PROBETA REPRODUCIDA NORMA ASTM D638-
7.5 FIABILIDAD DEL PROCESO DE LA APLICACIÓN DE INGENIERÍA INVERSA
2.2.1 Ect2 localizes predominantly to the central spindle and cell cortex
To examine the exact localization of Ect2 in HeLa S3 cells, indirect immunofluorescence microscopy was performed with anti-Ect2 antibodies. These antibodies predominantly stained the central spindle and midbody in mitotic cells (Fig. 20A, upper two rows) in accordance with a previous report (Tatsumoto et al., 1999). In addition, however, we could observe cortical staining of Ect2 throughout mitosis. In interphase cells Ect2 localized predominantly to the nucleus, but also a weak cortical staining could be observed especially at cell-cell contacts (data not shown). No such staining was observed with pre-immune IgG attesting to the specificity of the used anti Ect2-antibodies (data not shown).
To further confirm that this staining was specific for Ect2 protein, we depleted Ect2 in HeLa S3 cells by siRNA duplex specifically targeting Ect2 and indirect immunofluorescence microscopy was performed. In Ect2 siRNA transfected cells (48 hours), the central spindle, midbody and cell cortex staining was strongly reduced and also nuclear staining was clearly diminished in interphase cells (Fig. 20A bottom two rows). To confirm efficient depletion of Ect2, cell extracts were prepared from control (GL2) and Ect2 siRNA treated HeLa S3 cells after 24 and 48 hours of transfection. These cell extracts were separated by SDS-PAGE and after Western blotting, the membranes were probed with anti-Ect2 antibodies. As compared to the control (GL2), Ect2 levels were clearly diminished in Ect2 siRNA treated cells (Fig. 20B upper panel). As a loading control, α-tubulin was detected with anti-α-tubulin antibodies (Fig. 20B lower panel).
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Ec 2 t α-tubulin 24 hours 48 hours GL2 Ect2 GL2 Ect2A
GL2 RNAi Ect2 RNAiEct2 α-tubulin Merge+DNA
Western blotting
Figure 20. Ect2 localizes to the central spindle, midbody and cell cortex during mitosis.
(A) HeLa S3 cells were treated with GL2 or Ect2 siRNA duplexes for 48 hours and were subsequently fixed and permeabilized with paraformaldehyde/Triton X-100. These cells were stained with anti-Ect2 antibodies in red (left),anti-α-tubulin antibodies in green (middle) and DNA was labelled with DAPI in blue. Merged images are shown at the right. Scale bar 10 µm.
(B) HeLa S3 cells were transfected with GL2 or Ect2 siRNA duplexes and after 24 hours and 48 hours of transfection, Hepes lysis buffer extracts were made. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. These membranes were probed with anti-Ect2 antibodies (upper panel) and, as a loading control, with anti-α-tubulin antibodies (lower panel).
2.2.2 The amino-terminal BRCT domain is required for central spindle
targeting, whereas the carboxyl-terminal PH domain targets Ect2 to the
cell cortex
To analyze which domains target Ect2 to the central spindle and the cell cortex, different myc-tagged Ect2 fragments (Fig. 21A) were transiently expressed in HeLa S3 cells. As shown in Fig. 21B, full length myc-tagged Ect2 localized to both the central spindle and
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A
myc α-tubulin merge+DNA
myc-Ect2 myc-Ect2 1-420 myc-Ect2 1-333 myc-Ect2 1-288
C
myc-Ect2Figure 21. The intact BRCT domain targets Ect2 to the central spindle, whereas the PH domain is required for cell cortex localization.
(A) Schematic representation of different amino-terminal and carboxyl-terminal fragments of Ect2 and their presence at the central spindle and cell cortex is indicated with ‘+’, whereas the absence is indicated with ‘-’.
(B) HeLa S3 cells were transfected with 3x-myc tagged constructs of Ect2 for 36 hours and were subsequently fixed and permeabilized with paraformaldehyde/Triton X-100. These cells were stained with anti-myc antibodies in red (left), anti-α-tubulin antibodies in green (middle) and DNA was labelled with DAPI in blue. Merged images are shown at the right. Scale bar 10 µm.
(C) HeLa S3 cells were transfected with myc-tagged constructs for 36 hours and were simultaneously fixed and permeabilized with formaldehyde/Triton X-100. These cells were stained with anti-myc antibodies in red (left) and with FITC-phalloidin for staining of actin in green (middle). DNA was labelled with DAPI in blue. Merged images are shown at the right. Scale bar 10 µm.
320-883
myc-Ect2 414-630
cell cortex. Amino-terminal Ect2 fragments containing the full BRCT domain (two BRCT repeats, residues 1-420 and 1-333) also localized to the central spindle, but were absent from the cell cortex (Fig. 21B). However, another amino-terminal construct encoding myc-Ect2 1-288, which has a truncated second BRCT repeat, did not localize to the central spindle in anaphase cells (Fig. 21B, lower panel). This strongly suggests that an intact BRCT domain, consisting of two BRCT repeats, is required for targeting Ect2 to the central spindle.
Whereas the amino-terminal half of Ect2 was sufficient for central spindle targeting, the carboxyl-terminal half of Ect2 strongly associated with the cell cortex, but was absent from the central spindle (Fig. 21C). The carboxyl-terminus of Ect2 consists of a DH domain (Dbl Homology) which is a Rho GEF domain, followed by a PH domain (Plekstrin Homology). Further truncation analysis revealed that the PH domain was essential for targeting Ect2 to the cell cortex (Fig. 21C). Considering that PH domains constitute lipid-binding motifs (Blomberg et al., 1999), it is attractive to postulate that the cortex association of Ect2 reflects a direct interaction with the plasma membrane. The PH domain has been shown to be essential for the cell transforming activity of Ect2 in NIH/3T3 cells by an yet unknown mechanism (Miki et al., 1993; Solski et al., 2004), suggesting that the cortex-associated pool of Ect2 is critical for its oncogenic potential.
2.2.3
Ect2 is targeted to the central spindle via the MKlp1/MgcRacGAP
and Aurora-B/MKlp2 complexes
The central spindle is composed of non-kinetochore, anti-parallel MTs and many proteins required for cytokinesis are localized to this structure. Therefore we next asked which central spindle proteins were required for targeting Ect2 to the central spindle. Specifically, we depleted the kinesins MKlp1 and MKlp2, the mitotic kinase Aurora-B and the GTPase activating protein MgcRacGAP from HeLa S3 cells, using previously validated siRNA duplexes. In agreement with previous reports (Kitamura et al., 2001; Matuliene and Kuriyama, 2002; Neef et al., 2003; Terada et al., 1998), depletion of all
these proteins resulted in a significant increase in binucleated cells, indicative of cytokinesis defects (data not shown).
s Ect2 siRNA MKlp1 MgcRacGAP iRNA Ect2 α-tubulin merge+DNA
A B
α-tubulin merge+DNA Aurora-B MKlp2C
Western blottingFigure 22. Ect2 requires the MKlp1/MgcRacGAP and Aurora-B/MKlp2 complexes to localize to the central spindle.
(A) HeLa S3 cells were transfected with siRNA duplexes against MKlp1, MgcRacGAP, Aurora-B and MKlp2 genes for 36 hours and were subsequently fixed and permeabilized with paraformaldehyde/Triton X-100. These cells were stained with anti-Ect2 antibodies in red (left), anti-α- tubulin antibodies in green (middle) and DNA was labelled with DAPI in blue. Merged images are shown at the right. Scale bar 10 µm.
(B) HeLa S3 cells were transfected with an Ect2 siRNA duplex for 36 hours and were subsequently fixed and permeabilized with paraformaldehyde/Triton X-100. These cells were stained with anti- MKlp1, anti-MgcRacGAP (Cyk4), anti-Aurora-B and anti-MKlp2 antibodies in red (left), anti-α-tubulin antibodies in green (middle) and DNA was labelled with DAPI in blue. Merged images are shown at the
(C) HeLa S3 cells were transfected with siRNA duplexes against GL2, MKlp1, MgcRacGAP, MKlp2, Aurora-B and Ect2 genes for 36 hours. Cell extracts were made in Hepes lysis buffer and after SDS-PAGE, the separated proteins were blotted onto nitrocellulose membranes and probed with anti-Ect2 antibodies (upper panel) and anti-α-tubulin antibodies (lower panel) as loading control.
In MKlp2 and Aurora-B depleted cells, the central spindle was often strongly deformed, missing many of the anti-parallel MTs in this region. Also in MKlp1 and MgcRacGAP depleted cells central spindle formation was affected, but to a lesser extent than in Aurora-B and MKlp2 depleted cells. At present it is not clear if this reflects a stronger dependency of central spindle formation on MKlp2 and Aurora-B or if this reflects the efficiency of the depletion. These data are consistent with the view that all the above proteins contribute to central spindle formation (Fig. 22A). Importantly, depletion of each of these four proteins abolished the interaction of Ect2 with residual central spindle and midbody structures, but did not affect its localization to the cell cortex (Fig. 22A and data not shown). Since the absence of Ect2 from central spindle and midbody could not be explained by diminished Ect2 levels (Fig. 22B), we conclude that MKlp1, MKlp2, Aurora-B and MgcRacGAP were all required for proper localization of Ect2 to these structures. In Ect2 depleted cells, on the other hand, MKlp1, MKlp2, Aurora-B and MgcRacGAP localization to the central spindle was not affected. These results strongly suggest that Ect2 acts downstream of MKlp1, MKlp2, Aurora-B and MgcRacGAP and merely uses the central spindle to position itself.
We could not directly compare the respective localization of these endogenous proteins at the central spindle as most antibodies were generated in rabbits. Hence, we could only determine co-localization at the central spindle of all these proteins with Plk1 which was detected with a mouse monoclonal antibody. Although MKlp1, MKlp2, Aurora-B, MgcRacGAP, Plk1 and Ect2 all co-localize at the central spindle during early anaphase (Fig. 23A), close examination of late stage dividing cells revealed distinct localization patterns for some of these proteins. In late anaphase and telophase cells, MKlp2 co-localized with Aurora-B and Plk1 in two bands adjacent to the midbody structure (Fig. 23B), consistent with the notion that MKlp2 interacts with these two kinases (Gruneberg et al., 2004; Neef et al., 2003). In contrast, both MKlp1 and MgcRacGAP localized more closely to the midbody structure (Fig. 23B), in line with
their ability to form a stable complex, called central spindlin (Mishima et al., 2002; Mishima et al., 2004). When Ect2 localization was analyzed carefully, it clearly matched the localization of the MKlp1/MgcRacGAP complex near the midbody (Fig. 23B), suggesting that this complex could be involved in targeting Ect2 to the central spindle and midbody.
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Plk1 merge+DNA
Figure 23. Ect2 co-localizes with MKlp1 and MgcRacGAP in telophase.
(A) HeLa S3 cells were fixed and permeabilized with paraformaldehyde/Triton X-100 and stained with anti-Plk1 antibodies in red (left) and anti-Aurora-B, anti-MKlp2, anti-Ect2, anti-MKlp1 and anti-MgcRacGAP antibodies in green (middle). DNA was labelled with DAPI in blue. Merged images are shown at the right. Scale bar 10 µm. (B) HeLa S3 cells were fixed and processed as in A and stained with the above mentioned antibodies. Midbody structure localization at telophase is shown. Scale bar 3.3 µm.
2.2.4 Ect2 interacts with the MKlp1/MgcRacGAP complex via the amino-
terminal BRCT domain
Since Ect2 localization to the central spindle is dependent on the MKlp1 and MgcRacGAP proteins and Ect2 co-localizes with these proteins at the midbody, we next asked whether these proteins exist in a complex. To determine whether Ect2 interacts with the MKlp1/MgcRacGAP complex, co-immunoprecipitation experiments were performed. HeLa S3 cells were synchronized by an aphidicolin/nocodazole block release protocol and harvested when most cells were present in anaphase or telophase. Endogenous Ect2 was then immunoprecipitated using anti-Ect2 antibodies or pre- immune antibodies for control (Fig. 24A). The resulting immune complexes were probed after Western blotting for the presence of various central spindle components. The mitotic kinesin MKlp1 (Kuriyama et al., 2002; Matuliene and Kuriyama, 2002) and the GTPase activating protein MgcRacGAP (Hirose et al., 2001) could readily be detected in Ect2 immunoprecipitates, whereas the mitotic kinase Aurora-B (Terada et al., 1998) and the mitotic kinesin MKlp2 (Hill et al., 2000; Neef et al., 2003) were absent, and none of these proteins were observed in control immunoprecipitates (Fig. 24A). These data indicate that Ect2 interacts specifically with the MKlp1/MgcRacGAP complex. These results are in agreement with the dependency of Ect2 on MKlp1 and MgcRacGAP proteins for its central spindle and midbody localization (Fig. 22A). In addition, these results suggest that MKlp2 and Aurora-B might have an indirect regulatory function in targeting Ect2 to the central spindle.
Since Ect2 localization is mediated via the amino-terminal BRCT domain (Fig. 21B), we next investigated whether this domain could directly interact with the MKlp1/MgcRacGAP complex. To investigate this possibility, different amino-terminal myc-tagged fragments were transiently expressed in HeLa S3 cells and immunoprecipitated using anti-myc (9E10) antibodies after synchronization, as described above. As shown (Fig. 24B), the amino-terminal fragments comprising residues 1-420 and 1-333 were able to co-precipitate MKlp1 and MgcRacGAP. A smaller fragment with a truncated BRCT domain (residues 1-288) on the other hand was unable to co- precipitate MKlp1 and MgcRacGAP and the same was true for an unrelated control
protein, hWW45 (Fig. 24B and 25C) (Chan et al., 2005; Valverde, 2000). In agreement with Ect2’s requirement to localize to the central spindle (Fig. 21A and 21B), the minimal domain identified here that could still bind to the MKlp1/MgcRacGAP complex comprised again the complete BRCT domain. These results show that targeting of Ect2 to the central spindle is mediated via the interaction of its BRCT domain with the MKlp1/MgcRacGAP complex.
A B
Figure 24. The MKlp1/MgcRacGAP complex co-immunoprecipitates with Ect2.
(A) Hepes lysis buffer extracts were made of HeLa S3 cells synchronized in anaphase and telophase stages by an aphidicolin-nocodazole block and release protocol. Immunoprecipitation experiments were performed on these extracts using anti-Ect2 antibodies and IgG pre-immune antibodies as a control. These immunoprecipitates were separated on SDS-PAGE, transferred onto nitrocellulose membranes and probed with anti-Ect2, anti-MKlp1, anti-MgcRacGAP, anti-Aurora-B and anti-MKlp2 antibodies. (B) HeLa S3 cells were simultaneously synchronized and transfected with myc-Ect2 1-420, myc-Ect2 1- 333, myc-Ect2 1-288 and myc-hWW45 as a control. Immunoprecipitation experiments were done as described above with anti-myc antibodies. Blots were then probed with anti-myc, anti-MKlp1 and anti- MgcRacGAP antibodies.
2.2.5 The interaction between the BRCT domain of Ect2 and the
MKlp1/MgcRacGAP might be phosphorylation dependent
Recently, it was shown that the BRCT domain of BRCA1 constitutes a phosphopeptide- binding domain (Clapperton et al., 2004; Manke et al., 2003; Williams et al., 2004; Yu et al., 2003). The crystal structure of the BRCA1 BRCT domain in complex with the BACH1 (BTB and CNC homology 1) phosphopeptide has been determined (Fig. 25A). This structure revealed that two amino acid side chains of S1655 and K1702 in the BRCT domain directly interact with the phosphate group of the phosphopeptide and another direct interaction was made with an amide bond of glycine residue G1656. Given that the two side-chain interactions critical for phosphoprotein binding in BRCA1 are present and highly conserved in Ect2 (T153, K195), we considered the possibility that the interaction of Ect2 with the MKlp1/MgcRacGAP complex could require a phosphorylated docking site. Also, the Clustal-W sequence alignment of the first Ect2 BRCT repeat with BRCT repeats of BRCA1, BARD1, XRCC1, RFC1 and PTIP revealed that although the primary sequence of BRCT repeats is not well conserved, the critical residues that have been implicated in phosphopeptide binding are conserved in all the BRCT repeats analyzed (Fig. 25B). Therefore, we mutated the two highly conserved residues (T153, K195) in the Ect2 1-333 BRCT domain to alanine residues (Fig. 25B) (Clapperton et al., 2004; Williams et al., 2004). This myc-tagged mutant fragment (1-333, T153A/K195A) was transiently overexpressed in HeLa S3 cells and immunoprecipitated after synchronization in ana- and telophase of the cell cycle. As shown in Fig. 25C, the wild type 1-333 fragment readily co-precipitated MKlp1 and MgcRacGAP, but only low levels were bound to the mutant BRCT (T153A/K195A) domain. Immunofluorescence microscopy analysis revealed that the BRCT T153A/K195A mutant fragment was only present at low levels at the central spindle, in accordance with its weak interaction with the MKlp1/MgcRacGAP complex (Fig. 25D). Together, these data strongly suggest that the localization of Ect2 to the central spindle could be mediated via the BRCT dependent binding to the MKlp1/MgcRacGAP complex and probably involves a phosphorylation- site specific docking mechanism.
A
C
D
B
Figure 25. The MKlp1/MgcRacGAP complex interacts with the BRCT domain of Ect2 possibly in a phosphorylation dependent manner.
(A) Schematic representation of BACH1 phosphopeptide (blue) contacts with the tandem BRCT domains of BRCA1. The residues marked in red coloured boxes within the BRCT domain are highly conserved throughout the whole family of BRCT domains (see arrows in Fig. 25B). Dashed lines, hydrogen bonds; pink crescent, van der waals interactions; green circles, water molecules.Image adapted and modified from Clapperton et al, Nature Structural and Molecular Biology 11, 512-518 (2004).
(B) Clustal-W sequence alignment of the first Ect2 BRCT repeat with BRCA1 (Breast Cancer Carboxyl terminal), BARD1 (BRCA1 associated ring domain1), XRCC1 (X-ray repair complementing defective repair in chinese hamster cells 1), RFC1 (replication factor C1) and PTIP (pax transactivation domain- interacting protein). The BRCT domain number indicates the BRCT repeat used for alignment out of the total number of BRCT repeats present in each protein. Arrows indicate the highly conserved residues in this alignment.
(C) HeLa S3 cells were simultaneously synchronized and transfected with myc-Ect2 1-333, myc-Ect2 1- 333 T153A/K195A and myc-hWW45 (as a control), respectively. Anti-myc antibodies coupled to protein G sepharose beads were used to immunoprecipitate these proteins from cell extracts of cells synchronized in anaphase and telophase of the cell cycle. The immunoprecipitates were separated by SDS-PAGE, blotted and probed with anti-myc, anti-MKlp1 and anti-MgcRacGAP antibodies.
(D) U2OS cells were transfected with myc-Ect2 1-333 and myc-Ect2 1-333 T153A/ K195A plasmids for 36 hours and were subsequently fixed and permeabilized with paraformaldehyde/Triton X-100. These cells were stained with anti-myc in red (left) and anti-α-tubulin antibodies in green (middle). DNA was labelled withDAPI in blue. Merged images are shown at the right. Scale bar 10 µm.
2.2.6 Ect2 central spindle localization is not essential for cytokinesis
As shown above, the intact BRCT domain is necessary and sufficient for central spindle localization of Ect2. We next wondered if overexpression of this domain could displace endogenous Ect2 from the central spindle. To examine this, an antibody that detects a carboxyl-terminal epitope in Ect2 recognizing only endogenous Ect2 protein was used. Examination of cells overexpressing the amino-terminal fragments (1-333 and 1-420) showed that endogenous Ect2 was indeed displaced from the central spindle by these fragments (Fig. 26A). Expression of a shorter amino-terminal fragment (1-288) with a truncated BRCT domain (1-288) that does not localize to the central spindle did not interfere with endogenous Ect2 localization. This indicates that the BRCT domain is able to displace endogenous Ect2 from the central spindle, most likely as a result of competition with endogenous Ect2 for binding to the MKlp1/MgcRacGAP complex.
Despite displacement of endogenous Ect2 from the central spindle by the large amino-terminal Ect2 fragment (1-420), these cells did not show obvious cytokinesis defects (Fig. 26, right panel) as revealed by their mononuclear appearance (data not shown). This suggests that Ect2 central spindle localization is not absolutely required for cytokinesis and that the cortical pool of Ect2 might be sufficient to carry out cytokinesis in these cells. Surprisingly, though, cells expressing the smaller BRCT containing Ect2 fragment (1-333) became binucleated after 48 hours of expression, indicative of a cytokinesis failure in these cells (Fig. 26, right panel). Since both BRCT domain-
containing fragments displaced endogenous Ect2 equally well (Fig. 26), the cytokinesis defect upon overexpression of the 1-333 fragment is unlikely to be a result of Ect2 central