[Autophagy 5:2, 256-258; 16 February 2009]; ©2009 Landes Bioscience
Article Addendum
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Trypanosome TOR as a major regulator of cell growth and autophagy
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Antonio Barquilla and Miguel Navarro*
Instituto de Parasitología y Biomedicina “López-Neyra”; Consejo Superior de Investigaciones Científicas, CSIC; (Spanish National Research Council); Granada, Spain
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complexes or TORCs.2 This dual function of TOR kinases confers
the capability of governing spatial and temporal cell growth separately.3 TOR complex 1 (TORC1) controls temporal aspects of cell
growth through processes such as ribosome biogenesis, transcription,
translation and repression of autophagy, while TORC2 controls
spatial aspects of cell growth by actin cytoskeleton remodelling
(reviewed in refs. 4 and 5).
Recently, we described the growth of the highly proliferative
Trypanosoma brucei bloodstream form as being controlled by two
functional independent TOR kinases, TbTOR1 and TbTOR2.1
TbTOR1 and TbTOR2 bind to and control signaling through
TbTORC1 and TbTORC2, respectively. Whereas TbTOR2 regulates actin polarization towards the endocytic pathway, playing
a crucial role in endocytosis and cytokinesis, TbTOR1 function
promotes cell growth and proliferation by positively regulating
protein synthesis, cell size, cell cycle progression and RNA polymerase I localization to the nucleolus. All data suggested that overall
TOR functions were conserved in this early-branched eukaryote,
including the two distinct TOR complexes described in metazoans
and yeast. However, several new findings have emerged that differ
from those described for other eukaryotes. We found two additional
TOR-like proteins, TbTOR-like 1 and 2, with a similar domain
structure present in TOR proteins, although our results suggest
that they are not components of the classical TORC1 or TORC2.
Furthermore, rapamycin treatment of bloodstream trypanosomes
resulted in a pronounced reduction of cell proliferation but, interestingly, by inhibiting TORC2 signalling, rather than affecting TORC1
signalling as occurs in other eukaryotes. Thus, it is important to bear
in mind that rapamycin cannot be used for triggering the autophagic
process in T. brucei because TbTOR1 is rapamycin-insensitive.
Treatment of the T. brucei bloodstream form in the nanomolar range
inhibits TORC2, producing actin depolarization which interferes
with endocytosis and cytokinesis.1
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Trypanosomatid protozoa parasites are responsible for tropical
diseases, and undergo complex life cycles involving developmental
forms adapted to insect vectors and vertebrate hosts. During
their life cycle these parasites proceed through different forms in
response to dramatic environmental changes and/or developmentally regulated programs. Successful progression of the parasite
through its life cycle is highly dependent on the capacity of adaptation to distinct stresses involving processes such as autophagy.
In eukaryotes, target of rapamycin (TOR) protein kinases act as
a sensor, which integrates the nutritional and energetic status,
adjusting cell metabolism and growth. Compromising cell viability
in yeast and mammals leads to a reduction of TOR function, triggering processes aimed to overcome unfavorable conditions. This
is partly achieved by TOR-mediated regulation of protein synthesis
and recycling of cellular components by autophagy. In the last few
years, autophagy has been described during developmental differentiation processes in Trypanosomatidae. However, no link between
TOR signalling, autophagy, and differentiation has been described
so far. This addendum is a commentary to the work published by
our group,1 in which we discuss the possible role of TOR kinases,
as a controller of cell growth and autophagy, in the regulation of
differentiation processes during Trypanosomatids life cycles.
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Key words: Trypanosoma brucei, target of rapamycin, TORC1, developmental differentiation, autophagy
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Fitness of eukaryotic cells to variations in environmental or
metabolic cues is a crucial parameter for survival. The modulation
of cellular growth in response to nutritional or energetic status is
exerted through the regulation and coordination of a wide variety of
processes including metabolism, transcription and translation. There
is currently substantial work pointing to the target of rapamycin
kinase as being responsible for coupling nutrient availability to activation of anabolic processes leading to cell growth or, in contrast,
those processes leading to cell adaptation to stress situations. TOR
kinases function in two functionally and structurally multiprotein
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*Correspondence to: Miguel Navarro; Instituto de Parasitología y Biomedicina
“López-Neyra”; Consejo Superior de Investigaciones Científicas, CSIC; (Spanish
National Research Council); Avda. del Conocimiento s/n; Granada 18100 Spain;
Tel.: 34.958181651; Fax: 34.958181633; Email: [email protected]
Submitted: 12/05/08; Revised: 12/09/08; Accepted: 12/10/08
Previously published online as an Autophagy E-publication:
http://www.landesbioscience.com/journals/autophagy/article/7591
Addendum to: 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; PMID: 18796613; DOI: 10.1073/pnas.0802668105.
256
Autophagy is Triggered upon TbTOR1 Loss-of-Function
We asked whether TbTOR1 was involved in the regulation
of autophagy in this ancient eukaryote. To address this question,
cells depleted of TbTOR1 were examined by electron microscopy.
TbTOR1-depleted cells showed an increased number of doublemembrane structures containing cytoplasmic material, dilatation or
abnormal appearance of endoplasmic reticulum and membranous
cellular components that rearrange in membranous whorls called
myelin figures or multi-lamellar membrane (Fig. 1).1 This suggests
that an autophagic process takes place in the T. brucei bloodstream
Autophagy
2009; Vol. 5 Issue 2
Trypanosome TOR and autophagy
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form in situations where TbTOR1 (TORC1)
signaling is reduced, similar to what occurs in
other eukaryotes.
Characterization of autophagy requires, in
addition to ultrastructural analysis by electron
microscopy, further confirmation by a marker
such as the Atg8/LC3 protein. This marker
has been used successfully in the characterization of autophagy in the related organisms T.
cruzi and Leishmania.6,7 Unfortunately, on the
basis of our initial observations, the Atg8/LC3
T. brucei orthologues did not behave this way.
A yellow fluorescent protein fused with either
TbAtg8A or TbAtg8B (TcAtg8.1 orthologues)
did not show punctate distribution or cleavage
upon nutrient starvation or TbTOR1 depletion
in monomorphic bloodstream trypanosomes
(Barquilla A and Navarro M, unpublished data),
consistent with a previous report.8 Similarly, an
additional Atg8 orthologue, TcAtg8.2, did not
function as an autophagosome marker in T.
cruzi.6 Future work will be required to identify
an autophagosomal marker in T. brucei.
In other eukaryotes, TORC1 inhibition by
distinct stresses leads to an enhanced resistance
to unfavorable conditions. Accordingly, it is
reasonable that reduced protein synthesis and
activation of autophagy mediated by TORC1
inactivation would confer partial resistance to
unfavorable conditions in trypanosomatids. To
test whether TbTOR1 and TbRaptor (TORC1)- Figure 1. Ultrastructural (TEM) analysis of cellular structures (black arrows) reminiscent of autophagy
in the bloodstream form trypanosomes upon TbTOR1 RNA interference. (A) TbTOR1-depleted cells
depleted cells showed enhanced viability during contain abundant vesicles with a range of distinct morphologies similar to those of autophagic
stress situations, we cultured these cell lines and vacuoles (AV) in mammals and yeast. AV morphologies include double-membraned vesicles exhibitthe untransfected control without changing the ing heterogeneous intralumenal contents. A rare or dilated endoplasmic reticulum profile can also
medium, and followed the cell number daily be seen. (B) Vacuole containing membrane whorls. The punctate pattern of membranes resembles
(Fig. 2). Interestingly, TbTOR1- and TbRaptor- those of rough endoplasmic reticulum. (C) Detail of a double-membraned vacuole. (D) Multilamellar
bodies, another variant of AV, are also common upon TbTOR1 RNAi. Bars, 0.25 mm.
depleted cell lines, but not untransfected or
uninduced controls, exhibited enhanced resistance and survived for a longer period of time. At day 4, no living the vertebrate, and the procyclic and epimastigote form in the tsetse
cells were detected in controls, whereas induced RNAi cell lines fly. The preadapted nonproliferative stages are the stumpy bloodremained viable. This result suggests that reduction of TbTORC1 stream form in the vertebrate and metacyclic in the tsetse fly. It is
signalling confers partial resistance to stress situations, essential for tempting to speculate that TbTOR1 signalling may play an imporovercoming temporal unfavorable conditions.
tant role in these developmental transitions, as TOR inactivation
confers resistance to overcome unfavorable conditions.
Role of Autophagy during Developmental Differentiation in
During the last few years, autophagy has been characterized in life
Trypanosomatids
stages of Leishmania and T. cruzi characteristic of the invertebrate
6,7,11 In the insect vector, noninfective proliferating epimasTrypanosomatids exhibit extraordinarily complex life cycles host.
tigotes
differentiate to the infective metacyclic trypomastigotes by
involving an insect vector and a vertebrate host. Accomplishment
a
process
called metacyclogenesis, a process triggered by nutritional
of the life cycle requires accurate adaptation to distinct hosts and
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distinct compartments within the vector. Little is known about para- stress. It has been demonstrated that autophagy is triggered during
site adaptation to the drastic environmental changes encountered as metacyclogenesis, transition from the log phase to stationary phase of
the parasite traverses through the insect, which is accomplished by growth, and starvation periods. Moreover, a defect in the autophagic
modulating several processes, including metabolic and morpholog- process compromised cell viability upon these situations and interical readjustments.9 In addition, trypanosomatids undergo complex feres with the differentiation to the infective metacyclic form, a
developmentally-regulated differentiation processes as a mechanism crucial process for survival and infectivity in the mammalian host.7
for preadaptation to the successive host.10 The T. brucei life cycle Furthermore, during metacyclic to amastigote differentiation of L.
includes proliferation stages, the long-slender bloodstream form in mexicana in the mammalian host, autophagic processes also occur.11
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Autophagy
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Trypanosome TOR and autophagy
nitrogen-rich environment to a glucose-depleted and proline-rich
one. In this situation, it is conceivable that TbTOR1 activity may be
reduced as occurs in other eukaryotes, thus triggering an autophagic
process as occurs in mammals and yeast.15
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Acknowledgements
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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 Huges Medical Institute International Research Scholar
(HHMI-55005525).
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References
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Figure 2. Cells with reduced TORC1 signalling showed enhanced resistance to unfavorable conditions. Bloodstream form cell lines for inducible
TbTOR1 RNAi, TbRaptor RNAi and untransfected control were seeded at
2 x 105 cells per ml in order to achieve the maximum density (8–9 x 106
cells per ml) before cell cycle blockade occurs in induced RNAi cells. Cells
reached 2.5 x 106 cells per ml the first day, and 8–9 x 106 cells per ml in
the second, but cell density quickly drops afterwards. RNA interference was
induced by addition of doxycycline (1 μg/ml) to the cultures at day 0. (A)
Uninduced (DOX-) RNAi cell lines and the untransfected control quickly died
after reaching maximum density at day 3. No live cells were detected on
day 5. (B) Induced (DOX+) TbTOR1 and TbRaptor RNAi cell lines are more
resistant than the untransfected control. At day 4, cell density in RNAi cell
lines was approximately ten-fold higher than the untransfected control. At
day 5, RNAi cell lines were still viable, whereas no live cells were detected
in the untransfected control.
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1. 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.
2. Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, Bonenfant D, Oppliger W,
Jenoe P, Hall MN. Two TOR complexes, only one of which is rapamycin sensitive, have
distinct roles in cell growth control. Mol Cell 2002; 10:457-68.
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4. Martin DE, Hall MN. The expanding TOR signaling network. Curr Opin Cell Biol 2005;
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nutritional stress response and differentiation in Trypanosoma cruzi. J Biol Chem 2008;
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8. Herman M, Perez-Morga D, Schtickzelle N, Michels PA. Turnover of glycosomes during
life-cycle differentiation of Trypanosoma brucei. Autophagy 2008; 4:294-308.
9. Matthews KR, Ellis JR, Paterou A. Molecular regulation of the life cycle of African trypanosomes. Trends Parasitol 2004; 20:40-7.
10. Fenn K, Matthews KR. The cell biology of Trypanosoma brucei differentiation. Curr Opin
Microbiol 2007; 10:539-46.
11. Williams RA, Tetley L, Mottram JC, Coombs GH. Cysteine peptidases CPA and CPB
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13. Nolan DP, Rolin S, Rodriguez JR, Van Den Abbeele J, Pays E. Slender and stumpy bloodstream forms of Trypanosoma brucei display a differential response to extracellular acidic and
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During T. brucei infection, cells multiply in the bloodstream
where a constant and nutrient-rich milieu allows rapid proliferation
of the long-slender bloodstream form. Once parasitemia reaches
certain levels, trypanosoma differentiate to a nondividing short
stumpy bloodstream form by a quorum-sensing mechanism.10
Stumpy forms are metabolically preadapted for the drastic environmental change that occurs upon transmission to the fly’s midgut,
being more resistant to distinct stresses.13 When the insect ingests
the blood meal infected with trypanosomes, quiescent stumpy forms
are capable of differentiating rapidly and synchronously to procyclic
forms, activating cell cycle progression and establishing an effective
infection. Interestingly, Herman and coworkers verified in an elegant
study that glycosomes, peroxisome-like organelles required for
compartmentalization of enzymes involved in carbohydrate metabolism, are targeted for degradation by autophagy upon differentiation
programs in T. brucei.8 This autophagic process is triggered during
the differentiation from long-slender to short stumpy, but mainly
from short stumpy to procyclic forms. TOR function may have
a major role in this differentiation processes as occurs in fungi.14
Transition from the short stumpy bloodstream to the procyclic form
involves drastic changes in the source of energy from a glucose- and
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2009; Vol. 5 Issue 2