2236
Research Article
A MORN-repeat protein is a dynamic component of
the Toxoplasma gondii cell division apparatus
Marc-Jan Gubbels1,*, Shipra Vaishnava2, Nico Boot1,‡, Jean-François Dubremetz3 and Boris Striepen1,2,§
1
Center for Tropical and Emerging Global Diseases and 2Department of Cellular Biology, University of Georgia, Paul D. Coverdell Center, Athens,
Georgia 30602, USA
3
UMR CNRS 5539, Université de Montpellier 2, Montpellier, 34095, France
*Present address: Department of Biology, Boston College, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA
‡
Department of Biology, Faculty of Science Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
§
Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 20 February 2006
Journal of Cell Science 119, 2236-2245 Published by The Company of Biologists 2006
doi:10.1242/jcs.02949
Summary
Apicomplexan parasites divide and replicate through a
complex process of internal budding. Daughter cells are
preformed within the mother on a cytoskeletal scaffold,
endowed with a set of organelles whereby in the final stages
the mother disintegrates and is recycled in the emerging
daughters. How the cytoskeleton and the various
endomembrane systems interact in this dynamic process
remains poorly understood at the molecular level. Through
a random YFP fusion screen we have identified two
Toxoplasma gondii proteins carrying multiple membrane
occupation and recognition nexus (MORN) motifs.
MORN1 is highly conserved among apicomplexans.
MORN1 specifically localizes to ring structures at the
apical and posterior end of the inner membrane complex
and to the centrocone, a specialized nuclear structure that
organizes the mitotic spindle. Time-lapse imaging of tagged
Introduction
The protozoan phylum Apicomplexa is extremely species rich
and members of this group are found as parasites in virtually
every vertebrate and many invertebrate animals. Several
species cause life-threatening diseases in humans including
malaria (Plasmodium) and AIDS-associated encephalitis or
gastroenteritis
(Toxoplasma
and
Cryptosporidium).
Apicomplexans are intracellular parasites and each
reproductive cycle is initiated through a motile stage, the
zoite. This stage invades the host cell in a complex process
depending on the parasite’s gliding and secretory machinery
(Sibley, 2004). Although the zoite form is a structurally
conserved start and end point of each cycle, the course of
intracellular development has considerable morphological
and functional diversity across the phylum. The parasites
achieve this diversity through a remarkable flexibility of their
cell and division cycle. Toxoplasma divides in a way most
similar to animal cells: DNA replication is followed by
nuclear division and cytokinesis resulting in two new zoites
(Sheffield and Melton, 1968). Plasmodium and Eimeria
proceed through several rounds of DNA synthesis and nuclear
division prior to cytokinesis (Dubremetz, 1975). Sarcocystis
omits nuclear division and cytokinesis for multiple rounds
yielding a cell with a polyploid nucleus. This nucleus divides
and segregates concomitantly with the formation of multiple
MORN1 revealed that these structures are highly dynamic
and appear to play a role in nuclear division and daughter
cell budding. Overexpression of MORN1 resulted in severe
but specific defects in nuclear segregation and daughter cell
formation. We hypothesize that MORN1 functions as a
linker protein between certain membrane regions and the
parasite’s cytoskeleton. Our initial biochemical analysis is
consistent with this model. Whereas recombinant MORN1
produced in bacteria is soluble, in the parasite MORN1 was
associated with the cytoskeleton after detergent extraction.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/119/11/2236/DC1
Key words: Toxoplasma, Apicomplexa, Inner membrane complex,
MORN, Cell division, Parasite
daughter cells (Speer and Dubey, 1999; Vaishnava et al.,
2005).
The ability of the parasites to form varying numbers of
progeny (from two to thousands) is linked to their unique
budding mechanism. Daughter cells pre-form within the
mother as buds, are endowed with a set of organelles and
emerge from the mother cell. Throughout the diverse division
modes, budding is invariably linked to the final round of
mitosis. Both number and localization of buds depends on the
number and position of centrosomes (Dubremetz, 1975;
Morrissette and Sibley, 2002a; Vaishnava et al., 2005). A
critical step towards building daughter cells is the initiation of
a new inner membrane complex (IMC). These flattened
double-membranous cisternae delineate the forming daughters
and are homologous to the alveolae found in ciliates and
dinoflagellates (Cavalier-Smith, 1993). The IMC is structurally
organized by a meshwork formed by a group of proteins
weakly related to intermediate filaments (Gubbels et al., 2004;
Hu et al., 2002a; Mann and Beckers, 2001). Initially the IMC
is flexible allowing for growth but proteolytic processing of
IMC components is then thought to stabilize the structure in
the mature cell (Hu et al., 2002a; Mann et al., 2002). In addition
to providing a pellicle, the IMC also plays a critical role as an
anchor for the gliding apparatus of the parasite (Gaskins et al.,
2004).
Journal of Cell Science
Cell division in Toxoplasma
The IMC also interacts with a set of 22 subpellicular
microtubules. Both structures are in intimate physical contact
through an organized array of intramembranous particles
(Morrissette et al., 1997), and (at least initially) grow in a
coordinated fashion during budding (Hu et al., 2002a).
Whether microtubule growth drives IMC extension or
vice versa is less clear. Pharmacological ablation of
microtubules had strong effects on the formation of
daughter cells (Shaw et al., 2000). Budding is not completely
abolished but is uncoupled from nuclear and organellar
segregation. The extend to which budding is affected also
varies depending on the parasite species and drug
concentration (Morrissette and Sibley, 2002b; Vaishnava et
al., 2005).
Whereas the structural elements of daughter cell formation
have been characterized in some detail, how they interact and
how this interaction is controlled and timed is poorly
understood. In particular, how the various membranous
systems and organelles tie in with the complex cytoskeleton
remains elusive. In this study we describe a Toxoplasma
gondii protein that participates in the assembly of the IMC.
This protein, which is conserved among apicomplexans, caps
the anterior and posterior end of the IMC in a ring-like
structure. During cell division these rings are dynamic and
contract during the final phase of budding. Thus the
membrane occupation and recognition nexus (MORN)associated ring may divide and segregate nuclei and
organelles. Detergent fractionation experiments showed that
2237
this otherwise soluble protein is associated with the
cytoskeleton.
Results
Identification of two membrane occupation recognition
nexus (MORN) proteins in Toxoplasma gondii
To identify parasite genes based on the subcellular localization
of their products we have used a genetic strategy based on
expression cloning (Gubbels et al., 2004). This screen used a
library of T. gondii genomic DNA fragments fused to the
yellow fluorescent protein (YFP) coding region. Clone 52C19
was initially isolated as a tag associated with the plasma
membrane (data not shown). Rescue and sequence analysis
revealed an open reading frame encoding a protein with
similarity to membrane occupation recognition nexus
(MORN) repeat proteins. Searching the T. gondii genome
database (www.toxodb.org) with the cloned sequence
identified two related genes (one of them tagged in the screen).
Transcription of both loci was confirmed in tachyzoites by RTPCR and 5⬘ and 3⬘ RACE-PCR (see Materials and Methods
for details). Both proteins consist of 23 amino acid MORN
repeats (Fig. 1). MORN1 (accession number DQ181547) and
MORN2 (DQ181548, tagged in 52C19) encode predicted
proteins with 39.8% sequence similarity and 24.5% identity.
MORN1 shows a high level of sequence conservation across
the phylum of Apicomplexa and homologs were identified in
all species for which substantial sequence information is
available (Fig. 1). MORN2 does not appear to have clear
PFam MORN consensus: YeGewknGkrhGkGvytwadGdr
Repeat 2 of TgMORN1: YEGEFVFGKREGHGRFLYADGAT
Tgon
Eten
Cpar
Pfal
-M ES CH AY HG QI KD GL FH GK GT LI YS GN EK YE GE FV FG KR EG HG RF LY AD GA TY EG KW VE DR IH GQ GV AH FA S
MV ES CH AY HG QI KD GL FH GH GT LL YS GN EK YE GD FV FG KR EG NG RF EY AD GA TY QG KW VE DR IH GQ GV AV FA S
ME TS SH SY SG DI KG GL FH GR GV LI YS KN EK YE GD FV MG KR EG FG KF TY AD GA SY EG EW VD DK IH GQ GK AS FS S
MT EV TH CY NG NI KD GL FH GF GI LI YS QH EK YE GD FV YG KR EG RG KF TY AD GA TY EG EW VD DK IH GK GI AN FV S
1. .. .. .. .1 0. .. .. .. .2 0. .. .. .. .3 0. .. .. .. .4 0. .. .. .. .5 0. .. .. .. .6 0. .. .. .. .7 0. .
Tgon
Eten
Cpar
Pfal
GN RY EG QW EM GR IN GF GK LS YS NG DE YE GE WV DG KM HG RG TY RY AE GD VY TG EW RD DK RH GK GS VT YV SA KG GN VY EG QW DM GK IS GY GK LK YS NG DE YE GE WV DG KM HG RG TY RY KE GD VY TG EW RD DK RH GK GV VV YV GA KG GN TY EG QW EN GK IN GY GK LT FS NG DV YE GE WV DG KM HG RG VY KY VD GD IY SG EW RD DK RH GK GT VT YV SS TG D
GN IY EG EW EN GK IN GF GM LC YN NG DK YE GE WL DG KM HG RG TY TY ED GD VY IG EW KN DK RH GK GC VK YK GN EN .. .. .. 80 .. .. .. .. 90 .. .. .. .. 10 0. .. .. .. 11 0. .. .. .. 12 0. .. .. .. 13 0. .. .. .. 14 0. .. .
Tgon
Eten
Cpar
Pfal
SV VE KY EG DW VN GK MH GH GK YI YS DG GV YE GD WI DG KM HG KG TY VF PN GN VY EG EW AH DM KD GY GV LT YQ NG E
AI VE KY DG DW VN GK MH GH GK YT YS DG GE YE GD WM DG KM HG KG KY VF PN GN QY DG EW AN DM KE GY GV LT YH NG E
QI IE KY EG DW VN GK MH GH GK YV YV DS AV YE GD WF EG SM HG KG TY IF PC GN VY EG EW VN DV KE GY GV LT YQ NG E
KI AE TY EG DW VD GK MQ GR GT YF FA DG GI YE GD WV DG KM EG KG VY KY LN GN KY EG EW IN DM KN GY GT LA YV NG E
.. .1 50 .. .. .. .1 60 .. .. .. .1 70 .. .. .. .1 80 .. .. .. .1 90 .. .. .. .2 00 .. .. .. .2 10 .. .. .. .
Tgon
Eten
Cpar
Pfal
KY EG YW KQ DK VH GK GT LT YT RG DK YI GD WM DA KK DG EG EL IY AN GD RF KG QW AD DR AN GF GV FT YA NG NR YE G
KY EG YW KA DR VH GK GT LT YS RG DK YV GE WV DA KK HG EG EL IY SN GD RF KG EW VD DR AC GF GV FQ YA NG NK YE G
KY EG YW KD GK VN GK GT LT YS RG DK YV GD WL DA KK HG EG EL FY SN ND RF KG NW VA DK AC GF GV YT YA NG NR YE G
LY EG YW KN DK VH GK GT LT YS KG DK YI GE WK YA KK CG EG EL IY AS GD KF KG QW KN DK AN GY GI LL YN NG NK YE G
22 0. .. .. .. 23 0. .. .. .. 24 0. .. .. .. 25 0. .. .. .. 26 0. .. .. .. 27 0. .. .. .. 28 0. .. .. .. 29 0
Tgon
Eten
Cpar
Pfal
EW TD DK RH GR GV FY CA ED GS AY EG EF VG GR KE GN GI LR LA TG HQ LE GT WS GG QL VR VT SF VF AQ DS PW LN VD L
QW AD DK RV GL GT FY CA ED GS CY EG EF VQ GR KE GN GT LR FA TG HQ LE GV WA GG EL VR VT AF MF SP DS RW NN PD YW EN DR RH GK GI FY CA ED NN VY EG EW AN GR KD GK GI LR FA MG HS IQ GV WK DG VL SQ FH SL QF PP ES QW SN PN F
EW LD DH RH GM GT FT CK ED GT IY SG HF QF NR KH GK GT LT FV NG HI LQ GI WN SG LL EK VI NY EL TP SS PW ND PD L
.. .. .. .3 00 .. .. .. .3 10 .. .. .. .3 20 .. .. .. .3 30 .. .. .. .3 40 .. .. .. .3 50 .. .. .. .3 60 .. .
Fig. 1. MORN1 is highly conserved among apicomplexans. The MORN1 protein consists of 14 MORN repeats. (Top) The PFam (Protein
families database) consensus sequence is aligned with one of the repeats and identical residues are highlighted in black. (Bottom) T. gondii
(Tgon) MORN1 was aligned with homologous proteins identified in P. falciparum (Pfal; PF10_0306; www.plasmodb.org), C. parvum (Cpar:
EAK89899; www.cryptodb.org) and E. tenella (Eten; Contig3337, nt 6439-7527; www.sanger.ac.uk). Residues identical in at least three out of
four species are highlighted in black, those that are similar are in grey. Nucleotide sequence data are available in the GenBankTM, EMBL and
DDBJ databases under the accession numbers DQ181547-48.
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Journal of Cell Science 119 (11)
Journal of Cell Science
homologs. Based on its conservation MORN1 was
studied in further detail.
Antibodies raised against recombinant MORN1
label the apical and posterior end of the inner
membrane complex
MORN repeat proteins play a role in membranemembrane and membrane-cytoskeleton interactions
(Shimada et al., 2004; Takeshima et al., 2000). To
explore the function of MORN1 in apicomplexans
antisera were raised against purified recombinant
protein. Sera were tested by western blotting using
purified recombinant protein and T. gondii protein
Fig. 2. Antibodies to MORN1 label a structure at the anterior and posterior
lysate. In both a band with an apparent molecular mass
end of the inner membrane complex (IMC). (A) Anti-MORN1 antiserum
of 41 kDa was detected; this band was absent using
reacted specifically with purified HIS-tagged MORN1 (HM1, 42.8 kDa,
pre-immune sera and no reactivity towards
second band probably represents a dimer) but not MORN2 protein (HM2,
recombinant MORN2 (HM2) was detected (Fig. 2A).
51.5 kDa) in western blot analysis. A band of slightly smaller molecular mass
The observed mass matches the mass predicted for the
consistent with native MORN1 was detected in parasite lysates (RH, 40.9
MORN1 protein (40.9 kDa). To locate the protein
kDa); no reactivity was observed with the preimmune serum (PI). (B-D) In
immunofluorescence assays this antiserum (green) produces staining at the
within the parasite, fibroblast cultures infected with T.
apical and posterior end of the IMC (red) of the parasite as well as a dot midgondii tachyzoites were fixed and reacted with the
cell (anterior label indicated by arrowheads). (E-G) In dividing cells daughter
MORN1 antiserum. A complex staining pattern was
IMCs are equally labeled. An additional structure in the IMC, most probably
observed with MORN1 present at the posterior and
the micropore, is also detected (arrow).
anterior ends of the cell as well as in a single dot in
the mid-cell region (Fig. 2B). Labeling at the posterior
end was considerably stronger. In parasites
undergoing cell division, the complexity of the pattern
double labeling assays using an antibody to ␣-tubulin this
increased, as additional lines and dots were observed (Fig. 2E).
segment of the spindle colocalizes with the nuclear MORN1
To provide reference, cells were double labeled with an
(arrowheads in Fig. 3E-G). Spindles are readily labeled in the
antibody specific to IMC1, a structural element of the IMC
closely related coccidian parasite Sarcocystis neurona
(Mann and Beckers, 2001). The MORN1 structures precisely
(Vaishnava et al., 2005). As shown in Fig. S1 in supplementary
colocalize with the apical and posterior end of the IMC in
material, double labeling experiments in developing S. neurona
mature parasites and in the forming daughter cells (Fig. 2D,G,
schizonts localize MORN1 (green) to the nuclear envelope
arrowheads indicate the apical end of cells). Interestingly, weak
precisely coinciding with the positions of the multiple spindles
but reproducible labeling of an additional structure within the
(␣-tubulin, red) found in this organism. Sarcocystis merozoites
IMC was observed (arrow in Fig. 2G). This structure could
emerging at the end of the developmental process show
possibly represent the micropore, an opening in the pellicle and
labeling indistinguishable from T. gondii tachyzoites.
the site of endocytosis (Nichols et al., 1994). No robust
MORN1-YFP transgenic lines were established to study the
molecular markers are currently available to confirm this
function of MORN1 during parasite development. Initial
assignment.
attempts using transgenes driven by the strong tubulin
promoter failed to yield stable transformants (see discussion of
MORN1 is a component of the centrocone, a specialized
MORN1 over-expression below). Toxicity was not due to the
structure of the intranuclear mitotic spindle
bulky YFP tag as transfection with constructs employing
To identify the localization of MORN1 at mid-cell, DNA was
epitope tags were equally detrimental (data not shown). We
stained with DAPI to label the nucleus. MORN1 was present
reasoned that strong and/or constitutive expression might be
at the anterior side within the nucleus (Fig. 3A). In some cells
toxic and decided to introduce the native promoter upstream of
two nuclear MORN1 dots were observed (Fig. 3B). The DNA
the coding region to provide appropriate expression.
content of individual nuclei in DAPI-stained preparations was
Transfection with this construct yielded stable fluorescent
measured by image analysis (see Materials and Methods for
transformants at a low frequency, which were isolated by cell
details) and scored for the number of nuclear MORN1 dots per
sorting (Southern and western blot analyses showed nonnucleus. As shown in Fig. 3D, nuclei with two MORN1 dots
homologous insertion; data not shown).
harbor, on average, double the amount of DNA of single
MORN1-YFP transgenics had a normal growth rate and
MORN1 dot nuclei (n=105, P<0.0001). Furthermore, in Upresented YFP localization identical to the pattern observed
shaped nuclei undergoing division the two dots were associated
with the antibody (Fig. 3H). Optical sectioning, deconvolution
with the leading edge of the daughter nuclei (Fig. 3C). These
and three-dimensional (3D) projection revealed that MORN1
observations are consistent with MORN1 localization to the
structures at the posterior end of cells and buds were rings
centrocone. The centrocone is a specialized structure of the
rather than lines (see Movies 1 and 2 in supplementary
apicomplexan nucleus associated with the intranuclear mitotic
material). Rings could also be observed at the electron
spindle (Dubremetz, 1975; Sheffield and Melton, 1968). The
microscopic level. Fig. 3N shows a cross section through the
spindle directly adjacent to the centrosome can be detected
posterior end of a tachyzoite with a ring of gold particles as a
using tagged tubulin transgenes (Striepen et al., 2000). In
result of labeling with the MORN1 antibody. In higher
Cell division in Toxoplasma
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Journal of Cell Science
Fig. 3. MORN1 is associated with the nucleus and undergoes
cell cycle dependent changes. (A-C) Combined MORN1
antibody (green) and DNA staining using DAPI (blue) reveals
a structure (arrowhead) associated with the nucleus that
doubles (B, note higher DNA content) and associates with the
daughters during division (C). (D) DNA content of nuclei was
measured by image analysis of DAPI-stained preparations and
plotted against the number of nuclear MORN1 dots (n=105,
P<0.0001). (E-G) Co-staining of MORN1 with anti-␣-tubulin
antibody resulted in colocalization of nuclear MORN1
(arrowhead) and tubulin (F). (H-K) In vivo labeling using a
MORN1-YFP transgene (see 3D projection of this dataset as
Movies 1 and 2 in the supplementary material). These
parasites were transfected with (I) GRASP55-RFP (Golgi), (J)
FNR-RFP (apicoplast) and (K) centrin-RFP (centrosome).
(L-Q) Immunoelectron microscope analysis of MORN1
structures. (L) Gold particles mark the posterior end of a
tachyzoite (arrowheads), higher magnification of the same
area (M) shows the labeling at the inside of the IMC. In
parasite cross sections the MORN1 antibody labels a ring
structure (N). O shows a tachyzoite early in division. White
arrowheads highlight the newly forming daughter IMC apical
of the nucleus (N). The centrocone (black arrow) appears
electron dense in the lumen and below the membrane that
separates it from the nucleoplasm (see Fig. 7E for a schematic
view of this structure). (P,Q) Higher magnifications show gold
particles located with this dense material. The edge of the
daughter IMC (white arrowheads) is equally labeled.
centrocone (black arrow) as well as the outline of an
adjacent daughter bud (arrowheads) are visible. Fig. 3P
and Q show two higher magnifications of centrocones.
Labeling is consistently apparent in the triangular section
of the centrocone (arrow) as well as in the dense material
underlying the centrocone within the nucleoplasma
(double arrowheads). The dense material on both sides of the
membrane is of comparable density and structure, suggesting
a common composition supported by the MORN1 location.
Also note that the cisternae of early forming IMC (arrowheads)
show MORN1 labeling at their ends on the side of the
membrane facing the centrocone.
magnifications of the posterior end of tachyzoites gold labeling
is found on the inner (cytoplasmic) face of the IMC (Fig.
3L,M).
To provide further subcellular references additional
transgenes were introduced by transfection into the MORNYFP clone resulting in labeling with red fluorescent protein of
the nucleus (H2B-mRFP), apicoplast (FNR-RFP), centrosome
(centrin-RFP), and Golgi (GRASP-RFP). The Golgi, and
especially the apicoplast, show association with the
centrosome of the mitotic spindle during cell division (Pelletier
et al., 2002; Striepen et al., 2000; Vaishnava et al., 2005).
Consistent with centrocone localization of nuclear MORN1,
close proximity was observed between MORN1-YFP and
GRASP-RFP, FNR-RFP and centrin-RFP (arrows in Fig. 3IK). These observations were confirmed by electron
microscopy. Fig. 3O shows a longitudinal section through the
apical half of a tachyzoite. A central nucleus (N) with a
MORN1-associated structures are highly dynamic
during mitosis and budding
To gain further insight into the behavior of MORN1-associated
structures parasites double labeled with MORN1-YFP and
histone H2B-mRFP (nucleus) were time-lapse imaged.
Infected cultures were observed for 5 hours at 37°C and stacks
of optical sections were taken in both channels every 10
minutes. Fig. 4 shows a panel of selected frames; the entire
movie is available in the supplementary material (Movie 3).
Two double-labeled tachyzoites are visible in Fig. 4A, a single
dot of centrocone label is present in each nucleus (arrow in Fig.
4D) and two faint rings, the first signs of newly forming
daughter buds, are seen in close proximity (double arrows).
The centrocone label then increases in size and intensity and
stretches into a bar perpendicular to the longitudinal axis of the
parasite (Fig. 4E,F). The daughter rings move apart in
association with both ends of the bar. Next the bar splits into
two discrete dots (Fig. 4G). Concurrently the daughter rings
further increase in size and a central (conoidal) ring becomes
visible (arrow in Fig. 4G). The conoidal ring is pushed away
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Journal of Cell Science 119 (11)
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Fig. 4. Time-lapse analysis of the dynamic MORN1
localization during T. gondii cell division. (A) MORN1YFP (green) and H2B-mRFP (red, nucleus) transgenic
parasites were imaged every 10 minutes. The entire
dataset is available as Movie 3 in the supplementary
material. Distances between the daughter and mother cell
MORN1 structures (B) are plotted over time (C, average
of both daughters given). (D-K) Selected time points (as
indicated in C) are shown at higher magnification for one
cell (boxed in A). Arrow in (D) highlights centrocone,
double arrows the first sign of daughter buds. Centrally
forming apical ring is marked by arrow before (G) and
after extrusion (H). An additional weakly labeled MORN1
is apparent in I, arrow. Note ring constriction from J to K.
from the nucleus towards the apex of the mother cell thus
establishing the hollow cylinder shape of the IMC (arrows Fig.
4H). The posterior daughter rings then push back onto the
nucleus engulfing and dividing it into two equal halves in the
process (Fig. 4I,J, also see Movie 4 in the supplementary
material). Finally, both rings markedly constrict, coinciding
with fission of the nucleus into two daughter nuclei (Fig. 4K).
Deconvolution microscopy also revealed the presence of a
second MORN1 structure in the nucleus (highlighted by arrow
in Fig. 4I). Labeling was considerably weaker when compared
with the centrocone. This structure moves within the nucleus
during mitosis and finally comes to rest at a position in the
middle between the two emerging daughter nuclei.
Movements of MORN1 structures were quantified by image
analysis of each deconvolved stack. Distances between the
daughter centrocone and the apical (red) and posterior (green)
MORN1 ring of the daughter and the posterior ring of the
mother (black) as well as the diameter of the posterior daughter
ring (blue) were measured (see Fig. 4B,C) and plotted over
time (distances were recorded for both daughters and the
average is shown). The conoidal and the posterior MORN1 ring
start moving at the same time. After the nucleus slides back
initially as previously observed (Radke et al., 2001) it is held
at a constant mid-cell position. Note that constriction of the
ring coincides with the end of the longitudinal movements.
MORN1 interacts with the parasite’s cytoskeleton
The MORN1 ring is highly dynamic during daughter cell
formation, however, MORN1 does not contain any ATPase
domains suggesting that the force for this movement is most
probably generated through interaction with other proteins. We
therefore hypothesized that MORN1 might interact with
elements of the parasite’s cytoskeleton. The T. gondii
cytoskeleton is resistant to extraction with Triton X-100 and
deoxycholate (Mann et al., 2002; Morrissette et al., 1997) and
co-fractionation in extraction assays has been a hallmark of
proteins associated with the cytoskeleton (Gaskins et al.,
2004). MORN1 showed marked resistance to Triton X-100
extraction and 82% of the protein remained in the pellet
fraction (Fig. 5B, this was highly reproducible, n=3,
s.d.=8.4%). By contrast, deoxycholate solubilized 80% of the
MORN1. Note that recombinant MORN1 protein is soluble in
1% Triton X-100 up to a concentration of 100 ␮g/ml (data not
shown).
The force driving the ring constriction could potentially be
generated by the actin/myosin system. Several differentially
localized myosins have been characterized in T. gondii. Myosin
A is found at the periphery of the cell and is essential for
gliding motility and invasion (Meissner et al., 2002). Myosin
C (MyoC) has been previously suggested to have a role in
cytokinesis (Delbac et al., 2001). To test if myosin C could be
a candidate interactor and motor for the MORN1 ring, parasites
stably expressing a Myc-tagged version of MyoC (a kind gift
from D. Soldati, University of Geneva, Switzerland) were
double labeled with antibodies to MORN1 (red) and Myc
(green). In dividing tachyzoites MyoC forms distinct rings at
the posterior end of the daughter buds that precisely colocalize
with the MORN1 rings (arrows and insets in Fig. 5C,D,F). As
shown here and reported previously (Delbac et al., 2001),
MyoC staining is not limited to these rings.
Overexpression of MORN1 severely perturbs parasite
nuclear division and cytokinesis
Based on the morphological and functional characterization of
MORN1 we hypothesized that the overexpression toxicity
observed for this gene might be due to disruption of parasite
cell division. To test this hypothesis transient transfection
experiments were performed using MORN1 transgenes driven
by the strong tubulin promoter and parasites were subjected to
immunofluorescence or live cell microscopy 24-48 hours after
transfection (MORN1-RFP is shown but MORN1-YFP
transgenics behaved identically). Parasites overexpressing
MORN1-RFP grow to considerable size but fail to divide
(compare Fig. 6B and D, which are shown at the same
magnification). These cells contain a large unsegregated
nucleus (compare Fig. 6C with D upper panel). The DNA
Cell division in Toxoplasma
Journal of Cell Science
content of parasite nuclei was measured 48 hours after transient
transfection as described above and plotted against MORN1RFP expression (Fig. 6E). The mean DNA content of MORNRFP expressors (+) was on average 11-fold higher (n=120,
P<0.0001) than that of untransfected cells (–). This suggests
that MORN1 overexpression does not interfere with DNA
replication yet prevents nuclear segregation and budding. Cells
were double labeled with IMC3-YFP or YFP-TUB to highlight
IMC and microtubules. Some new IMC seemed to form in
MORN1 overexpressors, however, no discernable daughter
buds could be identified (compare Fig. 6G and I). By contrast,
Fig. 5. MORN1 interacts with the cytoskeleton. Parasites were
extracted using different detergents and separated into pellet (P) and
soluble (S) fraction. Controls consist of intact parasites (PBS, all
protein in pellet) and parasites solubilized by boiling in 1% SDS (all
proteins in supernatant). (A) Extracts were subjected to SDS-PAGE,
blotted and probed with antibodies. Cytoplasmic YFP-YFP (Gubbels
et al., 2003) served as fully soluble control. (B) MORN1 signals
were quantified and expressed as percentages of the pellet plus
supernatant sum. (C-F) Parasites expressing Myc-tagged myosin C
were labeled with antibodies to MORN1 (red, C) and Myc epitope
(green, D). A vacuole containing four dividing parasites is shown;
note posterior division rings (indicated by arrows and in inset)
labeled with both antibodies.
2241
overexpressors produced multiple well-formed microtubular
skeletons per cell [arrow in Fig. 6K highlights one of the
intensely labeled conoids (Hu et al., 2002b)]. However, the
subpellicular microtubules frayed broadly and were not cupped
as observed in wild-type parasites (see dotted lines in Fig.
6L,M). MORN1 in overexpressors typically localized to three
intensely fluorescent rings (Fig. 6A,F,J), which showed no
association with the newly formed microtubular skeletons but
were labeled with an IMC marker (arrows in Fig. 6G,H).
Pharmacological analysis of MORN1 dynamics
To further study the interactions of the MORN1 rings with the
cytoskeleton, pharmacological experiments were performed
using actin- or tubulin-disrupting drugs, cytochalasin D and
oryzalin, respectively. Cytochalasin-resistant host cells
[KB100 Cyt1 a kind gift from David Sibley, Washington
University (Dobrowolski and Sibley, 1996; Toyama, 1984)]
were infected for 2 hours with parasites and then treated with
0, 2.5, 5 and 10 ␮M cytochalasin D, fixed after 24 and 48 hours
and stained for MORN1. No difference in the number or
morphology of MORN1 rings was observed regardless of the
drug concentration (data not shown). Note that the
actin/myosin-dependent motility and invasion of T. gondii is
completely abolished by 1 ␮M cytochalasin (our controls)
(Dobrowolski and Sibley, 1996). T. gondii-infected cultures
were also treated with 0.05, 0.25 or 2.5 ␮M oryzalin. 2.5 ␮M
oryzalin completely abolished any intracellular parasite
development. At lower concentrations parasites grew to
considerable size as previously reported but no clear buds were
visible. However, individual sheets of IMC were formed, each
associated with a region of MORN1 staining (arrowheads in
Fig. 6N-P). Interestingly, the duplication of the centrocone was
highly susceptible to oryzalin treatment; only a single
centrocone (arrow in Fig. 6P) can be detected in the polyploid
nucleus. Taken together these observations suggest that
microtubules, but not actin, play a role in the biogenesis of the
MORN1 ring and IMC.
Discussion
MORN repeat domains are found in proteins of organisms
across the tree of life. MORN repeats are often combined with
additional enzymatic domains as in the phosphatidylinositol4-phosphate 5-kinases found in many plants (Ma et al., 2004)
or the human Rab5 guanine exchange factor ALS2 (Hadano et
al., 2001). Most importantly in the context of this work, MORN
motifs play critical roles in several proteins with roles in the
organization of membranous and cytoskeletal structures.
Examples are the junctophilins, which are critical for the tight
appositions of endoplasmic reticulum and plasma membrane
in excitable cells (Takeshima et al., 2000). Loss or mutation of
these proteins prevents junctional complex assembly and leads
to neuronal dysfunction (Holmes et al., 2001; Takeshima et al.,
2000). MORN proteins have also been implicated in the
biogenesis of the sperm flagellum (Ju and Huang, 2004; Satouh
et al., 2005). Since no enzymatic activity has been associated
with the MORN structure it has been suggested that MORN
repeats act as protein-protein or protein-phospholipid binding
domains. This would be consistent with the observation that
MORN proteins are frequently part of larger protein complexes
(Ju and Huang, 2004; Kunita et al., 2004; Satouh et al., 2005).
In this study we show that T. gondii MORN1 is associated
Journal of Cell Science
2242
Journal of Cell Science 119 (11)
Fig. 6. Overexpression of MORN1-RFP
abolishes parasite cell division. (A-D) Expression
of MORN1-RFP driven by the strong ␣-tubulin
promoter results in ring-shaped accumulations of
RFP signal (24 hours after transfection) and
morphological defects of the parasite. Note large
size of cell (B) and nucleus (C) along with
changes in shape when compared to
untransfected controls (D). (E) Nuclear DNA
content in transfected (+) and untransfected (–)
parasites was quantified using image analysis 48
hours after transfection (arbitrary units of DAPI
fluorescence; n=120, P<0.0001, error bar show
standard deviation). (F-M) Parasites expressing
IMC3-YFP (F-I, green) or YFP-tubulin (J-M,
green) were transiently transfected with
tubMORN1-RFP (red). Untransfected controls
are shown in I and M. Some IMC3-YFP
colocalized with MORN1 (arrows) but no
discernable buds were formed. Multiple daughter
tubulin baskets have been initiated and extended
in the mutant, however, microtubules are not
constricted at the posterior end as observed in the
wild type (dotted lines in L and M). (N-P) RHinfected cultures were treated with 0.05 ␮M
oryzalin and stained with anti-MORN1 (N) and
anti-IMC1 (O) antibody. Note multiple
overlapping MORN1 and IMC1 structures
(arrowheads) and a single centrocone in the
nucleus (arrow, P).
with a set of subcellular structures that play key roles in
parasite mitosis, daughter cell formation and cytokinesis.
These structures fall into two classes: the nuclear centrocone
and several IMC-associated rings (see Fig. 2 and schematic
representation in Fig. 7). This suggests a role for MORN1 in
A
E
B
C
D
capping and or anchoring the ends of the IMC cisternae. Such
a role is supported by the tight colocalization of MORN1 and
IMC (Fig. 2), and the coordinated movements of both
structures during intracellular development (Fig. 4).
Furthermore, disruption of the MORN1 rings through MORN1
overexpression prevents budding (Fig. 6).
MORN1 could bind directly to the IMC
membrane, however, MORN1 lacks a
membrane-spanning domain or consensus
motifs for lipid modification. Previous studies
on junctophilins have shown that MORN
repeats by themselves can provide attachment
to the cytoplasmic face of the ER through
F
C
MT
MT
CC
NE
K
IMC
MyoC
MORN
Fig. 7. MORN1 in apicomplexan cell division.
MORN1 is shown in green, tubulin and
centrosome in red, IMC in dark blue and the
nucleus in light blue. (A) The centrocone persists
through interphase. (B) The centrosome duplicates
and sets up the mitotic spindle, splitting the
centrocone into two. (C) Mitosis occurs and apical
and posterior MORN1 rings form for each
daughter. (D) During budding, subpelicular
microtubules push the MORN1 rings down while
the spindle pushes the centrocones up.
Microtubule driven movements are indicated by
red arrows, constriction is shown in green (C,D).
(E) Schematic representation of the centrocone: C,
centrosome; CC, centrocone; MT, microtubules;
NE, nuclear envelope; K, kinetochores. (F) A
model for the structure and function of the
posterior ring in daughter buds.
Journal of Cell Science
Cell division in Toxoplasma
interaction with phospholipids (Takeshima et al., 2000).
Alternatively, MORN1 could interact with a component of the
membrane skeleton that associates with the inner face of the
IMC. Our electron microscopic analysis localizes the MORN1
ring to the inside of the IMC where the membrane skeleton is
found (Gaskins et al., 2004; Mann and Beckers, 2001). Lastly,
an integral IMC membrane protein could serve as an adapter
analogous to TgGAP50, which anchors the actin/myosinA
gliding machinery to the IMC (Gaskins et al., 2004). The
current data do not favor one model over the other. Identifying
molecular interactors of MORN1 will help to solve this
question and is the focus of our future work.
The posterior MORN1/IMC ring is highly dynamic during
mitosis and cell division. It moves along the longitudinal axis
and constricts perpendicular to this axis at the end resulting in
nuclear division and cytokinesis (Fig. 4, red and green arrows
in Fig. 7D,F). How does the MORN1 ring move in the absence
of motor domains in the MORN1 sequence? The tubulin and
actin/myosin cytoskeletons of the parasite provide two obvious
candidates. The subpellicular microtubules are organized by
the apical polar ring and grow by addition of monomers at the
distal end (Russell and Burns, 1984). Microtubular outgrowth
seems critical to budding, as daughter cell formation is
impeded under oryzalin (Morrissette and Sibley, 2002b; Shaw
et al., 2000; Stokkermans et al., 1996). Daughter cell IMC still
forms under oryzalin but is present as flat sheets rather than the
usual hollow cylinders (Stokkermans et al., 1996) (Fig. 6).
Interestingly, the ends of these sheets are still associated with
MORN1 (see Fig. 6). Based on these observations we
hypothesize that subpellicular microtubules are not essential to
initiate new IMC and MORN1 complexes, however, they are
critical to shape and extend the structure. This idea is supported
by the MORN1 overexpression data where multiple intact
microtubular skeletons form in the absence of functional
MORN1 rings. However, in the absence of a functional
MORN1 rings the distal ends of the microtubules fray and are
no longer tied into the typical barrel shape.
Although microtubular growth provides a convincing model
for longitudinal movement of the MORN rings during mitosis
(see Fig. 7 for a schematic outline) it is unclear how
microtubules could be responsible for its constriction (Fig. 4).
Constrictive actin-myosin rings are well characterized
elements of the cytokinesis machinery (Glotzer, 2005).
Recently myosin C and actin were shown to localize to the
anterior and posterior end of the parasite (Delbac et al., 2001;
Patron et al., 2005). Furthermore over-expression of myosin
myoB/C-tail caused a cell division phenotype (Delbac et al.,
2001). In this study we have shown that MORN1 and MyoC
colocalize in the posterior division ring. When MORN1 rings
are disturbed by MORN1 over-expression we no longer detect
myosin rings (data not shown), and budding is inhibited. These
data are consistent with a model (Fig. 7F) for budding in which
MORN1 acts as a linker between the posterior end of the IMC
and an internal constrictive ring formed by MyoC. However,
MORN1 ring formation and budding are resistant to
cytochalasin D treatment [our data and a previous electron
microscopic study (Shaw et al., 2000)]. Although this result
does not support involvement of actin it also does not exclude
it. Several clearly actin-dependent processes appear to be
resistant to cytochalasin D; this includes the constriction of the
cleavage furrow in fission yeast (Pelham and Chang, 2002).
2243
Further work is needed to fully delineate the components and
mechanism of this constrictive ring.
MORN1 represents the first molecular marker for the
centrocone. The centrocone is a unique apicomplexan structure
associated with the intranuclear spindle that has been described
at the electron microscopic level (Dubremetz, 1973;
Dubremetz, 1975; Sheffield and Melton, 1968). It is cone
shaped and delineated by the nuclear envelope with an opening
at the apical end where the centrosomes are found on the
cytoplasmic side (see Fig. 7E for a schematic representation).
Depending on the stage in division the cone may be separated
from the nucleoplasm by a membrane, which is continuous
with the nuclear envelope. This membrane is perforated by
spindle microtubules, which emanate from the cone and are
organized by the centrosome. Electron-dense material fills the
cone in between the microtubules and underlies the centrocone
on the nucleoplasmic site where the kinetochores are attached
(Dubremetz, 1973). MORN1 is found in both of these locations
(Fig. 3O-N, Fig. 7E). MORN1 overexpression prevents the
formation of new centrocones as the cells progress through the
cell cycle. Formation of new centrocones is equally dependent
on microtubules. Whereas multiple IMC-MORN1 structures
are formed under oryzalin treatment only a single centrocone
is found within the large polyploid nucleus. This is consistent
with the view that the centrocone cannot be formed de novo
and requires duplication, which in turn requires a functional
mitotic spindle.
Spindles are generally restricted to mitosis. We were
therefore surprised to find a MORN1-labeled structure
consistent with the location of the centrocone in every
tachyzoite regardless of its stage in the cell and division cycle.
We have recently described persistence of short spindles and
centrocones through interphase in the closely related parasite
S. neurona (Vaishnava et al., 2005). As expected, MORN1 colocalizes with spindles in S. neurona (Fig. S1 in supplementary
material). The new observation in T. gondii suggests that
centrocone persistence (and with it potentially persistence of
kinetochore attachment) could be a general theme of
apicomplexan nuclear organization.
Materials and Methods
Parasites and cells
RH-strain T. gondii tachyzoites were passaged in confluent human foreskin
fibroblasts as described previously (Roos et al., 1994). Sarcocystis neurona
merozoites were passaged in primary bovine turbinate cells as described previously
(Vaishnava et al., 2005). MORN1-YFP parasites were isolated after transfection
with plasmid 5⬘morn1MORN1-YFP/DHFR/3⬘-morn1 under pyrimethamine
selection and enrichment using a MoFlo high-speed cell sorter (DAKO/Cytomation,
Fort Collins, CO, USA) as described previously (Gubbels et al., 2004). The
following lines have been described previously: IMC3-YFP (Gubbels et al., 2004),
YFP-␣-tubulin (Striepen et al., 2000) and Myc-myosin C (Delbac et al., 2001).
Sequence analysis
Recovered
sequences
were
searched
against
www.ToxoDB.org,
www.PlasmoDB.org, www.CryptoDB.org, www.GeneDB.org and the NCBI
non-redundant database. Sequences were aligned using ClustalW
(www.ebi.ac.uk/clustalw), and prepared for publication using BoxShade
(http://bioweb.pasteur.fr/seqanal/interfaces/boxshade.html). Identity and similarity
numbers were calculated using MatGat software Version 2.03 (Campanella et al.,
2003).
5⬘- and 3⬘-RACE of MORN1 and MORN2
Total RNA was extracted from RH tachyzoites using Trizol (Invitrogen). RT-PCR,
5⬘-RACE and 3⬘-RACE were performed using the SMART RACE cDNA
amplification kit (BD BioSciences) following the manufacturer’s instructions using
primer 1 and 2 (MORN1), and 3 and 4 (MORN2; see supplementary material Table
2244
Journal of Cell Science 119 (11)
1 for the sequences of primers used in this study). Amplified fragments were cloned
into pCR2.1 using the TOPO-TA cloning protocol (Invitrogen).
Journal of Cell Science
Plasmid construction
Plasmid tubMORN1-YFP/sagCAT was cloned by PCR amplification of the
MORN1 ORF from genomic DNA using primers 5 and 6 and replacement of the
first YFP in tubYFPYFP/sagCAT (Gubbels et al., 2003) by BglII/AvrII digestion.
5⬘morn1-MORN1-YFP/sagCAT was generated by amplifying the 1920 bp
preceding the start-codon using primers 7 and 8 and replacing the tub-promoter
in tubMORN1-YFP/sagCAT by PmeI/BglII digestion. Plasmid 5⬘morn1-MORN1YFP-3⬘dhfr/sagDHFR/3⬘morn1 was constructed by amplification of 5⬘morn1MORN1-YFP-3⬘dhfr using primers 9 and 10 and cloned at the ApaI/ClaI sites
into pKO-DHFR (see below) resulting in 5⬘morn1MORN1-YFP/DHFR. The
3⬘morn1 genomic region was amplified (primers 11 and 12) and inserted by
XbaI/NotI digestion into plasmid 5⬘morn1MORN1-YFP/DHFR resulting in
5⬘morn1-MORN1-YFP-3⬘dhfr/sagDHFR/3⬘morn1. pKO-DHFR was constructed
from the pKO plasmid (Martin Gastens, unpublished; this pKS-based plasmid
carries a T. gondii tub-CAT-sag1 cassette flanked on both sides by a multiple
cloning site); first the tub promoter was replaced with the sag promoter from the
sagCATsag cassette by HindIII/NsiI digestion. The CAT gene was replaced with
DHFR-TS m2m3 ORF using NsiI/PacI digestion [primers 14 and 15; the internal
BglII site within DHFR-TS was deleted using primer 13 in the megaprimer
approach (Lai et al., 2003)].
Plasmid morn1CentrinYFP/sagCAT was constructed by amplifying the centrin
ORF from cDNA (primers 16 and 17) and cloning via BglII/AvrII digestion into
morn1-MORN1-YFP/CAT. Plasmid tubH2B-mRFP/sagCAT was constructed by
replacing YFP (BglII/AvrII) in tubYFP-mRFP/CAT with H2B from tubH2BYFP/CAT [kindly provided by K. Hu and D. S. Roos, University of Pennsylvania
(Hu et al., 2004)]. The monomeric mRFP originated from the pDsRed-monomerN1 plasmid (BD BioSciences) and was PCR amplified (primers 18 and 19) deleting
the internal BglII site using the megaprimer approach (primer 20). The amplified
mRFP was cloned AvrII/AscI into a tubYFP-YFP(AscI)/sagCAT plasmid (Michael
Kurth, M.J.G. and B.S., unpublished). Plasmid tubGRASP55-RFP/sagCAT was
cloned by excision of GRASP55 from tubGRASP55-YFP/sagCAT (Pelletier et al.,
2002) (kindly provided by G. Warren, Yale University, USA) and replacing
ferredoxin NADH reductase (FNR) in tubFNR-RFP/sagCAT (Striepen et al., 2000).
Recombinant protein expression, antibody production and
affinity purification
The MORN1 ORF was amplified from genomic DNA using primers 21 and 22 and
cloned by ligation independent cloning into plasmid pAVA421 (Alexandrov et al.,
2004) as described previously (Aslanidis and de Jong, 1990) to generate a six Histagged N-terminal fusion. Full-length MORN2 was similarly cloned (primers 23
and 24). Recombinant fusion proteins were purified in the presence of 6 M urea on
Ni2+-NTA resin (Qiagen, Hilden, Germany). Polyclonal antibody was generated by
rabbit immunizations (Cocalico Biologicals, Reamstown, PA, USA). MORN1
antibodies were affinity purified against purified His-tagged protein cross-linked to
activated CNBr Sepharose 4B (Sigma, St Louis, MO, USA) as described previously
(Harlow and Lane, 1988).
Immunofluorescence and microscopy
Images were taken using a DM IRB inverted microscope (Leica) equipped with a
PL-APO 100⫻/1.4 NA oil emersion lens and a Hamamatsu C4742-95 camera and
adjusted for contrast using Openlab software (Improvision). For
immunofluorescence we used the following antibodies: affinity purified antiMORN1 (1:100), monoclonal 45.15 anti-IMC1 (1:1000; a kind gift from Gary
Ward, University of Vermont, USA (Mann and Beckers, 2001), rabbit anti-GFP
(1:5000; Torrey Pines Biolabs, CA, USA), monoclonal antibody 12G10 anti-␣tubulin (1:10; a kind gift from Jacek Gaertig, University of Georgia, USA) (JerkaDziadosz and Frankel, 1995), monoclonal anti-Myc epitope tag 9E10 (1:1000). Goat
anti-rabbit and goat anti-mouse antibodies conjugated with Alexa Fluor 488 or
Alexa Fluor 546 (Molecular Probes, Eugene, OR, USA) were used as secondary
antibodies at 1:200 dilution. DNA was visualized by staining with 4⬘,6-diamidino2-phenylindole (DAPI; 2 ␮g/ml in PBS) for 5 minutes. To measure the DNA content
of individual nuclei, the intensity of DAPI staining was measured in situ by image
analysis. Images were recorded with constant exposure time and within the linear
range of the CCD (the contrast was not adjusted). Nuclei were defined as objects
by generating binary image masks using Openlab software. The mean pixel intensity
was multiplied by the area for each mask to obtain a cumulative intensity
measurement in arbitrary units. More than 50 nuclei were measured for each sample
and statistical significance was evaluated using the Student’s t-test. For
deconvolution, specific time-lapse experiments (as indicated), were performed on a
DeltaVisionRT microscope (Applied Precision, Issaquah, WA, USA). SoftWoRx
software was used for image analysis and presentation.
Electron microscopy
Vero cells infected for 24 hours with T. gondii were resuspended using 5 mM EDTA
in PBS, pelleted, and fixed for 15 minutes at room temperature with 2%
paraformaldehyde and 0.1% glutaraldehyde in 0.2 M sodium phosphate buffer pH
7.4. The pellet was dehydrated in ethanol at –20°C and embedded in LRWhite
(London Resin Co, Berkshire, UK). Thin sections were collected on carbon-coated
grids and saturated for 30 minutes with 2.5% non fat dry milk and 0.1% Tween 20
in PBS (PBS-MT). The grids were floated successively for 1 hour each on affinity
purified rabbit anti-MORN1 serum diluted 1:40, followed by protein A 10 nm gold
diluted in PBS-MT, with PBS washes between each step. The grids were then
stained with uranyl acetate and lead citrate and observed with a Jeol 1200EX
electron microscope operated at 80 kV.
Detergent extractions
Detergent extractions were performed essentially as described previously (Gaskins
et al., 2004). Extracts were spun for 20 minutes at 4°C, supernatants were recovered
and pellets solubilized in SDS extraction buffer. Samples were run on 10% Bis-Tris
gels and transferred to nitrocellulose. Blots were probed with the following
antibodies: rabbit anti-MORN1 (1:1000), monoclonal 45:15 anti-IMC-1 (1:2000);
monoclonal 12G10 anti-␣-tubulin (1:500), rabbit anti-GFP (1:5000) followed by
either goat anti-rabbit or goat anti-mouse conjugated to horseradish peroxidase
(1:3000; Bio-Rad). Enzyme activity was visualized using enhanced
chemiluminescence (Pharmacia) and signals were recorded on X-ray film or directly
quantified on a GeneGnome (Syngene, Cambridge, UK).
This work was funded in part by grants from NIH-NIAID to B.S.
and a postdoctoral fellowship to M.J.G. from the American Heart
Association. We thank Julie Nelson for help with cell sorting,
Véronique Richard (SCME-UM2) for immunoelectron microscopy
specimen preparation. We are also grateful to D. Soldati, G. Warren,
D. S. Roos, L. D. Sibley, G. Ward, and J. Gaertig for antibodies,
plasmids and parasite strains, D. Soldati and L. D. Sibley for
discussion, and J. Gaertig for critical reading.
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