Research Article
947
Microneme protein 8 – a new essential invasion factor
in Toxoplasma gondii
Henning Kessler1, Angelika Herm-Götz1, Stephan Hegge1, Manuel Rauch1, Dominique Soldati-Favre2,
Friedrich Frischknecht1 and Markus Meissner1,*
1
Hygieneinstitute, Department of Parasitology, University Hospital Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany
Department of Microbiology and Molecular Medicine, University of Geneva, CMU, 1, rue Michel-Servet 1211, Geneva 4, Switzerland
2
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 7 January 2008
Journal of Cell Science 121, 947-956 Published by The Company of Biologists 2008
doi:10.1242/jcs.022350
Summary
Apicomplexan parasites rely on sequential secretion of
specialised secretory organelles for the invasion of the host cell.
First, micronemes release their content upon contact with the
host cell. Second, rhoptries are discharged, leading to the
formation of a tight interaction (moving junction) with the host
cell, through which the parasite invades. The functional
characterisation of several micronemal proteins in Toxoplasma
gondii suggests the occurrence of a stepwise process. Here, we
show that the micronemal protein MIC8 of T. gondii is essential
for the parasite to invade the host cell. When MIC8 is not
present, a block in invasion is caused by the incapability of the
parasite to form a moving junction with the host cell. We
Introduction
The phylum Apicomplexa consists of several pathogens of immense
medical and veterinary importance, such as those causing malaria,
toxoplasmosis or coccidiosis. Because they are obligate intracellular
parasites, a key step during their life cycle is the invasion of the
host cell. Apicomplexan parasites actively invade the host cell in
a complex, organised, stepwise process (Carruthers and Boothroyd,
2006) that involves specialised secretory organelles (the
micronemes, rhoptries and dense granules) and a unique form of
movement termed gliding motility (Soldati and Meissner, 2004).
Upon initial attachment to a surface, the parasite starts to glide in
a substrate-dependent manner. Gliding involves the discharge of
adhesive transmembrane proteins at the apical end by specialised
secretory organelles called micronemes. It has previously been
demonstrated that micronemal proteins of the TRAP family
(thrombospondin-related anonymous protein) are essential for
gliding motility and invasion (Huynh and Carruthers, 2006; Sultan
et al., 1997) by binding to host-cell receptors and bridging them to
the cytoplasmic actin-myosin motor of the parasite via the glycolytic
enzyme aldolase (Buscaglia et al., 2003; Jewett and Sibley, 2003;
Starnes et al., 2006). The next step in invasion is the recognition
of the host cell followed by rhoptry secretion and intimate
attachment to the host cell. A conditional-mutagenesis approach for
the micronemal protein AMA1 has demonstrated a distinct role for
a micronemal protein during invasion. In the absence of AMA1,
the parasite fails to form a moving junction (MJ) (Mital et al., 2005).
The formation of the MJ depends on the secretion of rhoptry neck
proteins (RONs) and their interaction with AMA1 (Alexander et
al., 2005; Lebrun et al., 2005). In the absence of AMA1, the RONs
are still secreted and associate with the host cell in loose patches
but do not form an MJ (Alexander et al., 2005; Boothroyd and
Dubremetz, 2008). Up until now, the events leading to rhoptry
furthermore demonstrate that the cytosolic domain is crucial
for the function of MIC8 and can not be functionally
complemented by any other micronemal protein characterised
so far, suggesting that MIC8 represents a novel, functionally
distinct invasion factor in this apicomplexan parasite.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/121/7/947/DC1
Key words: Apicomplexa, Invasion, Microneme, Rhoptry, Tet system,
Toxoplasma
secretion and subsequently to the formation of the MJ are not well
understood. The functional linkage between micronemes and
rhoptries led us speculate that additional micronemal proteins might
be responsible for the establishment of the MJ.
Micronemal protein 8 (MIC8) was previously identified by
sequence similarity to other micronemal proteins in the genome of
Toxoplasma gondii. Analysis of MIC8 has demonstrated that it is,
like other micronemal transmembrane proteins, secreted upon an
increase in intracellular calcium concentration and processed close
to its transmembrane domain (TMD). Furthermore, it has been
demonstrated that MIC8 interacts with the soluble micronemal
protein MIC3 during the transit through the secretory pathway
(Meissner et al., 2002a). Here, employing a conditional geneexpression system, we present a detailed functional characterisation
of this essential protein (Meissner et al., 2002b). We show that
invasion depends on the function of MIC8. Further detailed
characterisation of the resulting phenotype showed that this protein
does not contribute to gliding motility. Instead it appears that MIC8
plays an essential role in a step before the formation of an MJ. We
show, in a novel complementation strategy, that the cytosolic
domains (CTDs) of MIC2, PfTRAP or AMA1 cannot substitute the
function of the MIC8 CTD, whereas the CTD of the homologous
protein MIC8-like1 restores function. This indicates that this
transmembrane protein is involved in a novel invasion step leading
to formation of the MJ.
Results
Establishment of inducible conditional MIC8 knockout
We employed a tetracycline-inducible system to generate a
conditional knockout mutant for mic8. Parasites expressing the
tetracycline-dependent transactivator TATi1 (Meissner et al., 2002b)
were transfected with a construct encoding a recombinant Ty-tagged
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948
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version of MIC8 (MIC8Ty) under control of the Tet-inducible
promoters pT7S1 and pT7S4 (Meissner et al., 2001). We generated
three independent clones expressing mic8Ty at different levels when
grown in the absence of anhydrotetracycline (ATc) (Fig. 1A). The
strongest-expressing clone (T7S4-5) showed a partial localisation
of MIC8 in an apically localised compartment that corresponded
to early endosomes (Fig. 1B, Fig. 2D). By contrast, parasite strains
expressing the transgene at a lower level showed a normal
localisation of MIC8Ty in the micronemes. Interestingly, prolonged
culture in the absence of ATc resulted in loss of transgene expression,
indicating that overexpression of MIC8 is unfavourable for the
parasite (data not shown).
We removed endogenous mic8 in transgenic parasites (T7S1-4
and T7S4-1) via homologous recombination with the construct
MIC8KOCAT (see Materials and Methods) (Fig. 1C) and obtained
two independent clonal parasite populations, as shown in analytical
PCRs on genomic DNA (Fig. 1D). Both clones, MIC8KOi-T7S14 and MIC8KOi-T7S4-1, showed the same behaviour in phenotypic
assays (data not shown). We chose MIC8KOi-T7S1-4 for detailed
phenotypic analysis and refer to this parasite strain as MIC8KOi.
We analysed the expression of mic8Ty in MIC8KOi. Parasites
grown in the absence of ATc showed almost identical levels of
expression of MIC8 (~75%, as judged from immunoblots) compared
to wild-type parasites (Fig. 2A,B). Incubation of parasites for 48
hours in the presence of ATc led to almost complete ablation of
MIC8Ty (to ~2%) compared with parasites grown in absence of
ATc for the same amount of time, as judged from immunoblots
(Fig. 2A,B and see later). Unless otherwise indicated, parasites were
grown in the absence or presence of ATc for 48 hours before the
respective experiment was performed. We confirmed that the
observed downregulation in mic8 expression was specific, because
the expression levels of other micronemal proteins (MIC2 and
MIC3) or of alpha-tubulin were not affected (Fig. 2B).
MIC8 is not required for trafficking of MIC3 to the micronemes
Previously, we found that MIC8 and MIC3 are capable of interacting
with each other during the transit through the secretory pathway,
and we suggested that MIC8 could act as an escorter for MIC3
(Meissner et al., 2002a), in analogy to other identified micronemal
escorter proteins, such as MIC6 (Reiss et al., 2001) and MIC2
(Rabenau et al., 2001). To test whether MIC8 is required for correct
localisation of MIC3, we analysed localisation of MIC3 in parasites
depleted for MIC8. Surprisingly, we did not observe a
mislocalisation or an altered expression level of MIC3 (Fig. 2B,C).
Furthermore, we did not detect any accumulation of MIC3 in early
endosomes when parasites overexpressing MIC8 were analysed
(Fig. 2D).
Based on these results, we conclude that the interaction between
MIC8 and MIC3 is not essential for targeting of MIC3 to the
micronemes. To confirm that the observed accumulation of MIC8
in T7S4-5 corresponds to the early endosomes, we performed colocalisation analysis with the Golgi marker GRASP-RFP (Pfluger
Fig. 1. Establishment of a conditional knockout for MIC8. (A) Regulation of mic8Ty expression by ATc in three independent parasite strains: T7S4-1, T7S4-5 and
T7S1-4. Parasites were grown with (+) or without (–) ATc for 48 hours, fixed and stained with antibodies against Ty, or against MIC6 as a control. Pictures were
taken under identical exposure conditions. (B) Immunofluorescence analysis of parasite strains T7S4-1 and T7S4-5 grown for 48 hours in the absence of ATc, as
shown in A. Parasites were stained with the indicated antibodies. Strong overexpression of MIC8Ty results in partial accumulation of MIC8 within the secretory
pathway (arrows). Other micronemal proteins, such as MIC6, appear not to be affected by overexpression of MIC8. (C) Homologous recombination of endogenous
mic8 with a knockout construct in which a CAT-expression cassette is flanked by 1.6 kb (5′FS) and 2.5 kb (3′FS) results in a knockout locus that can be identified
using analytical PCR with specific oligonucleotides, as indicated by the arrows. (D) Analytical PCR analysis to verify homologous recombination of the knockout
construct in the recipient strains T7S1-4 and T7S4-1. Numbers on top of each lane indicate the respective oligonucleotide combination (see C) used in each PCR
reaction. Scale bars: 25 μm (A); 5 μm (B).
Role of MIC8 in T. gondii invasion
949
Phenotypic characterisation of MIC8KOi
To analyse the function of MIC8 in detail, we inoculated human
foreskin fibroblast (HFF) cells with parasites in the presence and
absence of ATc, and followed the propagation of parasites. Whereas
MIC8KOi parasites grown in the absence of ATc showed normal
growth when compared to wild-type parasites, no growth was seen
when the expression of mic8 was ablated (Fig. 3A).
When we examined infected host cells within 4 days, we
occasionally detected huge parasitophorous vacuoles containing
large numbers of parasites, indicating that parasites were capable
of replicating in the absence of MIC8 (see also supplementary
material Fig. S1). Occasionally, we observed lysed vacuoles with
freshly egressed motile parasites; these parasites appeared to be
incapable of invading neighbouring host cells (Fig. 3A). Several
days after the inoculation of cells with parasites, we failed to detect
any intracellular parasites, indicating that initially infected host cells
had been lysed and that released parasites were incapable of reinvading new host cells (Fig. 3B and data not shown).
Journal of Cell Science
Intracellular replication and host-cell egress are not affected by
the absence of MIC8
Fig. 2. MIC8 is not required for trafficking of MIC3 to the micronemes.
(A) Immunofluorescence analysis of MIC8KOi grown for 48 hours in the
presence (+) or absence (–) of ATc prior to fixation. Staining was performed
with alpha-MIC8, or with alpha-MIC2 as a control (same exposure time for all
micrographs). (B) Immunoblot analysis of MIC8KOi and RH parasites grown
in the presence or absence of ATc for 48 hours and probed with the indicated
antibodies. No significant difference in the expression levels of MIC2, TUB1
or MIC3 could be detected in MIC8KOi cells under the two conditions.
(C) Localisation of MIC3 in MIC8KOi parasites grown in the presence of ATc
for 48 hours. Parasites were stained with the indicated antibodies. Merged
images are shown on the right. Blue stain indicates DAPI (nuclei). (D) Colocalisation studies in the parasite strain T7S4-5, which overexpresses
MIC8Ty (see also Fig. 1B). Upper panels: co-localisation study of MIC8Ty
and GRASP-RFP (Pfluger et al., 2005) in the parasite strain T7S4-5 transiently
transfected with GRASP-RFP demonstrates accumulation of MIC8 in a postGolgi compartment. Middle panels: co-localisation of MIC8Ty and the
propeptide of M2AP (Harper et al., 2006) in early endosomes. Lower panels:
co-localisation study of MIC8Ty and MIC3 shows that MIC3 is not localised
in early endosomes because of overexpression of MIC8Ty. Right: enlargement
of boxed parasite(s) in the merged pictures. Scale bars, 10 μm.
et al., 2005) and found that the majority of retained MIC8 localised
apical to the Golgi (Fig. 2D). When we performed co-localisation
analysis of MIC8 with an antibody against the propeptide of M2AP,
which localises to early endosomes (Harper et al., 2006), we detected
retained MIC8 within the same compartment (Fig. 2D).
To analyse the function of MIC8 during the asexual cycle of T.
gondii, we generated MIC8KOi parasites constitutively expressing
GFP. We inoculated HFF monolayers with equal numbers of
freshly released MIC8KOi parasites that were grown in the absence
of ATc, and subsequently compared growth of the parasites and
lysis of initially infected host cells in presence or absence of ATc
over a 72-hour time-course (Fig. 3B). We did not see any difference
in non-induced host-cell egress: the number of initially infected host
cells decreased irrespective of whether ATc was added to the
medium or not. Whereas, at 24 hours post-infection, approximately
90% of initially infected host cells were intact, these numbers
decreased to approximately 55% and 10% after 48 and 72 hours,
respectively (Fig. 3B). However, under conditions of MIC8
depletion, no subsequent infection of neighbouring host cells was
detected. This indicates that this protein is specifically required
during host-cell invasion, whereas egress is not affected in parasites
depleted of MIC8.
Host-cell exit can be artificially triggered by the use of the
calcium-ionophore A23187 (Black et al., 2000). When we treated
intracellular MIC8KOi parasites grown in the presence or absence
of ATc with A23187 for 5 minutes, no significant reduction in egress
was detected (supplementary material Fig. S1).
Depletion of MIC8 does not affect parasite attachment or
microneme secretion
Initial attachment is the first step in the invasion of the host cell,
so we performed attachment assays on fixed host cells. MIC8KOi,
TATi1-expressing and wild-type (RH) parasites grown in the
presence or absence of ATc were allowed to attach to the host cell
for 15 minutes. After removal of non-attached parasites, the
number of parasites associated with the host cell was compared
between these groups. We found a slight reduction in attachment
when parasites expressing TATi1 were compared with wild-type
parasites. Efficiency in attachment appeared to be further
decreased in the case of MIC8KOi, irrespective of whether MIC8
was expressed (Fig. 3C). The observed reduction in attachment
could be due to the extensive genetic modifications of the parental
RH parasites, which required three independent stable
transfections in order to generate MIC8KOi. However, no
difference in initial host-cell attachment was observed in the case
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Fig. 3. MIC8 is required for parasite survival and is
essential during the invasion of the host cell.
(A) Upper panels: growth of MIC8KOi parasites
was compared to RHwt parasites using plaque assay.
Parasites were inoculated on HFF cells in the
presence or absence of ATc for 7 days prior to
GIEMSA staining. Lower panels: same experiment
as above. Parasites were analysed 72 hours postinfection. Note that in the case of MIC8KOi grown
in the presence of ATc, freshly egressed parasites
did not invade neighbouring host cells. (B) Analysis
of non-induced egress by quantification of intact
initially infected host cells. HFF cells were
inoculated with MIC8KOi-GFP parasites in the
presence or absence of ATc and the decrease in
initially infected host cells was analysed over time.
The depicted quantification is representative of the
results derived from three independent experiments.
(C) Attachment assay of freshly released parasites
grown in the presence or absence of ATc to fixed
cells. Equal numbers of parasites were allowed to
attach on host cells at 37°C for 15 minutes before
non-attached parasites were removed by consecutive
washing steps with cold PBS. Data are mean values
of three independent experiments ± s.d.
(D) Invasion assay of freshly egressed parasites
grown in the presence or absence of ATc as in C.
After the indicated time, non-invaded parasites were
removed by several washing steps. Intracellular
parasites were further incubated for 12 hours and the
number of parasitophorous vacuoles was compared.
ON, over night. Data shown are mean values of
three independent experiments ± s.d. (E) Reexpression of MIC8 in extracellular parasites
restores invasion capability. Invasion assay was performed similar to as in D. After MIC8KOi grown in the presence or absence of ATc were allowed to attach for
10 minutes, non-attached parasites were removed. Attached parasites were further incubated in the presence or absence of ATc (–/–: parasites constantly kept in the
absence of ATc; +/+: parasites constantly kept in the presence of ATc; +/–: parasites kept in the presence of ATc and further incubated in media without ATc). Data
represent mean values of three independent experiments ± s.d.
of MIC8KOi parasites grown in the presence or absence of ATc.
Therefore, MIC8 appears to be not involved in this invasion step
(Fig. 3C).
To exclude the possibility that secretion of micronemal proteins
was affected, we compared the level of secreted MIC2. We found
no difference in secretion of MIC2 in parasites grown in the presence
or absence of ATc. Both constitutive microneme secretion as well
as induced secretion showed no difference to wild type in the
absence of MIC8 (supplementary material Fig. S1). Similar results
were obtained with other micronemal proteins, such as MIC6 and
MIC4 (data not shown).
MIC8 is required for invasion and not during egress
Next, HFF cells were inoculated with MIC8KOi parasites in the
presence or absence of ATc. After host-cell lysis, equal numbers of
freshly egressed parasites were analysed for their capability to
invade host cells. Parasites were allowed to invade for different
durations before the removal of non-invaded parasites and were
further incubated in the presence or absence of ATc. We found that
parasites depleted of MIC8 were completely deficient in invasion.
Even a prolonged invasion time of more then 12 hours did not
increase invasion events significantly (Fig. 3D). A direct comparison
between MIC8KOi parasites grown in the presence or absence of
ATc showed that less then 0.1% of the parasites were invasive. To
confirm that MIC8 is a general, essential factor for host-cell
invasion, we performed analogous assays on different host-cell types
(green monkey kidney cells, HeLa cells and macrophages) and
obtained identical results (data not shown).
We were interested to know whether this protein is involved in
a signalling cascade during egress of the host cell that prepares the
parasite for the next round of invasion, because egress and invasion
are tightly linked (Hoff and Carruthers, 2002). If this were the case,
extracellular parasites expressing only nascent MIC8 would be
incapable of invasion, because no modification of MIC8 can occur
during egress. In order to differentiate between a role of this protein
during egress and invasion, we modified the above experiment. HFF
cells were inoculated for 10 minutes with parasites grown in the
presence of ATc. Non-attached parasites were then removed.
Subsequently, expression of mic8Ty was reactivated in attached
extracellular parasites by the removal of ATc for 12 hours before
the invasion of host cells was analysed. Reactivation of MIC8
expression in extracellular parasites restored the capability of
invasion (up to 20%) when compared with MIC8KOi parasites that
were constantly kept in absence of ATc (Fig. 3E). These results
indicate that MIC8 is exclusively involved in invasion and is not
required or modified during egress. As soon as a critical level of
MIC8 was reached in extracellular parasites, invasion competency
was restored. The observed reduction in invasion, when compared
to parasites constantly grown in the absence of ATc (Fig. 3E), is
probably due to reduced viability of parasites kept for a prolonged
time outside of the host cell.
MIC8 is dispensable for gliding motility
Gliding motility is a prerequisite for active invasion of the host
cell. Several types of movement can be detected in wild-type
parasites (Fig. 4A) (Hakansson et al., 1999). Recently, it has been
Journal of Cell Science
Role of MIC8 in T. gondii invasion
951
Fig. 4. MIC8 is not involved in gliding motility. (A) Three different forms of movement can be identified by quantitative image analysis: circular gliding (C),
helical gliding (H) and twirling (T). These forms give distinct patterns when the acquired images are projected. The projection (proj.) of 100 images acquired at 3second intervals represents an overall observation time of 5 minutes. All three forms of movements can be detected in the case of RHwt and MIC8KOi grown in the
presence or absence of ATc. (B) Comparison of overall motility. Although differences between RH and MIC8KOi parasites can be observed (~30 vs 12% overall
gliding motility, respectively), no significant reduction in gliding motility can be observed between MIC8KOi grown in the presence or absence of ATc.
(C) Comparison of the relative frequency of each gliding type of motile parasites. A direct comparison between MIC8KOi grown in the presence or absence of ATc
did not show any significant differences in the frequency of the three forms of gliding motility. Mean values of three independent experiments ± s.d. are shown.
shown that the micronemal protein MIC2 is essential for helical
and twirling motility but not for circular gliding (Huynh and
Carruthers, 2006). In the absence of MIC2, parasites show a
reduced ability to invade the host cell. To assess whether the
observed complete block in invasion upon depletion of MIC8 was
due to a reduction in gliding motility, we performed quantitative
live imaging. As described previously, three forms of gliding
motility can be detected in this assay: circular gliding, upright
twirling and helical rotation (Fig. 4A) (Hakansson et al., 1999).
We performed time-lapse microscopy on RH parasites and
MIC8KOi grown in the presence or absence of ATc. After image
acquisition (one frame/3 seconds for 5 minutes), overlaying
projections of the acquired frames revealed the respective motility
patterns. All three forms of gliding motility were identified in
parasites depleted of MIC8 (Fig. 4A). Although RH parasites
showed a higher overall gliding motility than MIC8KOi (~30 vs
12% overall gliding motility), no significant reduction in gliding
motility was observed between MIC8KOi grown in the presence
or absence of ATc (Fig. 4B). When the relative occurrences of the
three different forms of gliding were compared, no significant
difference between MIC8KOi grown in the presence or absence
of ATc was observed (Fig. 4C). The general reduction of gliding
motility in MIC8KOi parasites could be due to the extensive genetic
modifications of the parental RH parasites (similar to the effect
observed for attachment). However, because no difference in
gliding motility was evident in the case of MIC8KOi parasites
grown in the presence or absence of ATc, the total block in invasion
is unlikely to be due to reduced gliding motility in the absence of
MIC8.
T. gondii expresses two highly conserved homologous of MIC8
Two highly conserved homologous proteins, MIC8-like1 (accession
number 76.m01679) and MIC8-like2 (accession number
49m.03396) can be found in the available genome database of T.
gondii (www.toxoDB.org) (Cerede et al., 2005) (supplementary
material Fig. S2). We confirmed expression of MIC8-like1 and
MIC8-like2 in T. gondii tachyzoites on the RNA level by reverse
transcriptase (RT)-PCR (supplementary material Fig. S2). The
function of a micronemal transmembrane protein appears to be
determined by the interaction of the C-terminal cytosolic tail
domain (CTD). In the case of members of the TRAP family, it has
been demonstrated that the CTD interacts with the glycolytic
enzyme aldolase, which links this protein to the actin-myosin motor
essential for gliding motility (Jewett and Sibley, 2003; Buscaglia
et al., 2003). An alignment of the CTDs of different microneme
proteins indicates that most sequences shown to be essential for
interaction with aldolase (Starnes et al., 2006) are not present in
the CTD of the MIC8 family members (supplementary material
Fig. S2). This corresponds well with our phenotypic
characterisation of MIC8KOi that suggests a different function of
MIC8 during invasion.
Complementation analysis of MIC8KOi
We were interested to determine whether the CTD of different
micronemal proteins can be exchanged for the CTD of MIC8.
Therefore, we designed a complementation strategy, taking
advantage of the tight regulation and clear invasion phenotype of
MIC8KOi, that allowed us to use ATc selection to obtain stable
transfected parasites.
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the obtained parasite population was analysed for expression of
either the myc- or Ty-tagged version of MIC8. We observed three
different sub-populations that expressed MIC8Ty, MIC8myc or both
(Fig. 5A,B) in the presence of ATc, indicating that three independent
Journal of Cell Science
Transfection of a construct leading to constitutive expression of
a myc-tagged version of MIC8 served as a positive control. Stable
selection was performed in the presence of ATc to deplete the
parasite of the inducible Ty-tagged copy of MIC8. After 5-6 days,
Fig. 5. Complementation analysis of MIC8KOi using different chimeric proteins. (A) Schematic of the complementation strategy. Transfection of MIC8KOi with
complementation constructs under ATc selection results in three different integration events: (1) homologous recombination leads to a promoter exchange resulting
in constitutive expression of Ty-tagged MIC8 (stained in green); (2) a combination of both homologous and non-homologous recombination leads to constitutive
expression of both MIC8Ty (stained in green) and the respective myc-tagged complementation construct (stained in red); (3) only non-homologous recombination
leads to exclusive, constitutive expression of the myc-tagged complementation construct. This event indicates functional complementation. The
immunofluorescence analysis shown was performed on a representative pool of MIC8KOi parasites complemented with pMIC8MIC8mycTMCTDMIC8 after 6 days
under ATc selection. (B) Top: overview of the complementation constructs used in this study. The TMD and CTD of MIC8myc have been substituted as indicated.
In the case of MIC2, two constructs were generated (see text). In the case of MIC8mycGPI (GPI), the TMD and CTD of MIC8 was exchanged for the GPI
anchoring signal of the major surface antigen SAG1. Bottom: representative immunofluorescence analyses of MIC8KOi transfected with the indicated
complementation construct. Stable transfection has been achieved by application of ATc selection as shown in A. Numbers in each image indicate the average
percentage of parasites (± s.d.) in which random integration occurred in four independent experiments. Scale bars: 12.5 ␮m.
Role of MIC8 in T. gondii invasion
the case of the construct MIC8myc and in the case of the chimera
MIC8mycTMCTDMIC8-like1 (Fig. 5B). In all cases, we verified the
correct localisation of the respective chimeric protein in the
micronemes (Fig. 5B). Therefore, it appears that the correct targeting
of the extracellular domains of MIC8 is not sufficient to restore the
function of MIC8. Furthermore, we conclude that the CTD of MIC8
is functionally distinct and cannot be complemented by the CTD of
microneme proteins belonging to the TRAP family or of AMA1.
Interestingly, when we generated a mutant in which the highly
conserved C-terminal tryptophan of MIC8 was substituted for
alanine, we were able to achieve complementation, indicating that
this residue, although highly conserved in the MIC8 family (see
supplementary material Fig. S2), is not absolutely required for the
function of MIC8.
MIC8 is essential for formation of the moving junction
When parasites are treated with cytochalasin D (CD), which leads
to an arrest in invasion, an MJ is formed and the content of the
rhoptries is secreted into the cytosol of the host cell, leading to the
formation of evacuoles (Fig. 6A) (Hakansson et al., 2001). To
Journal of Cell Science
integration events of the complementation construct occurred:
exchange of the promoter pT7S1 for pMIC8 (because of
homologous recombination of the complementation construct
pMIC8MIC8myc with construct T7S1MIC8Ty), random integration
of the complementation construct, and a combination of random
integration and homologous recombination (Fig. 5A). Only random
integration, which resulted in exclusive expression of the myctagged complementation construct, indicated functional
complementation for MIC8Ty (Fig. 5A).
We generated several complementation constructs in which the
CTD and TMD of MIC8 were exchanged for the corresponding
domains of MIC2 (accession number AAB63303), AMA1 (accession
number AAB65410), PfTRAP (accession number AAA29776),
MIC8-like1 or the GPI-anchoring signal of SAG1 (Fig. 5B, upper
panel). In the case of MIC2, we generated two different
complementation constructs: one including sequences upstream of
the TMD that have previously been implicated in the correct
processing of MIC2 (Brossier et al., 2003) (Mic2a), and one
including only the TMD and CTD (Mic2b). Exclusive expression
of the respective complementation construct was only achieved in
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Fig. 6. MIC8 is essential for the formation of the moving junction. (A) Formation of evacuoles is abolished in parasites depleted of MIC8. MIC8KOi, grown in the
presence or absence of ATc, was incubated with host cells in the presence of CD. Formation of evacuoles (green) and an MJ (red) was analysed with monoclonal
antibodies against Rop2-5 and polyclonal antibodies against RON4. No evacuoles were observed when MIC8KOi was grown in the presence of ATc. Blue staining
indicates DAPI (nuclei). (B) Extracellular RON4 was detected by immunofluorescence analysis on non-permeabilised parasites expressing cytosolic GFP as a
viability marker. MIC8KOi parasites were allowed to attach to host cells in the presence or absence of CD for 20 minutes. Red indicates an MJ. (C) Quantification
of extracellular RON4, the presence of which indicates the formation of an MJ. Approximately 200 parasites were analysed in each assay (error bars indicate s.d.
obtained from five independent assays). The units on the y-axis are percent of parasites with RON4 signal. (D) Depletion of MIC8 does not affect RON4
expression levels. Immunoblots from MIC8KOi and RH parasites, grown for 48 hours in the presence (+) or absence (–) of ATc, show efficient downregulation of
MIC8 expression in the presence of ATc. No differences in RON4 expression levels could be detected. Scale bars: 15 ␮m (A); 20 ␮m (B).
Journal of Cell Science
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Journal of Cell Science 121 (7)
determine whether parasites depleted of MIC8 were capable of
forming an MJ and of secreting their rhoptries, MIC8KOi parasites
were grown in the presence or absence of ATc, treated with CD
and inoculated on HFF cells to determine evacuole formation. In
the absence of MIC8, no formation of evacuoles was detected (Fig.
6A). A similar phenotype has been reported for a conditional
knockout of the micronemal protein AMA1. Parasites depleted of
AMA1 still secrete RONs but fail to establish an MJ between the
parasite and the host cell (Alexander et al., 2005; Mital et al., 2005).
In this case, RONs associated with the MJ cannot be seen, but rather
they localise in loose patches on the surface of the host cell. We
were interested to determine whether a similar phenotype could be
observed upon depletion of MIC8.
To analyse secretion and localisation of RONs, we used RON4
as a marker. MIC8KOi parasites expressing GFP as a viability
marker were grown in the presence or absence of ATc. Parasites
were treated with or without CD before inoculation of HFF cells.
Subsequently, analysis of RON4 secretion was performed in
immunofluorescence assays on non-permeabilised parasites (Fig.
6B). In the absence of MIC8, the occurrence of a RON4 signal was
reduced by more then 90% (Fig. 6C) irrespective of whether the
parasites were treated with CD. Ablation of MIC8 had no influence
on the expression level of RON4 (Fig. 6D). Together, these results
demonstrate a crucial role of MIC8 during establishment of the MJ
that is upstream of AMA1.
Discussion
Host-cell invasion by apicomplexan parasites is a multi-step process,
requiring the sequential secretion of specialised secretory organelles
and the action of distinct sets of proteins (Carruthers and Boothroyd,
2006). Whereas secretion of micronemes has been demonstrated to
depend on a calcium-signalling cascade, the signals leading to
secretion of the rhoptries and formation of the MJ have not been
identified so far. Given the functional linkage of microneme and
rhoptry secretion, we speculated that micronemal proteins might
be implicated in this role.
To shed light on the function of MIC8, we generated a conditional
knockout for MIC8 by employing a tetracycline-inducible
transactivator system (Meissner et al., 2007). For the generation of
transgenic recipient strains with a second controllable copy of mic8,
it was necessary to keep expression of the transgene turned off to
prevent any deleterious effects due to overexpression of mic8Ty.
Interestingly, a strain overexpressing MIC8Ty showed a partial
accumulation of this protein in a post-Golgi compartment that
corresponded with early endosomes, as shown by co-localisation
with the pro-peptide of M2AP (Harper et al., 2006). It is likely that,
under conditions of MIC8 overexpression, a factor involved in
quality control or maturation of this protein is limiting, resulting in
partial accumulation of MIC8 in early endosomes. A similar
phenomenon was observed upon overexpression of MIC2 (Huynh
and Carruthers, 2006).
It has previously been demonstrated that MIC8 interacts with
MIC3 during the transit through the secretory pathway (Meissner
et al., 2002a) and it was speculated that MIC8 might act as an
escorter for MIC3, similar to other micronemal transmembrane
proteins (Rabenau et al., 2001; Reiss et al., 2001). However,
depletion of MIC8 in the established MIC8KOi parasites did not
result in any mislocalisation of MIC3, demonstrating that the
observed interaction between MIC8 and MIC3 is not essential for
the targeting of MIC3 to the micronemes. Currently, we can only
speculate on the mechanism involved in this targeting. It is possible
that MIC8 and MIC3 are sorted to the micronemes by interacting
with the same protein complex, that MIC3 contains sorting signals
that ensure its transport to the micronemes independently of MIC8
or that the escorter function of MIC8 is redundant. Further
experiments are required to clarify this complex issue.
We examined crucial events during the asexual life cycle of the
parasite and found that depletion of MIC8 explicitly affects the
ability of the parasite to invade the host cell. We did not observe
any effect on replication, egress or initial attachment to the host
cell. Interestingly, reactivation of MIC8 expression in extracellular
parasites led to restoration of invasion potency. This shows that
MIC8 is not required during egress of the host cell and that the
invasion of parasites depends on a critical level of MIC8 expression.
Furthermore, parasites depleted for this essential invasion factor
showed normal attachment and gliding motility, which is in sharp
contrast to the function determined for MIC2 (Huynh and
Carruthers, 2006).
We noticed that parasites depleted of MIC8 did not form any
evacuoles, similar to a conditional mutant for AMA1 (Alexander et
al., 2005; Mital et al., 2005). Parasites depleted for AMA1 are unable
to form an MJ and it has been demonstrated that interaction of AMA1
with RONs is essential for this invasion step (Boothroyd and
Dubremetz, 2008). However, in the absence of AMA1, RONs are
still released by the parasite and can be detected at the surface of the
host cell in loose patches (Alexander et al., 2005). In sharp contrast,
we were not able to detect any signal for RON4 when intact nonpermeabilised parasites were analysed for RON4 secretion. This
indicates that MIC8 functions in an essential step during invasion
that is downstream of MIC2 but upstream of AMA1 function.
The absence of RON4 on the surface of the host cell suggests
that MIC8 is either required for the initial association of RONs with
the surface of the host cell or for rhoptry secretion. In the first case,
this role could be facilitated by function of the extracellular
domains of MIC8. However, we demonstrated in a novel
complementation approach that correct targeting of chimeric
proteins consisting of the extracellular domains of MIC8 and the
TMD and CTD of different micronemal proteins, such as MIC2,
AMA1 and TRAP, did not restore the function conferred by wildtype MIC8. This demonstrates that correct targeting of the
extracellular domains is not sufficient to restore the function of
MIC8.
Having established that the CTDs of MIC8 and MIC8-like1
determine a novel functional role, we can now continue to identify
the cytosolic factors interacting with this domain to gain more
mechanistic insights into its function during the establishment of
the MJ. Given the clear phenotype of MIC8KOi in invasion, it
appears that MIC8-like1 and MIC8-like2, which we show here to
be expressed at the RNA level, cannot compensate for the loss of
mic8 in tachyzoites.
Previously, it has been shown that MIC8 is capable of binding to
the host cell upon dimerisation (Cerede et al., 2002). It is therefore
tempting to speculate that, upon dimerisation, MIC8 is involved in
a signalling cascade that ultimately leads to rhoptry secretion. Similar
mechanisms have been described in other eukaryotes for numerous
non-tyrosine kinase receptors that trigger receptor-mediated
endocytosis and subsequently a signalling cascade (Li, 2005). In fact,
we have recently demonstrated that Rab11A, a small G-protein
essential for recycling endocytosis and associated with the rhoptries
(Bradley et al., 2005), is required during invasion of the host cell
(Herm-Gotz et al., 2007), indicating a link between receptor-mediated
endocytosis, receptor recycling and rhoptry secretion.
Role of MIC8 in T. gondii invasion
In conclusion, here, we present evidence that the micronemal
protein MIC8 of T. gondii plays an essential role during a distinct
step of host-cell invasion required for the early formation of the
MJ. The characterisation of MIC8 as a key determinant for invasion
demonstrates that micronemal proteins are involved in several
independent steps during invasion that are likely to be conserved
in apicomplexan parasites. The identification of each individual
step will not only broaden our understanding of fundamental
apicomplexan biology but might also lead to the identification of
new drug and vaccine candidates. The challenging issue now will
be to further elucidate the mechanistic action of MIC8, especially
the role of the MIC8 CTD, which we show here to be essential for
its function.
Materials and Methods
Parasite strains, transfection and culture
T. gondii tachyzoites (RH hxgprt–) and TATi-1 (Meissner et al., 2002b) were grown
in HFF cells and maintained in Dulbecco’s modified Eagle’s medium (DMEM),
supplemented with 10% foetal calf serum (FCS), 2 mM glutamine and 25 μg/ml
gentamycin. Selection based on chloramphenicol resistance or pyrimethamine
resistance was performed as described previously (Kim et al., 1993; Donald and Roos,
1993).
Journal of Cell Science
Generation of the inducible MIC8 knockout
To introduce a Ty-tag into mic8 and generate a vector allowing inducible expression
of this gene, the cDNA of mic8 was cloned into the vector p7TetOS1 and p7TetOS4
downstream of the inducible promoter (Meissner et al., 2001). Next, inverse PCR
using oligonucleotides MIC8Ty-as (antisense) (5′-CTTAATTAAGATGCATCGAGGGGGTCCTGGTTGGTGTGGACTTCTGGTAAACACTGCAACTTTGACCC-3′) and MIC8Ty-s (sense) (5′-CCAATGCATCTACTGATCAGACAGATTTCG3′). The PCR fragment was subsequently digested with NsiI and re-ligated, resulting
in the expression vectors T7S1MIC8Ty and T7S4MIC8Ty, respectively. In order to
generate stable parasites with each construct, co-transfection of 60 μg T7S1MIC8Ty
or T7S4MIC8Ty with 30 μg pDHFR (Donald and Roos, 1993) was performed.
The knockout construct was based on the vector pT230CAT (Soldati and
Boothroyd, 1995), in which the 5′UTR was amplified using oligonucleotides MIC81 (5′-GGGGTACCACTCGTGGCGTCGCTG-3′) and MIC8-2 (5′-GGGGTACCTGGTGGTGGGCGGAAAG-3′) and the 3′UTR with MIC8-3 (5′-GCGGATCCTTAATTAAGCATAAGCTTTGTACCTGTGC-3′) and MIC8-4 (5′-TCCCCGCGGCATTGAACAACATGTATGCG-3′) and placed within KpnI and BamHI/NotI
restriction sites, respectively. 30 μg of the MIC8KOCAT construct was transfected
and, subsequently, CAT selection was performed. MIC8KOi parasites were isolated
from the obtained stable pool of transfectants via limiting dilution and identified
via genomic PCR using insert-, genome- and integration-specific oligonucleotide
combinations (see Fig. 2A). MIC8KOi parasites were then verified in immunoblots
and immunofluorescence assays to confirm inducible expression of MIC8Ty. For a
subset of studies, MIC8KOi was transfected with p5RT70GFP and GFP-expressing
parasites were isolated based on green fluorescence.
Generation of complementation constructs
For the generation of complementation constructs encoding the extracellular domains
of MIC8 fused to the TMD and CTD of MIC2, AMA1, PfTRAP, MIC8/2,
MIC8(W>A) and MIC8GPI, we exchanged the DNA sequence encoding the Ty-tag
in T7S1MIC8Ty for a myc-tag using complementary oligonucleotides MIC8myc-s
(5′-AATTCTTTCAAAGTTGCAGTGTTTACCTAGGGAGCAGAAGCTCATCTCCGAGGAGGACCTAGATGCATCTTAAT-3′) and MIC8myc-as (5′-TAAGATGCATCTAGGTCCTCCTCGGAGATGAGCTTCTGCTCCCTAGGTAAACACTGCAACTTTGAAAG-3′). In a second step, the TMD and CTD of MIC8 were reintroduced within NsiI and PacI restriction sites. Next, the promoter p7TetOS1 was
exchanged for p5RT70 and pMic using KpnI/EcoRI, resulting in the constructs
p5RT70MIC8myc and pMIC8MIC8myc, respectively. Amplification of pMIC8 was
achieved using oligonucleotides pMIC8-s (5′-CCCGGTACCTGGTGGTGGGCG3′) and pMIC8-as (5′-GCCGAATTCGAAACGAAAGATACCGCGACCAGC-3′).
Subsequently, the coding regions for different transmembrane CTDs were introduced
into MfeI/PacI restriction sites, resulting in different complementation constructs.
The oligonucleotides used for amplification of the TMCTD-encoding DNA
sequence were: MIC8CTD MIC8/2-tail: MIC8/2-sense2 (5′-GCGGCAATTGGACTGGCAACGGGAGCC-3′) and MIC8/2-as (5′-GCCTTAATTAATTATGACCACATTGAGCCTG-3′); MIC8CTD MIC8W>A tail: MIC8sense-MfeI
(5′-GCGGCAATTGCATTGGTGGTTGTGG-3′) and MIC8-W>Aas (5′-GCCTTAATTAAGACGCGATACCGCCCGAAGG-3′); PfTRAP-tail: PfTRAP-sense
(5′-GCGGCAATTGCAGGTGGAATAGCTGGAGG-3′) and PfTRAP-as (5′-GCCTTAATTAATTCCACTCGTTTTCTTCAG-3′); AMA1-tail: AMA1-s (5′-GCGGCAATTGCTGGACTCGCCGTAGGAG-3′) and AMA1-as (5′-GCCTTAATTAACT-
955
AGTAATCCCCCTCGACC-3′); MIC2tail-a: MIC2sense1 (5′-GCGGCAATTGAAGAGGGAGAACAGTCCAAGAAAG-3′) and MIC2as (5′-GCCTTAATTAACTACTCCATCCACATATCAC-3′); MIC2tail-b: MIC2sense2 (5′-GCGGCAATTGCGGGTGGCGTCATCGGCG-3′) and MIC2as (5′-GCCTTAATTAACTACTCCATCCACATATCAC-3′). See also alignment in Fig. 5B.
Plaque assay
The plaque assay was performed as described previously (Roos et al., 1994).
Monolayers of HFF, grown in six-well plates, were infected with 50-100 tachyzoites
per well. After 1 week of incubation at normal growth conditions (37°C, 5% CO2),
cells were fixed for 10 minutes with –20°C methanol 100%, dyed with Giemsa stain
for 10 minutes and washed once with PBS. Documentation was performed with a
Nikon SMZ 1500 binocular at 70-fold magnification.
Invasion assay
Monolayers of HFFs, grown in 24-well plates on glass slides, were infected with 107
parasites/well and incubated for 5, 10, 20 or 60 minutes, or overnight, at 37°C.
Subsequently, infected cells were washed four times with PBS and incubated for 12
hours in fresh medium before fixation and indirect immunofluorescence. Data were
compiled from more than three independent experiments each from counting six fields
of view per clone at 1000⫻ total magnification with a Leica DMIL microscope
(Type:090-135-002 I/05).
Attachment assay
Attachment assays were performed as described previously (Huynh et al., 2003).
Monolayers of HFFs, grown in 24-well plates on glass slides, were washed three
times in PBS (containing 1 mM CaCl2 and 1 mM MgCl2) and fixed with PBS
containing 2% glutaraldehyde for 5 minutes at 4°C. After three washing steps with
PBS, cells were blocked with 0.16 M ethanolamine for 24 hours at 4°C. Subsequently
to another washing step with PBS, 107 parasites were loaded on each well and allowed
to attach for 15 minutes at 37°C and rinsed three times with PBS before counting.
For each experiment, six fields of view at 400⫻ total magnification were analysed
with a Leica DMIL microscope (Type: 090-135-002 I/05).
Analysis of microneme secretion
For preparation of excretory-secretory antigens (ESA), approximately 108 tachyzoites
were washed and resuspended in 1 ml of HHE, stimulated to discharge micronemes
by addition of ethanol to a final concentration of 1.0% and incubated at 37°C for 30
minutes, as described previously (Carruthers et al., 1999). Cells were removed by
centrifugation at 2000 g and the supernatant and pellet were analysed on immunoblot.
Analysis of moving-junction formation
Analysis of evacuoles was performed as described previously (Hakansson et al., 2001).
Freshly released parasites were incubated in Endo buffer [44.7 mM K2SO4, 10 mM
Mg2SO4, 106 mM sucrose, 5 mM glucose, 20 mM Tris, 0.35% (wt/vol) BSA, pH
8.2] containing 1 μM CD for 10 minutes at room temperature, before adding them
on confluent layers of HFFs. 3⫻106 parasites were added per well and kept for 3
minutes at room temperature. After 20 minutes incubation at 37°C, medium was
replaced with pre-warmed Hanks/HEPES/1% FCS/1 μM CD media and incubated
for 15 minutes at 37°C. After two washing steps with PBS, the cells were fixed with
methanol at –20°C for 10 minutes before the immunofluorescence assay was
performed.
Analysis of RON4 secretion was performed as above with modifications, employing
GFP-expressing parasites to distinguish intact parasites from disrupted parasites. After
incubation in the presence or absence of CD, parasites were fixed with 4%
paraformaldehyde and immunofluorescence analysis was done without
permeabilisation of parasites to distinguish between secreted and non-secreted
RON4. Equal numbers of GFP-positive parasites (~200) were analysed for RON4
secretion per experiment.
Live video microscopy gliding assay
Glass slides in a 24-well plate were coated with PBS including 50% FCS for 24
hours at 4°C. The glass slides were washed once with PBS before adding 106 parasites
in a total volume of 300 μl pre-warmed Hank’s medium containing 20 mM HEPES
and 1 mM EGTA. The parasites were then imaged (37°C/5% CO2) using an inverted
Zeiss microscope (Axiovert 200 M with incubator XL-3 and CO2 control) using 16⫻
magnification (objective 10⫻, Optovar 1.6⫻) and differential interference contrast
(DIC). Videos were captured at one frame per 3 seconds using Axiovision AxioVs40
V 4.6.3.0 software and the type of gliding/movement was counted by observing every
individual parasite. On average, 447 parasites/movie (101-1052 parasites/movie) were
monitored and enumerated for all strains. Using ImageJ 1.34r software, projections
of the standard deviation and of the maximum of the fluorescent intensity between
frames revealed motility patterns over a 5-minute period.
Immunofluorescence and immunoblot
All manipulations were carried out at room temperature if not mentioned otherwise.
Tachyzoite-infected HFF cells on glass coverslips were fixed with 4% formaldehyde
in PBS for 20 minutes. Fixed cells were permeabilised with 0.2% Triton X-100 in
956
Journal of Cell Science 121 (7)
PBS for 20 minutes and blocked in 3% bovine serum albumin in PBS for 20 minutes.
The cells were then stained with the primary antibodies, as indicated in the text, for
1 hour followed by Alexa-Fluor-594-conjugated goat anti-rabbit or Alexa-Fluor-488conjugated goat anti-mouse antibodies (Molecular Probes). Images were taken using
Zeiss (Axiovert 200M) and processed using ImageJ 1.34r software.
SDS page was performed as described previously (Laemmli, 1970). Freshly
egressed parasites were harvested by centrifugation at 300 g for 10 minutes and lysed
in RIPA buffer (150 mM NaCl 1% Triton X-100, 0.5% DOC, 0.1% SDS, 50 mM
Tris, pH 8.0, 1 mM EDTA). Equal numbers of parasites were applied per line on a
10% polyacrylamide gel. Transfer to nitrocellulose and immunoblot analysis was
performed as described previously (Reiss et al., 2001).
The following previously described primary antibodies have been used in these
analysis: α-MIC2 (6D10) (Wan et al., 1997), α-MI3 (T82C10) (Garcia-Reguet et al.,
2000), α-Ron4 (t5 4H1) (Lebrun et al., 2005), α-RON4 (Alexander et al., 2006), αMIC6 (Reiss et al., 2001), α-MIC8Nt (Meissner et al., 2002a) and α-Rop2,3,4 (T3
4A7) (Sadak et al., 1988).
Sequence analysis of micronemal proteins
Journal of Cell Science
DNA and predicted proteins homologous to MIC8 were identified using the BLAST
programme provided at ToxoDB (http:/ToxoDB.org). Multiple sequence alignments
were performed with the help of ClustalW. Expression of MIC8, MIC8-like1 and
MIC8-like2 was confirmed by RT-PCR (Titan one tube RT-PCR; Roche) on mRNA
(isolated with mRNAeasy; Biochain) using oligonucleotide pairs MIC8-s (5′GCGGCAATTGCATTGGTGGTTGTGG-3′), MIC8as (5′-GCCTTAATTAAGACGCGATACCGCCCGAAGG-3′), MIC8/2-s (5′-GCGGCAATTGGACTGGCAACGGGAGCC-3′), MIC8/2-as (5′-GCCTTAATTAATTATGACCACATTGAGCCTG-3′), MIC8/3-s (5′-GGCCAATTGCCGGCTGCGTTGTCGGC-3′), MIC8/3as (5′-GTGTTGTCGCCTTCTCGC-3′), Tub-s (5′-GGAGCTCTTCTGCCTGGAACATGGTATC-3′) and Tub-as (5′-AGAGTGAGTGGAAAGGACGGAGTTGTACG-3′).
We are indebted to John Boothroyd, David Sibley, Vern Carruthers,
Gary Ward, Jean-Francois Dubremetz and Maryse Lebrun for sharing
reagents and helpful discussions. We thank Petra Rohrbach, Vern
Carruthers and Kai Matuschewski for helpful discussions and critical
comments on the manuscript. This work was funded by the SFB544
of the German research foundation (DFG) and the ‘BiofutureProgramm’ of the German Ministry of Science and Education (BMBF).
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