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Trypanosome TOR complex 2 functions in cytokinesis
Antonio Barquilla and Miguel Navarro*
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Instituto de Parasitología y Biomedicina “López-Neyra”; Consejo Superior de Investigaciones Científicas; CSIC; (Spanish National Research Council); Avda. del Conocimiento
s/n; Granada, Spain
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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
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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.
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Key words: Trypanosoma brucei, TOR, cytokinesis, TORC2, developmental differentiation
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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].
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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
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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
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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
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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
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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.
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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
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Trypanosome TORC2 in cytokinesis
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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.
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Speculations on TOR Signaling During the Trypanosome
Life Cycle
References
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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).
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