JA, Perez-Morga D, Gijon P, Dilbeck V, Pays E, Nolan DP. A differential role for actin during the life cycle of Trypanosoma brucei. EMBO J 2004; 23:780-9. 9. Diaz-Troya S, Florencio FJ, Crespo JL. Target of rapamycin and LST8 proteins associate with membranes from the endoplasmic reticulum in the unicellular green alga Chlamydomonas reinhardtii. Eukaryot Cell 2008; 7:212-22. 10. Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 2006; 22:159-68. 11. Fenn K, Matthews KR. The cell biology of Trypanosoma brucei differentiation. Curr Opin Microbiol 2007; 10:539-46. 12. Williams RA, Tetley L, Mottram JC, Coombs GH. Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana. Mol Microbiol 2006; 61:655-74. 13. Alvarez VE, Kosec G, Sant’Anna C, Turk V, Cazzulo JJ, Turk B. Autophagy is involved in nutritional stress response and differentiation in Trypanosoma cruzi. J Biol Chem 2008; 283:3454-64. 14. Herman M, Perez-Morga D, Schtickzelle N, Michels PA. Turnover of glycosomes during life-cycle differentiation of Trypanosoma brucei. Autophagy 2008; 4:294-308. NO T preventing the association with TORC2 partners and, thus, exclusively affecting the TbAVO3-TbTOR2 interaction, which is essential for TORC2 signaling. Sarbassov and colleagues observed that mTORC2 integrity was affected upon prolonged treatment with rapamycin, but this only occurred in some cell lines tested, with higher concentrations than those used for TORC1 inhibition and during prolonged incubation times.10 Since then, several authors questioned whether this was an indirect effect due to prolonged TORC1 inhibition. Both the functional independence of TbTOR proteins and TbTOR1 rapamycin insensitivity allowed us to demonstrate that TORC2 inhibition by rapamycin indeed occurs in the presence of functional TORC1 signaling, discarding a possible indirect effect due to prolonged TORC1 inhibition. Acknowledgements This work was funded by Spanish Ministry of Science Grant SAF2006-01763 and Red de Investigación de Centros de Enfermedades Tropicales Grant (RD06/0021/0010) M.N. is a Howard Hughes Medical Institute International Research Scholar (HHMI-55005525). www.landesbioscience.com Cell Cycle 699