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Vol. 14, Issue 5, 1769-1779, May 2003
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||School of Biological Sciences, University of Manchester, Manchester, M13 9PT England
Submitted February 22, 2002;
Revised January 8, 2003;
Accepted January 30, 2003
Monitoring Editor: Paul Matsudaira
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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,
1913). The positions of the
flagellum and kinetoplast have become the central features in classifying the
different life cycle stages of kinetoplastids
(Hoare and Wallace, 1966;
Vickerman, 1976). Electron
microscopy revealed the kinetoplast to be a highly complex disk-shaped
structure located within a distended portion of the mitochondrial matrix
adjacent to the flagellum basal bodies
(Vickerman, 1973). The T.
brucei kinetoplast consists of multiple copies of topologically
interlocked circular DNA molecules termed maxicircles and minicircles, along
with specific kinetoplast proteins. The maxicircle composition of the T.
brucei network is 25–50 copies and represents 10% of the network
mass (each maxicircle is 20 kb and all have identical DNA sequence).
Maxicircles encode the mitochondrial proteins and some guide RNAs (gRNAs),
whereas the minicircles are present in several thousand copies (1 kb in
length), are heterogeneous in sequence, and encode gRNAs. The gRNAs are
essential for the RNA editing process whereby the maxicircle encoded protein
transcripts are modified by the addition or removal of uridine residues to
produce the final mRNA molecules (Simpson
and Maslov, 1999; Simpson
et al., 2000;
Cruz-Reyes et al.,
2001; Schnaufer et
al., 2002; Stuart et
al., 2002; Stuart and
Panigrahi, 2002).
T. brucei is characterized throughout its life cycle by the
possession of a flagellum. The flagellar axoneme is subtended by a basal body
and exits the cell body through the flagellar pocket. Cells early in the cell
cycle possess a single flagellum and a single basal body with an associated
probasal body. During G1/S, the probasal body matures to nucleate the assembly
of the new flagellum, and two new probasal bodies are elaborated. The new
flagellum extends and the basal bodies move apart and adopt particular
positions, ultimately influencing the cell cleavage axis. Trypanosome flagella
are characterized by the possession of a paraflagellar rod (PFR). This highly
organized structure runs alongside the axoneme from the point of emergence
from the cell body (Gull,
1999). The kinetoplast DNA molecules are replicated in a single
periodic mitochondrial S phase that is temporally coordinated with nuclear DNA
synthesis (Cosgrove and Skeen,
1970; Woodward and Gull,
1990) but the replicated kinetoplast DNA (kDNA) is segregated
before mitosis begins. We have shown that this kinetoplast segregation process
is mediated by movement apart of the flagellar basal bodies; one daughter
kinetoplast moving with the basal body of the old flagellum and one with the
basal body of the new flagellum (Robinson
and Gull, 1991).
The molecular mechanisms of trypanosome mitochondrial genome replication
and its expression by RNA editing are known in some detail. Maxicircles and
minicircles replicate simultaneously, but maxicircles seem to replicate within
the kinetoplast structure, whereas minicircles leave the kinetoplast and
reattach at the two opposing poles of the network. These antipodal sites have
also been shown to contain some of the enzymes necessary for kDNA replication
(Klingbeil et al.,
2001; Morris et al.,
2001). Thus, although we have a rapidly growing appreciation of
kinetoplast replication we have very little insight to the features
responsible for the segregation of the daughter kinetoplasts. We do know that
segregation is mediated by the separation of the flagellar basal bodies
(Robinson and Gull, 1991;
Robinson et al.,
1995). In essence, we know how the process functions but we have
no information on the structures involved, even though unequivocal evidence
for the existence of some form of high-order structural link between the
kinetoplast and flagellum has been demonstrated
(Robinson and Gull, 1991).
We have now addressed this issue, and the application of particular electron microscopical fixation regimes has allowed the characterization of a tripartite attachment complex (TAC). Our results provide the structural explanation for the fidelity of kinetoplast position and segregation. We suggest that this highly ordered interaction of a mitochondrial "nucleoid" with the cytoskeleton may be an extreme, but informative, case of a more general structural interaction of mitochondria and plastids with elements of their respective cell's cytoskeleton.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Crithidia
fasciculata strain ATTC 1145 was cultivated and prepared for electron
microscopy as described by Russell et al.
(1984).
Transmission Electron Microscopy
Whole cells were prepared according to Tooze
(1985). Briefly, a mid-log
phase culture (5 x 106 cells/ml) of cells was fixed in 2%
(vol/vol) glutaraldehyde, 2% (wt/vol) paraformaldehyde in 0.1 M sodium
cacodylate buffer (SCB), pH 7.2. The cells were then washed in SCB, postfixed
with 2% (wt/vol) osmium tetroxide in SCB, stained with 0.5% aqueous magnesium
uranyl acetate, dehydrated, and embedded in Spurr's resin
(Spurr, 1969). Some samples
were prepared by a modification of a protocol
(Begg et al., 1978)
whereby cells were harvested as described above and washed in 0.1 M phosphate
buffer, pH 7.0. They were then fixed for 30 min at room temperature in 2.5%
(vol/vol) glutaraldehyde containing 1% tannic acid in 0.1 M phosphate buffer,
postfixed in 0.5% osmium tetroxide (wt/vol) in 0.1 M phosphate buffer, pH 6.0,
for 20 min at on ice. Samples were then stained with 1% aqueous magnesium
uranyl acetate and dehydrated/embedded in resin as detailed above.
Cells were also prepared by simultaneously fixing and extracting with 1%
glutaraldehyde, 1% formaldehyde in 0.1% Nonidet P-40 in 0.05 M PIPES, 1 mM
EGTA, 0.5 mM MgSO4, pH 6.9, for 1 h at room temperature. All other
steps were identical to the modified
(Tooze, 1985) procedure
described above except en bloc staining with 1% aqueous magnesium uranyl
acetate was used. All blocks were sectioned at 50- to 70-nm thickness and
stained in 5% (wt/vol) uranyl acetate in 1% acetic acid and 0.4% lead citrate
in 0.1 N NaOH. Sections were examined on a 420 transmission electron
microscope (Philips).
Flagella Preparation
EDTA was added to 5 ml of a mid-log phase culture to a final concentration
of 5 mM. Cells were harvested by centrifugation, washed once in
phosphate-buffered saline (PBS), pH 7.2, and extracted on ice for 10 min in
0.5% Triton X-100, 10 mM NaH2PO4, 150 mM NaCl, 1 mM
MgCl, pH 7.2. Cytoskeletons were harvested, washed in extraction buffer, and
resuspended on ice for 45 min in 10 mM NaH2PO4, 150 mM
NaCl, 1 mM MgCl, pH 7.2, containing 1 mM Ca2+. The
flagella preparation was then washed twice in PBS and resuspended in 500 µl
of PBS and used for immunofluorescence studies.
Immunofluorescence
Immunofluorescence was performed as described by Robinson and Gull
(1991). Essentially, cells or
kinetoplast–flagella complexes were fixed in methanol, rehydrated in
PBS, pH 7.2, and probed with a 1:1 mixture of monoclonal antibodies ROD1 and
BBA4 (Woods et al.,
1989) followed by fluorescein isothiocyanate-conjugated secondary
antibodies. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) and
preparations were visualized using an Axioscope (Carl Zeiss, Jena,
Germany).
5-Bromodeoxyuridine (BrdU) Incorporation and Labeling of
kDNA–Flagella Complexes
BrdU incorporation into cells and immunofluorescence were performed
according to Robinson and Gull
(1991) flagellum isolation was
performed according to Robinson and Gull
(1994).
Drug Treatment
Acriflavine was added to T. brucei cultures to a final
concentration of 50 ng/ml. Samples were taken every 4 h and processed for
imunofluorescence and DAPI staining. Counts of 300 cells were made at each
time point to determine the distribution of cell types in the population.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). A physical connection between the kinetoplast and the basal
body can be demonstrated by isolating the flagella by using detergent and
Ca2+ treatment. In such preparations, the mitochondrial
DNA in the kinetoplast is still firmly attached to the basal body of the
flagellum (Figure 1,
E–G). The attachment of the flagellum to the kinetoplast
after detergent and Ca2+ treatment confirms the presence
of a highly organized connection system.
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Given this evidence for a physical connection between the flagellar basal
body and the kinetoplast, we questioned whether we could detect any form of
ultrastructural links between these two organelles. Thin section electron
micrographs of T. brucei cells were produced after fixation by a
variety of individual protocols. A defined set of structures was detected
using this spectrum of fixation regimes but can be exemplified most distinctly
by a fixation regime using tannic acid in a modified protocol of Begg et
al. (1978).
Figure 2A shows an image of a
T. brucei procyclic cell, sectioned close to the flagellum pocket
area. The section reveals the basal body and a cross section of the
disk-shaped kinetoplast within the mitochondrion. Two novel filament systems
are defined in this image. First, a set of filaments runs between the proximal
end of the basal body and the adjacent outer mitochondrial membrane (large
bracket). This set of filaments, which we designate exclusion zone filaments,
occupy this defined area and their presence excludes cytoplasmic ribosomes.
The plane of the kinetoplast is orthogonal to the longitudinal axis of the
flagellum/basal body and so the kinetoplast is seen in transverse section in
this figure. The second set of filaments is present only on one face of the
kinetoplast and links it with the inner mitochondrial membrane (small brackets
and arrows). These filaments, which we designate the unilateral filaments, are
always present only on this single face of the kinetoplast. This area is
densely packed with fibrous material and this maintains the gap between the
kDNA network and the inner mitochondrial membrane with a constant spacing
(small brackets and arrows). We observe that the mitochondrial membrane in
this zone between the basal body and the kinetoplast exhibits a rather linear
profile with few, if any, corrugations. Moreover, cristae were never observed
in this zone of the mitochondrion. Cristae, however, were often present
projecting into the mitochondrial lumen on the other side of the kinetoplast
in Figure 2, A, B, and D. We
will refer to this tripartite structure, the exclusion zone filaments, the
differentiated mitochondrial membranes, and the unilateral filaments, as the
TAC.
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Figure 2B shows a thin
section of the flagellar pocket area of a cell, which is some way into its
cell cycle because it possesses two flagella. The kinetoplast of this cell is
characterized by the occurrence of fibrous lobes at each end of the
kinetoplast. Both terminal fibrous lobes have a different texture and electron
density to the main body of the kinetoplast. Our interpretation of this
micrograph (two flagella in one flagellum pocket with basal bodies not very
far apart) suggests that it is likely to be at a stage between 0.4 and 0.7 of
the unit cell cycle (Woodward et
al., 1995). This period encompasses S phase, and it seems
likely that these two antipodal fibrous lobes on the kinetoplast are
associated with kinetoplast replication
(Abu-Elneel et al.,
2001; Drew and Englund,
2001). This form of kinetoplast still exhibits the full TAC
however, and we note that the unilateral filaments still connect to the main
body of the kinetoplast but not totally to the antipodal lobe structures. This
image (Figure 2B) shows the
early replication processes of the TAC. Two ribosome-free regions are observed
and each region subtends a flagellum basal body. Also, two distinct zones of
mitochondrial membrane are present between in the region between the basal
bodies and the kinetoplast. Each zone of mitochondrial membrane exhibits a
linear profile with few corrugations. Thus, TAC replication occurs while a
connection is maintained to the replicating kinetoplast.
After replication the kinetoplast increases in size. Two examples of this form are seen in Figure 2, C and D. The cell in Figure 2C possesses two flagella and their associated basal bodies and probasal bodies. Figure 2C shows an oblique section of the two basal bodies and the two probasal bodies. All three components of the TAC are present at this stage of the cell cycle. The cell in Figure 2D again exhibits two sets of basal and probasal bodies. However, it occurs later in the segregation process because the two sets are further apart. Moreover, in this image the kinetoplast has a shallow "V" configuration, and the differentiated area of mitochondrial membranes that look thickened and more electron dense maintain a linear profile and lack cristae. As such the mitochondrial membranes now clearly form two discrete zones. Likewise, a separate set of exclusion zone filaments connects each of these areas of membrane to their particular basal body set. Figure 2E shows an image of a C. fasciculata cell where the flagellum has been sectioned longitudinally. The TAC can clearly be seen in this cell. The C. fasciculata TAC includes exclusion zone fibers that exclude cytoplasmic ribosomes (large bracket). Also, the mitochondrial membrane exhibits a linear profile in the region adjacent to the exclusion zone fibers (small brackets and arrows), and the unilateral filaments are also present in this trypanosome but seem to be more loosely organized in comparison with those of T. brucei.
Having obtained detailed data from standard electron microscopy preparations of T. brucei whole cells, we sought improvements in fixation of detergent-extracted cytoskeletons that might allow us even clearer demonstrations of the TAC components when cytoplasmic material was removed by detergent. One particular procedure proved highly informative. Thin section analysis after simultaneous fixation and detergent extraction of cells provided a novel view of the basal body/kinetoplast relationship (Figure 3). This combined detergent/fixation extraction protocol resulted in cytoskeletons where all of the membranous organelles and cytoplasmic components had been removed. However, the components of the TAC, including the specialized area of mitochondrial inner and outer membrane were preserved under these operational conditions. Figure 3, A–E, shows examples of basal bodies with their associated TAC/kinetoplasts. The exclusion zone filaments are more clearly seen in these preparations as 5- to 10-nm electron dense filaments emanating from the proximal end of the basal body (arrow) toward (and linking to) the remnant of the outer mitochondrial membrane. Although all of the normal mitochondrial membranes had disappeared (by detergent extraction), a double line of electron dense material survived at the precise junction between the unilateral filaments and exclusion zone filaments (arrowhead). Remarkably, these mitochondrial membrane remnants remain intact only between the basal bodies and the kinetoplast, whereas no remnants of mitochondrial membrane were observed outside of this region. The unilateral filament set was seen as a tightly packed mass between the inner mitochondrial membrane remnant and the kinetoplast (white arrow). The unilateral filaments retain their packed appearance and membrane-kinetoplast DNA interaction after extraction-fixation treatment. In these preparations, the kinetoplast retains its fibrous, striated, and compact organization that is normally visualized in whole cells. Figure 3E represents an image of this TAC area in a cell where the basal bodies have duplicated (there are two sets of a basal body and probasal body seen in the oblique section). This micrograph shows that at this stage each set of basal bodies is associated with a complete TAC reminiscent of the situation seen in a whole cell preparation (Figure 2D). Interestingly, however, the detergent/fixation protocol emphasizes the distinct difference between the differentiated mitochondrial membrane of the TAC and that of the normal mitochondrial membranes. A distinct gap is seen between the two nascent TACs, where the normal membrane has been removed. We interpret this as indicative of a late stage in the process in TAC segregation.
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It is important to ask when in the cell cycle the TAC-mediated connection
is present, when is it remodeled, and is the kDNA permanently attached or does
remodeling involve DNA detachment and reattachment? Our electron microscopy
analysis strongly suggests that the kinetoplast/basal body connection is
maintained via the TAC during all stages of the cell cycle, but it is
important to provide corroborative evidence by using different approaches. The
situation during S phase is particularly intriguing because kDNA replication
is known to involve the release of at least one component (minicircles) from
the kinetoplast complex (Abu-Elneel et
al., 2001; Drew and
Englund, 2001; Klingbeil
et al., 2001; Morris
et al., 2001). This raises the question of whether the
kinetoplast matrix structure as a whole is still firmly attached to the basal
bodies during S phase by means of the TAC. Cells were treated with the
thymidine analog BrdU, thus allowing the replicating kinetoplasts to
incorporate this proliferation marker. Cells were then detergent extracted and
the subpellicular microtubules depolymerized with calcium. These kDNA/flagella
preparations were processed for immunofluorescence and probed with anti-BrdU
antibodies. We searched for kinetoplasts that had incorporated the BrdU into
the replicating network and analyzed the association of the flagella to the
spread kDNA networks. Figure 4
illustrates such a BrdU-labeled kDNA/flagellum preparation. DAPI staining and
phase contrast imaging (Figure
4A, area under large bracket) show the kinetoplast network to be
in mid-to-late stages of replication as observed by the characteristic bilobed
appearance, typified by such a replicative form
(Robinson and Gull, 1991).
Figure 4B shows the same
network observed with fluorescent anti-BrdU labeling and phase contrast
imaging. This network has two antipodal lobes of BrdU incorporation, located
180o apart (areas under small brackets). Attachment of the
flagella, via their basal bodies, to the replicating network is clearly
observed at the outermost poles of the network, 180o apart and
within the sites where replicated DNA occurs. We conclude from this result
that the TAC is present and maintained during kDNA replication. Furthermore,
these data show that kinetoplast replication and segregation occurs
simultaneously and that replicated DNA occurs within the sites of physical
attachment of the flagellum basal bodies to the kDNA network.
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The above-mentioned results show that the TAC is a coherent structure
linking the kinetoplastid-located mitochondrial genome to the flagellar basal
bodies throughout the cell cycle. The BuDR experiment shows that the new TAC
complex associated with the new flagellum is able to gain kDNA at S phase. We
next addressed the issue of whether this connection would be disturbed by
inhibition of kDNA synthesis and if so, what would be the cellular
consequences? Acriflavine is an attractive compound for such studies because
it is known to have a selective effect on kDNA replication and has well
studied effects on mitochondrial DNA in many other cell types
(Simpson, 1968;
Tarrago-Litvak et al.,
1978; Gillham et al.,
1987; Matagne et al.,
1989; Agbe and Yielding,
1995). The most striking feature of acriflavine treatment is the
rapid rise through 4 and 8 h treatment of a cell type that we term 1K1N
[PDB]
(old)
and 1K1N
[PDB]
(new) (Figure 5). These cells represent >70% of the cell types in the 8-h drug-treated
population (Ogbadoyi, 1997).
Although these cells have only one kinetoplast, they are, judging by the
presence and length of the new flagellum, clearly in an advanced stage of the
cell cycle (Figure 5). Although
the basal bodies have replicated and segregated and the new flagellum has
formed, the mass of kDNA is associated with only one basal body complex (arrow
in E and H). In the majority of cases (6:1 ratio at 8 h), this is the basal
body subtending the old flagellum and we term these cells 1K1N
[PDB]
(old)
(Figure 5, G, H, and I). In
some cases, mainly at 4-h treatment, the kDNA is stretched between basal
bodies (included in counts but not shown). The stretched kDNA supports the
previous conclusion that the kinetoplast DNA is attached to the flagellum
basal bodies during replication and segregation by means of the TAC; the
stretched phenotype being explained by a lack of kDNA decatenation after drug
treatment, thus blocking network separation. The kinetoplast is thus pulled or
stretched between the segregating basal bodies while attached to the flagellum
basal bodies via the TAC. The consequence of the asymmetric segregation of the
kinetoplast in the 1K1N
[PDB]
(old) and 1K1N
[PDB]
(new) cells is the production upon
division of a 1K1N
[PDB]
sibling and a 0K1N sibling
(Figure 5, D–I). These
latter cell types are known as dyskinetoplastic trypanosomes and have long
been recognized as a feature of DNA-intercalating drug treatment of
trypanosomes (Guttman and Eisenman,
1965; Kusel et al.,
1967; Laub-Kupersztejn and
Thirion, 1969; Simpson,
1972; Zaitseva et
al., 1977; Shapiro et
al., 1989). Our observations now show that these
dyskinetoplastic trypanosomes can form at the very first cell division by
asymmetric segregation of the kDNA. Taken with our previous points, this
strongly suggests that the TAC/kinetoplast DNA connection is normally
remodeled at S phase to take account of the new TAC and replicated kDNA and
that this remodeling can be perturbed leaving the kDNA associated with one
(usually their old TAC complex). The lack of a mitochondrial genome is clearly
lethal in the longer term for these procyclic cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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,b;
Berger and Yaffe, 2000). Genes
identified from mutational analysis of mitochondrial inheritance encode
proteins that fall into two main groups: those likely to be associated with
the cytoskeleton and those associated with the outer mitochondrial membrane
(Jones and Fangman, 1992;
Guan et al., 1993;
Sogo and Yaffe, 1994;
Berger et al., 1997;
Hales and Fuller, 1997;
Shepard and Yaffe, 1999;
Fekkes et al., 2000).
In some cases, the evidence of a cytoskeletal association points to proteins
that influence mitochondrial shape and transmission to microtubules and at
other times to actin microfilaments
(McConnell et al.,
1990; McConnell and Yaffe,
1993; Hermann et al.,
1997; Otsuga et al.,
1998; Sesaki and Jensen,
1999). Moreover, there is evidence that mitochondrial outer
membrane proteins such as Mmm1p are also involved in processes that control
the distribution and stability of the DNA nucleoid at the face of the inner
mitochondrial membrane (Hobbs et
al., 2001). Also, the intermembrane space protein Mgm1p is
implicated in mitochondrial structure, fusion, and inheritance
(Wong et al., 2000).
Such macromolecular complexes involving crosstalk between mitochondrial
membranes and the mitochondrial genome are reminiscent of the structural
features of the TAC that we describe in trypanosomes. The T. brucei
genome project is, as yet, incomplete. However, there is good evidence for the
existence of homologs of proteins such as Mgm1p and others in the T.
brucei GSS database (our unpublished observations), suggesting wide
evolutionary conservation of such components. Although genetic evidence for molecular components influencing links between the cytoskeleton and the mitochondrion has increased, our results provide a reciprocal view whereby such discrete structural linkages have been visualized in the electron microscope.
Exclusion Zone Filaments of TAC
The proximity of the flagellum basal body and kinetoplast was noted in
classical descriptions in the early years of the last century and now forms a
definition of the stages of trypanosomatids
(Hoare and Wallace, 1966).
Traditional fixation techniques showed little structure at the most proximal
region of the flagellum basal bodies. Indeed, Vickerman
(1969) remarked "a
definite structural nexus between the basal body of the flagellum and the
mitochondrial envelope of the kinetoplast has not become apparent by electron
microscopy even though the two are linked in morphogenesis." Vickerman
(1973) recognized the fact
that some physical material might be present because he noted a "zone of
exclusion," whereby ribosomes were not present between the mitochondrion
and basal bodies. Souto-Padron et al.
(1984) used quick-freeze
deep-etch approach to visualize filaments in this general area. Our approach
using different fixation regimes has allowed us to visualize for the first
time a defined substructure of filaments within this area and we have termed
them the exclusion zone filaments. Our results showing that these filaments
are present under conditions of detergent extraction is consistent with the
idea that it is this set of filaments that forms and maintains the
connection.
Unilateral Kinetoplast Filaments of TAC
Because the kinetoplast is maintained at a specific position yet the single
mitochondrion extends along the length of the cell
(Vickerman, 1965;
Simpson, 1972;
Vickerman et al.,
1988), there is a strong suggestion that some structure links the
kDNA within the mitochondrial lumen to a particular position on the inner
mitochondrial membrane. Moreover, this structure should presumably be
positioned directly opposite the basal bodies. The presence of the filament
system, which we have termed the unilateral kinetoplast filaments, provides
the structural explanation for this positioning mechanism. The identification
of the unilateral kinetoplast filaments also raises the possibility that such
a "local" differentiated face of the kinetoplast disk may play a
role in organizing and directing structural features of kDNA replication.
Differentiated Mitochondrial Membrane of TAC
There are four distinct features of the mitochondrial membrane in the area
between the basal bodies and the kinetoplast. Both membranes often seem to be
more parallel than in other areas of the mitochondrion. Also, this zone
exhibits no cristae, and the membrane "survives" detergent
extraction. Finally, the area emerges as two separated zones when the
replicated basal bodies separate. The nature of the detergent-resistant link
between the two filament components of the TAC present on either side of the
mitochondrial membranes is unknown. One can rehearse possible architectures
ranging from a "molecular rivet" spanning both mitochondrial
membranes to an oligomeric protein/lipid raft complex. Whatever the nature of
the transmembrane connection, it must allow maintenance of a discrete membrane
potential that is required for protein import and is vital to the
parasite.
Replication and Segregation
The TAC is present at all parts of the cell cycle and duplicates along with
the basal bodies at the periodic kDNA S phase. Our electron microscopy reveals
two dense structures, possibly akin to the "fibrous knots"
described in another context by Rudzinska and Vickerman
(1968) at either end of the
kinetoplast during this S phase. Although the unilateral filaments of the TAC
are still present along the main kinetoplast, they do not seem to connect so
directly with these two terminal structures, which we term the fibrous lobes.
Our correlation of the known cell cycle markers in T. brucei and the
presence of these fibrous lobes place their occurrence within S phase.
Replication of the T. brucei kDNA network occurs with maxicircles
replicating within the structure, whereas minicircles leave and reattach at
the two opposing poles of the network
(Ferguson et al.,
1994; Robinson and Gull,
1994; Abu-Elneel et
al., 2001; Drew and
Englund, 2001; Klingbeil
et al., 2001; Morris
et al., 2001). In addition to replicated minicircles,
these antipodal sites contain some of the enzymes necessary for kDNA
replication. We suggest that the fibrous lobes are the ultrastructural
correlates of these antipodal sites and are part of the mechanism whereby
replicated kDNA is reincorporated and connected with the new and old TACs.
This raises the question of whether the TAC components are inherited in a
conservative or semiconservative manner. One rationale for probasal body
association with the TAC in pre-S phase cells is that it will therefore be
connected to the internal unilateral filaments (and therefore kinetoplast)
before it matures to form the new flagellum.
Inheritance and Dyskinetoplastic Cells
Dyskinetoplastic strains of trypanosomes have been described both as
natural occurrences and as the result of experimental insults
(Guttman and Eisenman, 1965;
Kusel et al., 1967;
Laub-Kupersztejn and Thirion,
1969; Riou and Saucier,
1979; Schnaufer et
al., 2002). However, recent results show that knockdown of
certain mitochondrial functions (such as RNA editing) is a lethal event even
in bloodstream forms (Schnaufer et
al., 2001). Simpson
(1972) noted that in
Leishmania tarentolae low concentrations of acriflavine produced a
selective inhibition of kDNA synthesis for a few generations whereon one
daughter trypanosome retained all of the remaining kDNA. However, in higher
concentrations of the drug, trypanosomes produced a one-step "all and
none" type of kinetoplast segregation. He hypothesized that in such
cases the dye interferes with the distribution of kDNA between daughters as
well as inhibiting DNA replication
(Simpson, 1972). We support
this view and suggest that the TAC provides an explanation of these events. We
envisage the all and none form of division resulting from a failure of
replication and segregation such that one basal body segregates without an
attached kinetoplast. This form of division would result because the exclusion
zone filaments have been formed in the previous cell cycle, but new unilateral
filaments would need to be formed (or remodeled) in the present cell cycle.
The production (within one cell cycle) and cellular consequences of
missegregation reinforces our view that segregation of the basal bodies as
well as kinetoplast replication and segregation are major events influencing
cell cycle checkpoint control (Ploubidou
et al., 1999).
Replicating free minicircle intermediates of C. fasciculata have
been visualized almost exclusively situated at the flagellum face of the
kinetoplast (Drew and Englund,
2001). Some kDNA binding proteins are localized to the whole
kinetoplast network, whereas others such as a DNA primase and a minicircle
binding protein localize to specific zones
(Abeliovich et al.,
1993; Hines and Ray,
1998; Johnson and Englund,
1998; Abu-Elneel et al.,
1999,
2001). The unilateral filaments
may provide the basis for a structural anisometry plus a focused matrix
necessary for ordered enzymatic functions required for replication of the
network.
Kinetoplast Positioning and Segregation Are Mediated by TAC
The existence of some "link" between the kinetoplast and basal
body of the flagellum has been speculated upon in many previous descriptions
of trypanosome cell biology. Our use of specific fixation regimes has allowed
us to visualize the TAC, a cytoplasmic filament system, a differentiated zone
of mitochondrial membrane, and a lumenal mitochondrial filament system that we
suggest represents a highorder system responsible for both kinetoplast
positioning and segregation (Figure
6). This transmembrane structure provides the explanation for why
the kinetoplast maintains its position close to the basal body in the various
trypanosomes. Our view is that this complex may represent an extreme form of a
more generalized mitochondrion/cytoskeleton interaction and as such is
informative in its component structure and complexity. We suggest that the
complex TAC is seen at an extreme level in trypanosomes because of their need
to ensure absolute efficiency in the segregation of their single mass of
mitochondrial DNA. The TAC is a component of the kinetoplast positioning and
segregation machinery as seen by our electron microscopy and
immunofluorescence studies on whole cells, cytoskeletons, and
kinetoplast/flagellum complexes. We suggest that this structure is present in
many, if not all, pathogenic trypanosomes as well as nonpathogenic
kinetoplastida such as C. fasciculata.
|
Our electron microscopy shows a novel mitochondrial DNA/cytoskeleton
interaction, the TAC. We show that the basal body complexes of the flagellum
cytoskeleton are firmly attached to the kinetoplast via the TAC. We also show
that this TAC linkage must undergo some form of remodeling during kinetoplast
S phase, because acriflavine inhibition results in basal bodies that segregate
without any associated kDNA. Given the need for an efficient bipolar,
kinetoplast segregation mechanism then the evolution of a TAC link, not merely
to elements of the cytoskeleton but thence to the unit flagellar basal bodies
and their replication/segregation cycle, seems to be an elegant example of the
hitch-hikers guide to the cytoskeleton
(Gull, 2001). We suggest that
this complex may represent an extreme form of a more generally occurring
mitochondrion/cytoskeleton interaction.
|
ACKNOWLEDGMENTS |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: kDNA, kinetoplast DNA; TAC, tripartite attachment complex.
* These authors share first authorship on this article. 
Present address: Department of Biological Sciences, Federal University of
Technology, Bosso Rd., P.M.B. 65, Minna, Nigeria
Present address: Laboratoire de Parasitologie Molelulaire, Unité
Mixte Recherche-Centre National de la Recherche Scientifique 5016,
Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux
Cedex, France
Present address: Sir William Dunn School of Pathology, University of
Oxford, South Parks Rd., Oxford OX1 3RE, United Kingdom.
|| Corresponding author. E-mail address: keith.gull{at}pathology.ox.ac.uk.
|
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