Extra-glycosomal localisation of Trypanosoma brucei hexokinase 2.

Abstract

The majority of the glycolytic enzymes in the African trypanosome are compartmentalised within peroxisome-like organelles, the glycosomes. Polypeptides harbouring peroxisomal targeting sequences (PTS type 1 or 2) are targeted to these organelles. This targeting is essential to parasite viability, as compartmentalisation of glycolytic enzymes prevents unregulated ATP-dependent phosphorylation of intermediate metabolites. Here, we report the surprising extra-glycosomal localisation of a PTS-2 bearing trypanosomal hexokinase, TbHK2. In bloodstream form parasites, the protein localises to both glycosomes and to the flagellum. Evidence for this includes fractionation and immunofluorescence studies using antisera generated against the authentic protein as well as detection of epitope-tagged recombinant versions of the protein. In the insect stage parasite, distribution is different, with the polypeptide localised to glycosomes and proximal to the basal bodies. The function of the extra-glycosomal protein remains unclear. While its association with the basal body suggests that it may have a role in locomotion in the insect stage parasite, no detectable defect in directional motility or velocity of cell movement were observed for TbHK2-deficient cells, suggesting that the protein may have a different function in the cell.

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Extra-glycosomal localisation of Trypanosoma brucei hexokinase 2
April C. Joicea, Todd L. Lydaa,1, Andrew C. Saycea,2, Emilie Verplaetseb, Meredith T. Morrisa,
Paul A.M. Michelsb, Derrick R. Robinsonc, James C. Morrisa,⇑
aDepartment of Genetics and Biochemistry, Clemson University, Clemson, SC, USA
bResearch Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Université catholique de Louvain, Brussels, Belgium
cCNRS, University of Bordeaux 2, Bordeaux, France
a r t i c l e i n f o
Article history:
Received 6 January 2012
Received in revised form 16 February 2012
Accepted 20 February 2012
Available online 14 March 2012
Keywords:
Trypanosoma brucei
Hexokinase
Flagellum
Glycosome
Glycolysis
a b s t r a c t
The majority of the glycolytic enzymes in the African trypanosome are compartmentalised within perox-
isome-like organelles, the glycosomes. Polypeptides harbouring peroxisomal targeting sequences (PTS
type 1 or 2) are targeted to these organelles. This targeting is essential to parasite viability, as compart-
mentalisation of glycolytic enzymes prevents unregulated ATP-dependent phosphorylation of intermedi-
ate metabolites. Here, we report the surprising extra-glycosomal localisation of a PTS-2 bearing
trypanosomal hexokinase, TbHK2. In bloodstream form parasites, the protein localises to both glyco-
somes and to the flagellum. Evidence for this includes fractionation and immunofluorescence studies
using antisera generated against the authentic protein as well as detection of epitope-tagged recombi-
nant versions of the protein. In the insect stage parasite, distribution is different, with the polypeptide
localised to glycosomes and proximal to the basal bodies. The function of the extra-glycosomal protein
remains unclear. While its association with the basal body suggests that it may have a role in locomotion
in the insect stage parasite, no detectable defect in directional motility or velocity of cell movement were
observed for TbHK2-deficient cells, suggesting that the protein may have a different function in the cell.
? 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction
In the African trypanosome, Trypanosoma brucei, glycolytic en-
zymes have been localised to peroxisome-like organelles called
glycosomes (Opperdoes and Borst, 1977). This organisation, which
is thought to serve as a means of regulating the pathway (Haanstra
et al., 2008), is unusual as glycolysis is a cytosolic process in most
cells. Even in systems with cytosolic glycolysis, glycolytic enzymes
have been found in subcellular compartments distinct from the
cytosol, although the function of these proteins in the alternative
compartments may not be in the glycolytic pathway. These addi-
tional localisations include the sarcoplasmic reticulum in rabbit
muscle tissue (Xu and Becker, 1998), chloroplasts (reviewed in
Lunn (2007)), the Toxoplasma gondii apicoplast (Fleige et al.,
2007), and sometimes (in some protists) in mitochondria (Liaud
et al., 2000; Kroth et al., 2008). Glycolytic enzymes have also been
found in the nucleus where, for example, GAPDH is involved in
DNA repair or on the cell surface (e.g., aldolase serves as a plasmin-
ogen receptor in many pathogenic microorganisms) (Kim and
Dang, 2005; Avilan et al., 2011).
A number of species localise glycolytic and other metabolic
enzymes near the flagella. Chlamydomonas reinhardtii has three
glycolytic enzymes (phosphoglycerate mutase, enolase and pyru-
vate kinase) associated with the flagellum to produce ATP (Mitchell
et al., 2005). In mammals, a hexokinase (HK1) has been found at-
tached to the fibrous sheath that surrounds the axoneme and outer
dense fibres of sperm flagellum, suggesting a role in extra-
mitochondrial energy production (Travis et al., 1998; Miki et al.,
2004; Nakamura et al., 2008).
Kinetoplastid metabolic enzymes have been found proximal to
the flagellum. For example, three isoforms of adenylate kinase
localise to either the flagellar axoneme or paraflagellar rod (PFR)
via an N-terminal extension in the proteins (Ginger et al., 2005).
In Leishmania, a HK (which was characterised as a haemoglobin
receptor) has been localised to the flagellar pocket, suggesting that
it may serve multiple functions depending upon localisation
(Krishnamurthy et al., 2005).
The T. brucei genome encodes two 98% identical HK polypep-
tides (TbHK1 and TbHK2) that are expressed in both bloodstream
form (BSF) and insect stage (procyclic stage, PF) parasites. These
proteins form hexamers that in vitro have distinct biochemical
0020-7519/$36.00 ? 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijpara.2012.02.008
⇑Corresponding author. Address: Department of Genetics and Biochemistry,
Clemson University, 214 BRC, 51 New Cherry Street, Clemson, SC 39634, USA. Tel.:
+1 864 656 0293; fax: +1 864 656 0393.
E-mail address: jmorri2@clemson.edu (J.C. Morris).
1Current address: Laboratory of Parasitic Diseases, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, 9000 Rockville Pike, Building 4,
Room B1-16, Bethesda, MD 208920-0425, USA.
2Current address: Oxford Glycobiology Institute, Department of Biochemistry,
University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
International Journal for Parasitology 42 (2012) 401–409
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International Journal for Parasitology
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Page 2

properties depending on the ratio of TbHK1 and TbHK2 included in
the oligomers (Chambers et al., 2008b). Proteomic analysis of puri-
fied glycosomes has revealed that both proteins are expressed in
BSF and PF glycosomes (Colasante et al., 2006). While the function
of these polypeptides is currently unresolved, genetics-based stud-
ies have confirmed that both are essential to BSF parasites (Albert
et al., 2005; Chambers et al., 2008a).
The glycosomal localisation of the TbHKs has been attributed to
the presence of a N-terminal peroxisomal targeting sequence
(PTS2), as this sequence has been shown to be responsible for
the import of other glycosomally-targeted proteins (Blattner
et al., 1995). Here, we report the unexpected life cycle-dependent
dual localisation of TbHK2. In BSF parasites, TbHK2 localises to gly-
cosomes and the flagellum, while in PF parasites TbHK2 localises to
glycosomes and regions proximal to basal bodies.
2. Materials and methods
2.1. Subcellular fractionation of trypanosomes
Subcellular fractionations were performed using BSF and PF line
449 of Trypanosoma brucei brucei (a genetically modified cell line
derived from strain Lister 427) (Biebinger et al., 1997). BSF para-
sites were cultured in HMI-9 medium containing 10% heat-inacti-
vated FBS (Invitrogen, USA) at 37 ?C under water-saturated air with
5% CO2. PF trypanosomes were grown in SDM-79 medium (Brun
and Shonenberger, 1979) supplemented with 15% FBS at 28 ?C with
5% CO2. Cultures were always harvested prior to entering the sta-
tionary phase, i.e., at densities lower than 2 ? 106cells/mL for BSFs
or 2 ? 107cells/mL for procyclic cells, by centrifugation at 700g for
10 min.
Fractionation of cell compartments was performed using
increasing concentrations of digitonin as follows: BSF parasites
(108cells) and procyclic trypanosomes (2 ? 108cells) were washed
twice in ice-cold buffer (25 mM HEPES, pH 7.4, 250 mM sucrose
and 1 mM EDTA) and then resuspended in 0.5 mL of the same buf-
fer. The cell suspension was divided with each aliquot containing
?100 lg of protein. HBSS buffer (Gibco, USA) was added to adjust
aliquot volume to 100 lL. Digitonin dissolved in dimethylformam-
ide was then added followed by incubation (4 min at room temper-
ature, RT). Untreated cells and those completely permeabilised
(total release, the result of incubation in 0.5% Triton X-100) were
used for comparison. After centrifugation of the suspensions
(12,000g for 2 min), the supernatant (released fraction) was probed
by western blotting for cytosolic or glycosomal resident proteins.
Western blotting was performed on samples resolved by 12%
SDS–PAGE followed by transfer to a nitrocellulose support. The
membrane was incubated in block (1% non-fat milk, 10 mM Tris–
Cl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) and probed with appro-
priate antibodies. These included: rabbit polyclonal antisera raised
against T. brucei pyruvate kinase (aPYK, antiserum used at a dilu-
tion of 1:100,000), T. brucei hexokinase (aTbHK, at 1:100,000), T.
brucei enolase (aENO, at 1:150,000), or T. brucei glycerol kinase
(aGK, at 1:100,000) as well as anti-haemagglutinin (aHA,
1:1,000, Rockland Immunochemicals, Gilbertsville PA, USA). Pri-
mary antibodies were detected with anti-rabbit IgG conjugated
to horseradish peroxidase (Rockland Immunochemicals) and were
visualised using the ECL Western Blotting System (Pierce, USA).
2.2. Immunofluorescence microscopy on whole cell and cytoskeletal
preparations
Immunofluorescence (IF) assays were performed using a proto-
col modified from Field et al. (2004). In short, parasites were har-
vested by centrifugation (800g, 5 min), washed with ice-cold
Voorheis’s modified PBS (vPBS; 137 mM NaCl, 3 mM KCl, 16 mM
Na2HPO4, 3 mM KH2PO4, 46 mM sucrose, 10 mM glucose, pH 7.6),
and then fixed (10 min BSF; 1 h PF) in an equal volume of 6% para-
formaldehyde and vPBS on ice. Cells were washed with vPBS, al-
lowed to settle on poly-lysine slides, and then permeabilised
with 0.1% Triton X-100 in PBS (137 mM NaCl, 3 mM KCl, 16 mM
Na2HPO4, 3 mM KH2PO4) for 10 min. After washing in PBS, block
(1% BSA and 0.25% Tween in PBS) was added (1 h, RT), followed
by addition of the appropriate primary antibody. For localisation
of TbHKs,aTbHK (1:500) was used. For localisation of TbHK2, affin-
ity-purified TbHK2 polyclonal antisera (aTbHK2, 1:1 or 1:10 for
BSF; 1:100 for PF), which was raised against a peptide correspond-
ing to the C-terminal end of TbHK2 (CGVGAALISAIVADGK), was
used (Morris et al., 2006). Endogenously tagged TbHK2 was local-
ised using an affinity purified amyc polyclonal antibody (1:50,
Rockland Immunochemicals, Gilbertsville PA, USA), while HA-
tagged polypeptides were localised with an aHA antibody (1:100,
Rockland Immunochemicals). The monoclonal antibody Mab25
was used to detect the axoneme (Absalon et al., 2007), and
Mab22 was used to detect the tripartite attachment complex
(TAC) fibres of the basal bodies (Bonhivers et al., 2008). Primary
antibodies were detected with either FITC- or TexasRed-conju-
gated goat anti-mouse or goat anti-rabbit (1:100, Rockland Immu-
nochemicals) and visualised on a Zeiss Axiovert 200M using
Axiovision software version 4.6.3 for image analysis.
Cytoskeletons were isolated from cells washed in vPBS followed
by incubation for 10 min with 0.5% Triton X-100 in MME (10 mM
MOPS (3-(N-morpholino) propane sulfonic acid), 2 mM EGTA and
1 mM MgSO4) buffer as previously described (Robinson et al.,
1991). Samples were washed with vPBS, allowed to settle on
poly-lysine coated slides and visualised as previously described
(Robinson et al., 1991).
2.3. Expression of tagged TbHK2
To further explore localisation, a constitutively expressed HA-
tagged version of TbHK2 was generated using a pXS6 plasmid for
expression in trypanosomes. This vector drives expression with
the T. brucei rRNA promoter, typically yielding robust expression
(Alexander et al., 2002). For cloning into this vector, TbHK2 was
amplified to yield a product with appropriate restriction sites for
ligation.
In order to generate an endogenously tagged TbHK2, a linear-
ised construct containing the 30end of the TbHK2 open reading
frame (ORF) fused to a myc epitope, an abtubulin linker, the phleo-
mycin resistance gene, and the 30untranslated region of TbHK2 was
generated by PCR. Both constructs were used to transfect BSF strain
Lister 427 cells (2.5 ? 107cells) using the Amaxa Human T Cell
Nucleofector Kit (Lonza, Basel, Switzerland) as previously de-
scribed (Burkard et al., 2007). For the PF transfections, linearised
constructs were introduced by electroporation as previously de-
scribed (Wang et al., 2000).
2.4. Motility assays
For motility assays, cells were imaged at maximum capture
speed using the Zeiss AxioCam MRm camera and a Zeiss Axiovert
200M inverted microscope equipped with a 20? differential inter-
ference contrast (DIC) objective (Carl Zeiss MicroImaging, Inc.,
Thornwood, NY, USA) for 30 s. Enough fields were monitored so
that at least 200 traces of individual cells could be generated yield-
ing at least 10 productively motile cell traces available for analysis
for each cell line. The position of the anterior end of each produc-
tively motile cell was plotted on each frame and the distance be-
tween each of these anterior positions was used to determine the
incremental distance travelled. For each cell, total distance trav-
402
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elled was calculated by summing the incremental distances, and
effective distance travelled was calculated by measuring the dis-
tance between the initial and final position of the anterior end of
the cell. The directional motility coefficient of each productively
motile cell was calculated as:
DMC ¼deffective
dtravelled
where dtravelledis defined as the sum of all incremental distances
travelled for a given cell and deffectiveis defined as the linear distance
between the initial and final position of the anterior end of the same
cell.
All statistical analyses were performed using the KaleidaGraph
4.03 software package (Synergy Software, Reading, PA, USA). A
box and whisker plot of incremental speed for each cell line was
constructed where the 25th percentile, median and 75th percentile
are represented. The limit of each whisker is defined by the small-
est or largest value observed within a distance of 1.5 times the
interquartile range from the end of each quartile with observations
beyond this range indicated by open circles. DMC values generated
for each cell line were used to generate a dot plot which was over-
laid with a percentile plot with lines indicating the 5th percentile,
25th percentile, median, 75th percentile and 95th percentile. Sta-
tistical significance of differences in cell motility and directionality
were determined using a Wilcoxon–Mann–Whitney rank sum test
for unpaired data. The shape of the DMC distributions was assessed
by calculation of skewness and kurtosis. Briefly, for a given distri-
bution, skewness indicates the relationship of the mean and med-
ian values such that a skewness of zero indicates equality of the
values while negative skewness indicates a median greater than
the mean and positive skewness indicates a median less than the
mean. For the same distribution, kurtosis can be calculated as a
measure of the source of the variance. Leptokurtic distributions
(represented by positive kurtosis values) are centrally clustered
with a few extreme values contributing the majority of observed
variance, while the variance of platykurtic distributions (negative
kurtosis) is derived from many values.
3. Results
3.1. Fractionation studies of BSF parasites suggest extra-glycosomal
TbHK localisation
Initial studies on the organisation of glycolytic enzymes in T.
brucei revealed that most TbHK activity was associated with glyco-
somes (Misset et al., 1986). Following completion of genome
sequencing, it was recognised that T. brucei harbours two 98% iden-
tical HK genes (TbHK1 and TbHK2) that are predicted to yield poly-
peptides with N-terminal peroxisomal targeting sequences (PTS2)
that would localise expressed proteins to glycosomes. Indeed, pro-
teomic analysis of glycosomes has confirmed that both polypep-
tides are components of the organelles in PF and BSF parasites
(Colasante et al., 2006).
Earlier fractionation studies that scored TbHK activity may have
described only the compartmentalisation of TbHK1, as the function
(and activity) of TbHK2 in vivo is not clear. In vitro, recombinant
TbHK1 has HK activity while recombinant TbHK2 lacks detectable
enzyme activity, behaviour that has been experimentally attrib-
uted to protein sequence difference in the C-termini of the proteins
(Morris et al., 2006). To further explore TbHK distribution, parasite
lysates were fractionated and analysed by western blotting using
antibodies to glycosomal-or cytosolic-resident proteins. These in-
cluded an antiserum raised against native TbHK purified from
BSF trypanosomes that is likely a mixture of both TbHK1 and
TbHK2 (aTbHK) (Misset et al., 1986) (Fig. 1A). Interestingly, TbHK
was released at ?0.1 mg digitonin/mg protein, a concentration that
was found to liberate cytosolic proteins such as enolase (ENO) and
pyruvate kinase (PYK) but not the glycosome-resident protein
glycerol kinase (GK). To release GK, ?0.5 mg digitonin/mg protein
was required, a value consistent with the reported concentration
required for compromising the glycosomal membrane (Hannaert
et al., 2003).
The extra-glycosomal distribution of the TbHK in the fraction-
ation warranted further investigation to identify the compart-
ment(s) that the proteins occupy. Using IF microscopy to
resolve localisation, the aTbHK serum yielded a largely punctate
signal consistent with the expected glycosomal localisation of the
TbHKs (Fig. 1B). These apparently conflicting results led us to
consider distribution of the individual TbHKs, with a focus on
TbHK2.
3.2. IF microscopy using TbHK2-specific antisera reveals flagellar
localisation in BSF parasites
Because the aTbHK antisera likely detects both TbHK1 and
TbHK2, an affinity-purified antibody generated against the C-ter-
minal tail of TbHK2 (aTbHK2) was used to resolve the contribution
of TbHK2 to the unanticipated fractionation results. (Note: the dif-
ferent affinity-pure antisera used lack sufficient sensitivity for use
in the western blots used in the fractionation studies (Morris et al.,
2006)). By IF microscopy using the aTbHK2 antisera, TbHK2 local-
ised to the flagellum in BSF parasites (Fig. 2A), with faint labelling
of putative glycosomes. These findings were reproducible using
different blocking schemes (either 20% FBS or 1% BSA supple-
mented with 0.25% Tween-20) or antisera raised in different organ-
isms (mice and rabbit, Fig. 2A a and b, respectively). While flagellar
staining was intense, glycosomal staining was not as readily obvi-
A
B
PYK
TbHK
ENO
GK
0 0.01 0.05 0.1 0.25 0.5 0.75 1 T.R
mg dig/mg BSF protein
DAPI TbHK
Axoneme Merge
Whole BSF Cells
a
b
c d
Fig. 1. Subcellular distribution of glycolytic enzymes in bloodstream form (BSF)
Trypanosoma brucei brucei parasites analysed by digitonin fractionation and
immunofluorescence (IF) microscopy. (A) BSF trypanosomes were incubated with
increasing concentrations of digitonin and release of enolase (ENO), glycerol kinase
(GK), hexokinase (TbHK) and pyruvate kinase (PYK) monitored by western blots
using enzyme-specific antisera including an aTbHK antiserum. Total release (T.R.)
lanes correspond to cells incubated with 0.5% Triton X-100 to permeabilise all
membranes. (B) Fixed BSF parasites were visualised by IF microscopy using an
aTbHK antiserum (b) and a monoclonal antibody to the axoneme (c). DAPI (a) was
added with anti-fade reagent to stain the nucleus and kinetoplast DNA (kDNA) in all
samples. All images are an extended focus produced from Z-stack layers. Scale
bar = 5 lm.
A.C. Joice et al./International Journal for Parasitology 42 (2012) 401–409
403

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ous, although a single image from a Z stack layer revealed punctate
staining consistent with glycosomal staining (Fig. 2A, d white ar-
rows). Additional experimental controls were pursued to confirm
the specificity of the antisera. For example, flagellar signals were
eliminated when the primary antibodies were incubated with the
peptide used to generate the antibody (prior to addition of the
sample) (Fig. 2A e and f).
We speculated that the discrepancies between the two IF as-
says (using aTbHK, Fig. 1B, versus aTbHK2 antisera, Fig. 2A) could
be the result of the faint extra-glycosomal signal in the former
experiment being obscured by the intensity of the signal emanat-
ing from reactions with both TbHK1 and TbHK2 in the glyco-
somes. In order to eliminate the signal resulting from the TbHK
(namely TbHK1) that is highly expressed in the BSF glycosomes,
cells were fractionated to enrich for cytoskeletal components. This
procedure eliminates glycosome-resident proteins and was fol-
lowed by IF microscopy using aTbHK. Under these conditions,
TbHK co-localised with the axoneme (Fig. 2B), indicating that both
sera react with an antigen found to be associated with the
flagellum.
3.3. Flagellar localisation of tagged TbHK2
While three different antisera (mouse and rabbit aTbHK2 and
rabbit aTbHK) indicated that TbHK2 localised to the flagellum,
these observations could be the result of the three antisera cross-
reacting with a flagellar antigen that is immunologically similar
to the TbHKs. To rule out this possibility, recombinant TbHK2 bear-
ing an antigenic tag was expressed in the parasites and localised.
First, a TbHK2 chimera bearing a C-terminal HA tag was expressed
from an integrated copy of the constitutively expressed vector
pXS6:HK2HA. As with the aTbHK, the over-expressed TbHK2:HA
localised predominantly to glycosomes (Supplementary Fig. S1A).
Longer integration time revealed flagellar staining (white arrows),
again suggesting that the intense glycosomal foci that result from
over-expression were masking the less intense flagellar signal.
Supporting this possibility, cytoskeletal preparations demon-
strated that TbHK2:HA co-localised with an axonemal component
(Supplementary Fig. S1B).
Because over-expression could potentially alter localisation we
next introduced, through allelic exchange, an endogenous tag
(myc) in frame with the 30end of the TbHK2 ORF. IF microscopy
using anamyc mAb to probe TbHK2 localisation in the endogenous
tag cell line yielded findings consistent with those from the
aTbHK2 antibody, with co-localisation of TbHK2:myc and the axo-
nemal-reactive antibody (Fig. 3).
3.4. Lifecycle stage differences in the distribution of TbHK2
PF parasites differ from BSF parasites in a number of ways,
including having the capacity to use both glycolysis and amino
acid metabolism for ATP production. Digitonin fractionation of
PF parasites followed by western blotting using the pan-specific
aTbHK revealed a TbHK distribution distinct from BSF parasites,
with signal limited to fractions requiring ?0.5 mg digitonin/mg
protein for release (Fig. 4A), consistent with the concentration re-
quired for compromising the glycosomal membrane (Hannaert
et al., 2003).
To confirm the difference in distribution of the TbHKs in PF par-
asites, fixed parasites and PF cytoskeletal preparations were
probed with the aTbHK antisera (Fig. 4B). In whole cells, the distri-
bution of signal is punctate, consistent with glycosomal localisa-
tion. Unlike the BSF parasites, extraction of the cytoskeleton did
not yield flagellar-associated signal.
Probing fixed parasites and cytoskeletal preparations with the
aTbHK2 antisera again yielded localisation that differed from the
BSF parasites. In whole cells, signal was detected in foci, consis-
tent with glycosomes in PF parasites. However, two intense areas
of staining proximal to the mtDNA (the kDNA) were unantici-
pated. When focusing on a basal body marker (which partially
obscures the glycosomal staining), there was close association
of the TbHK2 signal with the basal body marker (Fig. 5B). In this
case, cells harbouring newly divided kDNA have both basal
bodies and TbHK2 associated with the new structure. Probing
cytoskeletal preparations with both the TbHK2 antisera and the
basal body mAb suggested that the signals were very close to
one another.
Using PF parasites expressing an endogenously myc-tagged
TbHK2, we corroborated these findings. First, distribution of the
myc signal in whole cells differs from what was found with BSF
parasites, with no detectable flagellar labelling but foci consistent
with glycosomes evident in fixed parasites (Fig. 6A). Extraction of
cytoskeletons followed by IF microscopy using basal body-specific
antisera indicated close localisation of the anti-myc signal and the
basal body (Fig. 6B.)
Fig. 2. Immunofluorescence (IF) microscopy of bloodstream form (BSF) Trypano-
soma brucei brucei parasites reveals localisation proximal to the flagellum. (A) IF
microscopy was performed using affinity-purified polyclonal sera raised against the
C-terminus of T. brucei hexokinase 2 (TbHK2) aTbHK2 antisera, from mouse (a) or
rabbit (b) detected with a species-specific FITC conjugated secondary antibody. A Z-
stack layer of aTbHK2 antisera stained BSF 90-13 cells (d) and the extended focus
image that included the stack (c) harbour glycosome-like bodies (arrows). Pre-
incubation of aTbHK2 antisera with peptide (4 ?C, 1 h) reduced the staining pattern
in BSF parasites (e and f). (B) Analyses of two examples of cytoskeletal preparations
by IF microscopy usingaTbHK antisera (a and d) and axoneme (b and e) monoclonal
antibodies. Scale bar = 5 lm.
404
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3.5. TbHK2?/?PF parasites are as motile and directional as the
parental parasites
The role of extra-glycosomal TbHK2 is not clear but its extra-
glycosomal localisation to either the flagellum or the basal bodies
suggested that the protein could have some role in the biology of
the flagellum. While TbHK2 is essential to BSF parasites (limiting
our ability to score the impact of knockout on this life-cycle stage),
PF cells lacking TbHK2 have been generated (Morris et al., 2006).
The PF TbHK2-deficient cells are slightly larger, have increased cel-
lular HK activity and have a modest delay in doubling time (Morris
et al., 2006). The new finding of TbHK2 proximal to the base of the
DAPI Myc Axoneme Merge
BSF 427
Endogenously
Tagged BSF
a b c d
e f g h
i j k l
Fig. 3. Immunofluorescence (IF) microscopy of endogenously tagged Trypanosoma brucei hexokinase 2 (TbHK2) bloodstream form (BSF) parasites. BSF strain Lister 427
parasites expressing TbHK2 bearing a myc tag from an authentic allele probed for both myc (b, f, and j) and axoneme (c, g, and k) antigens. DAPI (a, e, and i) signal was also
included for all samples. The white arrows point out areas of co-localisation. All images are an extended focus produced from Z-stack layers. Scale bars = 5 lm.
Fig. 4. Subcellular distribution of glycolytic enzymes in procyclic form (PF) Trypanosoma brucei brucei parasites suggest altered extra-glycosomal localisation of T. brucei
hexokinase 2 (TbHK2). (A) Enolase (ENO), glycerol kinase (GK), and hexokinase (TbHK) release was monitored by western blotting using aENO, aGK and aTbHK antisera,
respectively, after treating PF 29-13 trypanosomes with increasing digitonin concentrations. (B) Fixed parasites (a–d) and cytoskeletons (e–g) were stained with aTbHK
antiserum (b and e) and either basal body (c) or axoneme (f) monoclonal antibodies. Scale bar = 5 lm.
A.C. Joice et al./International Journal for Parasitology 42 (2012) 401–409
405

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flagellum in the PF parasites led us to consider that the protein
could participate in one of the functions of the flagellum, possibly
(but not limited to) motility.
To explore this, the motility of parental PF 29-13 cells and
TbHK2?/?PF parasites was observed through time-lapse micros-
copy and cell velocity and directional motility were scored. Try-
panosomes from both populations displayed a range of motility,
including cells that were nearly immobile, cells that were non-pro-
ductively motile (moving in a circular motion), and cells that
moved linearly over the length of the time lapse capture. Cell speed
analysis of the motile portion of the population revealed that both
the parental cells and TbHK2?/?parasites were similarly motile.
The cells exhibited maximum speeds of 16.3 and 13.2 lm/s, mean
speeds of 6.2 and 4.9 lm/s, and median speeds of 4.9 and 4.7 lm/s,
for parental and knockout cells, respectively (Fig. 7B). Although all
values observed for parental parasites marginally exceeded those
observed for TbHK2?/?parasites, the populations did not have sig-
nificantly different motility (P = 0.8768), with values similar to
those previously observed for PF parasites (Hutchings et al.,
2002; Rodriguez et al., 2009).
While the knockout cell line displayed similar speed to the
parental line, motion without direction could lead to a phenotype
distinct from the parental line. To assess this, the directional ten-
dency of individual cells was assessed by calculation of the direc-
tional motility coefficient (DMC) (Fig. 7C). While the mean and
median DMC of the parental and knockout parasites were not sta-
tistically different (means of 0.883 and 0.776 and medians of 0.948
and 0.776, for parental 29-13 and TbHK2?/?parasites, respectively;
P = 0.2225), the data sets displayed considerable differences in
shape. While both data sets are similarly skewed (?1.2851 and
?0.43748, respectively, for parental 29-13 and TbHK2?/?), the nat-
ure of the variation observed among the DMC distributions differs
(29-13 kurtosis of 0.68861, TbHK2?/?kurtosis of ?1.1514). Taken
together, these results suggest that no differences in directional
motility exist between the two cell lines, but greater variation in
individual cell directional motility occurs in TbHK2 deficient
parasites.
4. Discussion
The association of glycolytic proteins with flagellum is not
without precedence, as enzymes involved in glucose metabolism
have been found in the flagellum from a range of organisms,
Fig. 5. Immunofluorescence (IF) microscopy of procyclic form (PF) Trypanosoma brucei brucei parasites using a T. brucei hexokinase 2 (TbHK2)-specific antibody. (A) PF 29-13
cells probed with the TbHK2-specific affinity-purified antisera aTbHK2, (b). This image is an extended focus of Z-stack layers. (B) PF 29-13 parasites assayed with both
aTbHK2 antisera (b and f) and basal body monoclonal antibody (c and g) with the Z-stack optimised for depth of the basal bodies within the cell. (C) PF 29-13 cytoskeleton
preparations stained with aTbHK2 antisera (a and d) and basal body antibody (b and e). Scale bars = 5 lm.
406
A.C. Joice et al./International Journal for Parasitology 42 (2012) 401–409

Page 7

including green algae and mammalian spermatozoa (Travis et al.,
1998; Mitchell et al., 2005). The role of these proteins has primarily
been postulated to be in the production of ATP for the flagellum. Is
TbHK2 serving a similar role in the African trypanosome flagellum?
TbHK2 is competent for HK activity when organised with TbHK1
into oligomers (Chambers et al., 2008b), and TbHK1 has been iden-
tified in the flagellar proteome (Oberholzer et al., 2011), suggesting
that an extra-glycosomal interaction of the two proteins may
occur.
It is important to emphasise that the observations described
here regarding TbHK2 localisation do not rule out the possibility
of TbHK1 also localising to the flagellum. The two proteins, how-
ever, may have different levels of distribution between the flagel-
lum and glycosome. This suggestion is supported by the intense
staining of the flagellum and weak labelling of glycosomes using
the aTbHK2 antisera compared with the largely glycosomal signal
observed with the aTbHK antisera. Seven of the 10 amino acid dif-
ferences between TbHK1 and TbHK2 reside in the C-terminal tails
of the proteins, suggesting that this region may in part be respon-
sible for this difference in distribution.
If TbHK2 is indeed oligomerised with TbHK1 in the flagellum, it
may be playing some role in the regulation of TbHK activity (as
seen in vitro, (Chambers et al., 2008b)). To yield ATP, this model
would require that other components of the glycolytic pathway re-
side near the flagellum. Consistent with that, pyruvate phosphate
dikinase, glycerol-3-phosphate dehydrogenase and glyceralde-
hyde-3-phosphate dehydrogenase have all been found associated
with the trypanosome flagellum (Broadhead et al., 2006; Oberhol-
zer et al., 2007), but no indications exist to date for a functional fla-
gellar glycolytic pathway capable of ATP production.
The reported lack of regulation of several glycolytic enzymes
(including TbHK, phosphofructokinase (PFK), and glycerol kinase
(GK)) has been demonstrated to be incompatible with cytosolic
localisation (Bakker et al., 2000; Haanstra et al., 2008). It has been
proposed that compartmentalisation of the majority of the en-
zymes inside of glycosomes overcomes the potentially toxic effects
of this lack of regulation (Bakker et al., 2000; Haanstra et al., 2008).
If indeed the enzymes function in the flagellum, then there should
exist a compensatory mechanism in that cellular compartment,
particularly if pyruvate kinase (PYK) is present, as any net ATP syn-
thesised could be consumed by the unregulated glycolytic kinases
in the production of phosphorylated metabolites. By connecting
ATP synthesis directly to ATP-consuming cellular processes (such
as flagellar movement), the parasite may avoid the consequences
of unregulated kinase activity. Alternatively, flagellar TbHK2 may
participate in the metabolism of sugar nucleotides to generate
ATP – an activity that has been demonstrated in vitro with other
HKs (Gamble and Najjar, 1955), but which remains undetected
with recombinant TbHKs.
The mechanism behind the delivery of TbHK2 to the flagellum
remains to be resolved. A comparison of known and predicted fla-
gellar-resident T. brucei protein sequences with the TbHK2 se-
quence does not reveal an obvious conserved sequence that
could direct localisation. Additionally, comparison with sequences
from flagellar-associated glycolytic proteins from other organisms
such as green algae and mammals does not yield any notable con-
served sequences (Travis et al., 1998; Mitchell et al., 2005). How-
ever, boar GAPDH exists as a hexamer when associated with
sperm flagellum but not in muscle tissue, suggesting oligomeri-
sation may play a role in targeting (Westhoff and Kamp, 1997).
Fig. 6. Immunofluorescence (IF) microscopy of endogenously-tagged Trypanosoma brucei hexokinase 2 (TbHK2) in procyclic form (PF) Trypanosoma brucei brucei parasites.
Parasites expressing an endogenously myc tagged TbHK2 were fixed (A) or used to prepare cytoskeletons (B) and then probed for the myc epitope (A, b and f; B, a, d, and g) for
localisation with the axoneme (A, c and g;) or basal bodies (B, b, e, and h). Scale bars = 5 lm.
A.C. Joice et al./International Journal for Parasitology 42 (2012) 401–409
407

Page 8

The first indication that TbHK2 might have an extra-glycosomal
localisation was based on the finding that TbHKs were released by
concentrations of digitonin sufficient to liberate cytosolic proteins
(Fig. 1A). In contrast, some TbHK2 signal remained associated with
the flagellum in detergent-extracted cytoskeletons, suggesting that
within a life-cycle there exist distinct inter-flagellar pools of the
protein. In BSF parasites, for example, a portion of the protein re-
sides in the flagellar compartment but is subject to liberation by
low concentrations of detergent, while the remaining fraction is
more tightly associated with the cytoskeleton, based on sensitivity
of the signal to detergent extraction.
The life-cycle stage-dependent distribution of TbHK2 suggests
that the protein may have different functions during distinct
developmental phases. In PF parasites, TbHK2 basal body associ-
ation could result from docking and binding via transitional fi-
bres prior to entry into the flagellum in a relationship similar
to that which has been observed in Chlamydomonas between
IFT52 proteins and transitional fibres of basal bodies (Deane
et al., 2001). The association with the basal body in PF parasites
could position the protein for mobilisation down the length of
the BSF flagellum. Because the PF cell has a dynamic metabolism
that is not entirely dependent on glycolysis for ATP generation, it
is possible that TbHK2 localisation along the length of the flagel-
lum in BSF parasites reflects the demand of the organelle for ATP,
which in that life-cycle stage can only be provided by glucose
metabolism.
The lack of a detectable phenotype in TbHK2-deficient cells,
however, suggests TbHK2 may not be limited to a metabolic func-
tion in the flagellum, but rather the protein may play a role in
sensing of glucose and other hexoses. Glucokinases and HKs from
other systems including yeast, plants and animals, have all been
shown to be central in conveying information to the cell regarding
environmental glucose availability that allows the cell to respond
(Rolland et al., 2001). In other kinetoplastids, hexose transporters
have been found associated with the flagellum (Snapp and Land-
fear, 1999), suggesting that uptake of glucose may occur proximal
to the flagellar positioning of TbHK2, positioning it to serve in an
environmental glucose sensing role. In T. brucei, signalling en-
zymes associated with the flagellum are not without precedence,
as an adenylate cyclase (ESAG4) has been found associated with
the flagellum in both PF and BSF parasites (Paindavoine et al.,
1992).
Acknowledgements
The authors would like to thank Dr. James Bangs (University of
Wisconsin-Madison, USA) for pXS6 and Dr. Stephen Hajduk and
Rudo Kieft (University of Georgia, USA) for their assistance with
transfections. Additionally, we thank Dr. Philippe Bastin for his
helpful comments on the manuscript and Drs. Marcia Hesser and
Heidi Dodson for their technical assistance. This work was sup-
ported in part by US National Institutes of Health Grant
2R15AI075326-02 to JCM.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ijpara.2012.02.
008.
Cell Motility
PF 29-13
TbHK2-/-
0
5
10
15
20
Speed (µ µm/s)
Directional Motility
PF 29-13
TbHK2-/-
0.00
0.20
0.40
0.60
0.80
1.00
DMC
PF 29-13
TbHK2-/-
A
BC
Fig. 7. Impact of Trypanosoma brucei hexokinase 2 (TbHK2) knockout on single cell motility. Cell motility and direction of parental procyclic form (PF) 29-13 and TbHK2?/?
trypanosomes were assessed based on 30 s time-lapse capture sequences. (A) Representative images used to assess 29-13 and TbHK2?/?cell line motile properties. Position of
the anterior end of actively motile trypanosomes at each time point over the entire time course is indicated by the white dots in each of the five images. The directional
motility of all trypanosomes in these images was high (directional motility coefficient (DMC) ?1) with the exception of the TbHK2?/?cell indicated in the lower right corner
of each image that moved in an ‘‘L’’-shaped pattern (DMC = 0.572). Black circles enclose non-productively motile cells in both images. Black scale bar equals 50 lm. (B)
Incremental speed of productively motile cells represented by a box and whisker plot. (C) Directional motility of each cell line was assessed by plotting the DMCs observed for
each actively motile cell. A dot plot of these values is overlaid with a percentile plot.
408
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Page 9

References
Absalon, S., Kohl, L., Branche, C., Blisnick, T., Toutirais, G., Rusconi, F., Cosson, J.,
Bonhivers, M., Robinson, D., Bastin, P., 2007. Basal body positioning is controlled
by flagellum formation in Trypanosoma brucei. PLoS ONE 2, e437.
Albert, M.A., Haanstra, J.R., Hannaert, V., Van Roy, J., Opperdoes, F.R., Bakker, B.M.,
Michels, P.A., 2005. Experimental and in silico analyses of glycolytic flux control
in bloodstream form Trypanosoma brucei. J. Biol. Chem. 280, 28306–28315.
Alexander, D.L., Schwartz, K.J., Balber, A.E., Bangs, J.D., 2002. Developmentally
regulated trafficking of the lysosomal membrane protein p67 in Trypanosoma
brucei. J. Cell Sci. 115, 3253–3263.
Avilan, L., Gualdron-Lopez, M., Quinones, W., Gonzalez-Gonzalez, L., Hannaert, V.,
Michels, P.A., Concepcion, J.L., 2011. Enolase: a key player in the metabolism
and a probable virulence factor of trypanosomatid parasites-perspectives for its
use as a therapeutic target. Enzyme Res. 2011, 932549.
Bakker, B.M., Mensonides, F.I., Teusink, B., van Hoek, P., Michels, P.A., Westerhoff,
H.V., 2000. Compartmentation protects trypanosomes from the dangerous
design of glycolysis. Proc. Natl. Acad. Sci. USA 97, 2087–2092.
Biebinger, S., Wirtz, L.E., Lorenz, P., Clayton, C., 1997. Vectors for inducible
expression of toxic gene products in bloodstream and procyclic Trypanosoma
brucei. Mol. Biochem. Parasitol. 85, 99–112.
Blattner, J., Dorsam, H., Clatyon, C.E., 1995. Function of N-terminal import signals in
trypanosome microbodies. FEBS Lett. 360, 310–314.
Bonhivers, M., Landrein, N., Decossas, M., Robinson, D.R., 2008. A monoclonal
antibody marker for the exclusion-zone filaments of Trypanosoma brucei.
Parasite Vectors 1, 21.
Broadhead, R., Dawe, H.R., Farr, H., Griffiths, S., Hart, S.R., Portman, N., Shaw, M.K.,
Ginger, M.L., Gaskell, S.J., McKean, P.G., Gull, K., 2006. Flagellar motility is
required for the viability of the bloodstream trypanosome. Nature 440, 224–
227.
Brun, R., Shonenberger, M., 1979. Cultivation and in vitro cloning of procyclic
culture forms of Trypanosoma brucei in a semi-defined medium. Acta Trop. 36,
289–292.
Burkard, G., Fragoso, C.M., Roditi, I., 2007. Highly efficient stable transformation of
bloodstream forms of Trypanosoma brucei. Mol. Biochem. Parasitol. 153, 220–
223.
Chambers, J.W., Fowler, M.L., Morris, M.T., Morris, J.C., 2008a. The anti-
trypanosomal agent lonidamine inhibits Trypanosoma brucei hexokinase 1.
Mol. Biochem. Parasitol. 158, 202–207.
Chambers, J.W., Kearns, M.T., Morris, M.T., Morris, J.C., 2008b. Assembly of
heterohexameric trypanosome hexokinases reveals that hexokinase 2 is a
regulable enzyme. J. Biol. Chem. 283, 14963–14970.
Colasante, C., Ellis, M., Ruppert, T., Voncken, F., 2006. Comparative proteomics of
glycosomes from bloodstream form and procyclic culture form Trypanosoma
brucei brucei. Proteomics 6, 3275–3293.
Deane, J.A., Cole, D.G., Seeley, E.S., Diener, D.R., Rosenbaum, J.L., 2001. Localization of
intraflagellar transport protein IFT52 identifies basal body transitional fibers as
the docking site for IFT particles. Curr. Biol. 11, 1586–1590.
Field, M.C., Allen, C.L., Dhir, V., Goulding, D., Hall, B.S., Morgan, G.W., Veazey, P.,
Engstler, M., 2004. New approaches to the microscopic imaging of Trypanosoma
brucei. Microsc. Microanal. 10, 621–636.
Fleige, T., Fischer, K., Ferguson, D.J., Gross, U., Bohne, W., 2007. Carbohydrate
metabolism in the Toxoplasma gondii apicoplast: localization of three glycolytic
isoenzymes, the single pyruvate dehydrogenase complex, and a plastid
phosphate translocator. Eukaryot. Cell 6, 984–996.
Gamble, J.L., Najjar, V.A., 1955. Studies of the kinetics of the reverse reaction of yeast
hexokinase. J. Biol. Chem. 217, 595–601.
Ginger, M.L., Ngazoa, E.S., Pereira, C.A., Pullen, T.J., Kabiri, M., Becker, K., Gull, K.,
Steverding, D., 2005. Intracellular positioning of isoforms explains an unusually
large adenylate kinase gene family in the parasite Trypanosoma brucei. J. Biol.
Chem. 280, 11781–11789.
Haanstra, J.R., van Tuijl, A., Kessler, P., Reijnders, W., Michels, P.A., Westerhoff, H.V.,
Parsons, M., Bakker, B.M., 2008. Compartmentation prevents a lethal turbo-
explosion of glycolysis in trypanosomes. Proc. Natl. Acad. Sci. USA 105, 17718–
17723.
Hannaert, V., Albert, M.A., Rigden, D.J., da Silva Giotto, M.T., Thiemann, O., Garratt,
R.C., Van Roy, J., Opperdoes, F.R., Michels, P.A., 2003. Kinetic characterization,
structure modelling studies and crystallization of Trypanosoma brucei enolase.
Eur. J. Biochem. 270, 3205–3213.
Hutchings, N.R., Donelson, J.E., Hill, K.L., 2002. Trypanin is a cytoskeletal linker
protein and is required for cell motility in African trypanosomes. J. Cell Biol.
156, 867–877.
Kim, J.W., Dang, C.V., 2005. Multifaceted roles of glycolytic enzymes. Trends
Biochem. Sci. 30, 142–150.
Krishnamurthy, G., Vikram, R., Singh, S.B., Patel, N., Agarwal, S., Mukhopadhyay, G.,
Basu, S.K., Mukhopadhyay, A., 2005. Hemoglobin receptor in Leishmania is a
hexokinase located in the flagellar pocket. J. Biol. Chem. 280, 5884–5891.
Kroth, P.G., Chiovitti, A., Gruber, A., Martin-Jezequel, V., Mock, T., Parker, M.S.,
Stanley, M.S., Kaplan, A., Caron, L., Weber, T., Maheswari, U., Armbrust, E.V.,
Bowler, C., 2008. A model for carbohydrate metabolism in the diatom
Phaeodactylumtricornutum
deduced
analysis. PLoS ONE 3, e1426.
Liaud, M.F., Lichtle, C., Apt, K., Martin, W., Cerff, R., 2000. Compartment-specific
isoforms of TPI and GAPDH are imported into diatom mitochondria as a fusion
protein: evidence in favor of a mitochondrial origin of the eukaryotic glycolytic
pathway. Mol. Biol. Evol. 17, 213–223.
Lunn, J.E., 2007. Compartmentation in plant metabolism. J. Exp. Bot. 58,
35–47.
Miki, K., Qu, W., Goulding, E.H., Willis, W.D., Bunch, D.O., Strader, L.F., Perreault, S.D.,
Eddy, E.M., O’Brien, D.A., 2004. Glyceraldehyde 3-phosphate dehydrogenase-S, a
sperm-specific glycolytic enzyme, is required for sperm motility and male
fertility. Proc. Natl. Acad. Sci. USA 101, 16501–16506.
Misset, O., Bos, O.J., Opperdoes, F.R., 1986. Glycolytic enzymes of Trypanosoma
brucei. Simultaneous purification, intraglycosomal concentrations and physical
properties. Eur. J. Biochem. 157, 441–453.
Mitchell, B.F., Pedersen, L.B., Feely, M., Rosenbaum, J.L., Mitchell, D.R., 2005. ATP
production in Chlamydomonas reinhardtii flagella by glycolytic enzymes. Mol.
Biol. Cell 16, 4509–4518.
Morris, M.T., DeBruin, C., Yang, Z., Chambers, J.W., Smith, K.S., Morris, J.C., 2006.
Activity of a second Trypanosoma brucei hexokinase is controlled by an 18-
amino-acid C-terminal tail. Eukaryot. Cell 5, 2014–2023.
Nakamura, N., Shibata, H., O’Brien, D.A., Mori, C., Eddy, E.M., 2008. Spermatogenic
cell-specific type 1 hexokinase is the predominant hexokinase in sperm. Mol.
Reprod. Dev. 75, 632–640.
Oberholzer, M., Bregy, P., Marti, G., Minca, M., Peier, M., Seebeck, T., 2007.
Trypanosomes and mammalian sperm: one of a kind? Trends Parasitol. 23,
71–77.
Oberholzer, M., Langousis, G., Nguyen, H.T., Saada, E.A., Shimogawa, M.M., Jonsson,
Z.O., Nguyen, S.M., Wohlschlegel, J.A., Hill, K.L., 2011. Independent analysis of
the flagellum surface and matrix proteomes provides insight into flagellum
signaling in mammalian-infectious Trypanosoma brucei. Mol. Cell Proteomics 10
(M111 010538).
Opperdoes, F.R., Borst, P., 1977. Localization of nine glycolytic enzymes in a
microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett. 80,
360–364.
Paindavoine, P., Rolin, S., Van Assel, S., Geuskens, M., Jauniaux, J.C., Dinsart, C., Huet,
G., Pays, E., 1992. A gene from the variant surface glycoprotein expression site
encodes one of several transmembrane adenylate cyclases located on the
flagellum of Trypanosoma brucei. Mol. Cell. Biol. 12, 1218–1225.
Robinson, D., Beattie, P., Sherwin, T., Gull, K., 1991. Microtubules, tubulin, and
microtubule-associated proteins of trypanosomes. Methods Enzymol. 196, 285–
299.
Rodriguez, J.A., Lopez, M.A., Thayer, M.C., Zhao, Y., Oberholzer, M., Chang, D.D.,
Kisalu, N.K., Penichet, M.L., Helguera, G., Bruinsma, R., Hill, K.L., Miao, J., 2009.
Propulsion of African trypanosomes is driven by bihelical waves with
alternating chirality separated by kinks. Proc. Natl. Acad. Sci. USA 106,
19322–19327.
Rolland, F., Winderickx, J., Thevelein, J.M., 2001. Glucose-sensing mechanisms in
eukaryotic cells. Trends Biochem. Sci. 26, 310–317.
Snapp, E.L., Landfear, S.M., 1999. Characterization of a targeting motif for a flagellar
membrane protein in Leishmania enriettii. J. Biol. Chem. 274, 29543–29548.
Travis, A.J., Foster, J.A., Rosenbaum, N.A., Visconti, P.E., Gerton, G.L., Kopf, G.S., Moss,
S.B., 1998. Targeting of a germ cell-specific type 1 hexokinase lacking a porin-
binding domain to the mitochondria as well as to the head and fibrous sheath of
murine spermatozoa. Mol. Biol. Cell 9, 263–276.
Wang, Z., Morris, J.C., Drew, M.E., Englund, P.T., 2000. Inhibition of Trypanosoma
brucei gene expression by RNA interference using an integratable vector with
opposing T7 promoters. J. Biol. Chem. 275, 40174–40179.
Westhoff, D., Kamp, G., 1997. Glyceraldehyde 3-phosphate dehydrogenase is bound
to the fibrous sheath of mammalian spermatozoa. J. Cell Sci. 110 (Pt 15), 1821–
1829.
Xu, K.Y., Becker, L.C., 1998. Ultrastructural localization of glycolytic enzymes on
sarcoplasmic reticulum vesticles. J. Histochem. Cytochem. 46, 419–427.
fromcomparativewhole genome
A.C. Joice et al./International Journal for Parasitology 42 (2012) 401–409
409