To determine the mechanism through which the absence of hMps1 might impair checkpoint signaling, mock-transfected and hMps1 siRNA transfected cells were examined for kinetochore localization of different checkpoint proteins. This approach allowed us to ask whether there is any dependency between hMps1 kinase and checkpoint proteins with respect to their kinetochore localization.
In a first series of experiments, we focused on the hMad1/hMad2 complex. As mentioned previously, budding yeast Mps1p kinase is able to phosphorylate Mad1p in vitro (Hardwick et al., 1996), and in Xenopus egg extracts, XMps1 is required for kinetochore recruitment of the Mad1/Mad2 complex (Abrieu et al., 2001). These data suggest that Mps1 kinases may function upstream of Mad1/Mad2 in the checkpoint signaling cascade. To test this directly in human cells, hMps1 was silenced in HeLa S3 cells for 48 hours with the hMps1 siRNA duplex and kinetochores of mitotic cells were stained by indirect immunofluorescence with antibodies specific for the checkpoint proteins hMad1 and hMad2. As shown in Fig. 26A, the siRNA-mediated depletion of hMps1 abolished the kinetochore association of hMad1, whereas in mock-transfected cells hMad1 could readily be detected at kinetochores. In addition, hMad2, that forms a tight complex with hMad1, (Luo et al., 2002; Sironi et al., 2001) was not detected at kinetochores anymore (Fig. 26B), although strong kinetochore staining was seen in control cells (Fig. 26B). In parallel, cell extracts were prepared of control and hMps1-depleted cells and probed by Western blotting with antibodies specific for hMps1, hMad1 and hMad2, respectively. As shown in Fig. 26C, hMps1 was selectively depleted from cells, whereas the levels of the other checkpoint proteins remained unchanged, indicating that their displacement from the kinetochores is not due to protein degradation. Thus, hMad1 and hMad2 are clearly mislocalized in cells depleted for hMps1, and we conclude that hMps1 has an important function in mediating kinetochore association of the Mad1/Mad2 complex.
These data fall in line with previous reports, that Mps1 kinases function upstream of Mad1/Mad2 in the pathway regulating the mitotic spindle checkpoint in different organisms (Hardwick et al., 1996; Abrieu et al., 2001) and was recently confirmed by Liu et al. (2003).
Figure 26: Depletion of hMps1 by siRNA prevents binding of hMad1 and hMad2 to kinetochores.
(A) HeLa S3 cells were mock-transfected (upper panel) or transfected with hMps1 siRNA duplex (lower panel) for 48 hours. Cells were fixed and permeabilized simultaneously with formaldehyde/Triton-X-100 and analysed by indirect immunofluorescence microscopy using anti-hMps1 mAb N1 (left row) and rabbit anti-hMad1 antibody (middle row). DNA was stained using DAPI (right row). Bar = 10µm.
(B) HeLa S3 cells were transfected and fixed as described in (A). After fixation, cells were stained for hMps1 (left row) and hMad2 (middle row), respectively. DNA was stained using DAPI (right row).
Bar = 10µm.
(C) Immunoblots demonstrating selective depletion of hMps1 by siRNA. HeLa S3 cells were treated with appropriate siRNA duplexes as described in (A). Total HeLa S3 cell extracts were probed with antibodies specific to hMps1 (upper lane), hMad1 (2nd lane), and hMad2 (3rd lane). Antibodies to α-tubulin (lower lane) were used to demonstrate equal loading.
hMps1 is not required for kinetochore recruitment of Hec1, hBub1, hBubR1, CENP-E, CENP-F and CENP-B
In order to test whether the observed displacement of the hMad1/hMad2 complex upon hMps1 siRNA was specific, we tested a series of other checkpoint proteins for their kinetochore localization in hMps1-depleted cells. In particular, we concentrated on checkpoint components that, based on results from several groups obtained in different organisms, also influenced the Mad1/Mad2 localization. Thereby we could determine the position of hMps1 in the kinetochore assembly pathway compared to the known checkpoint proteins. The following proteins were chosen for analysis:
-Hec1: Hec1 is the homolog of budding yeast Ndc80p, which is required for chromosome segregation in yeast (Wigge et al., 1998). The coiled-coil protein Hec1 interacts with hMad1 in a yeast two-hybrid assay and siRNA of Hec1 in HeLa S3 cells displaces hMad1/hMad2 from the kinetochores, similar to what we observed in hMps1-depleted cells (Martin-Lluesma et al., 2002). Thus, Hec1 might function in the same pathway as hMad1/hMad2 and hMps1.
-hBub1 and hBubR1: immunodepletion of the protein kinases hBub1 and hBubR1 in Xenopus extracts demonstrated, that these two proteins are required for kinetochore association of XMad1/XMad2 (Sharp-Baker and Chen, 2001; Chen, 2002). Moreover, fission yeast Mph1p is required for Mad3p (BubR1-homolog) recruitment to kinetochores (Millband and Hardwick, 2002). Therefore, it is important to know whether hMps1 is required for kinetochore localization hBub1 and hBubR1 in human cells.
-CENP-E and CENP-F: immunodepletion of CENP-E in Xenopus extracts abrogated kinetochore association of XMad1/XMad2 (Abrieu et al., 2000). Moreover, immunodepletion of XMps1 revealed that XMps1 is partially required for CENP-E to be kinetochore localized (Abrieu et al., 2001). Therefore, XMps1 might function upstream of CENP-E. CENP-F was shown to interact with CENP-E in a yeast two-hybrid assay (Chan et al., 1998), and might also be a downstream target of XMps1.
Our assay allows us to test whether the XMps1-CENP-E link with regard to kinetochore localization is conserved in human cells.
-CENP-B: structural component of the centromere (Kitagawa and Hieter, 2001).
CENP-B serves as a control in these experiments in order to ensure that the assembly of structural centromeric proteins is not affected in checkpoint protein-depleted cells.
As shown in Fig. 27, elimination of hMps1 did not interfere with kinetochore association of the checkpoint proteins Hec1 (Fig. 27A), hBub1 (Fig. 27B), and hBubR1 (Fig. 27C). Furthermore, CENP-E (Fig. 27D) was still localized to kinetochores in hMps1-depleted cells, which is in contrast to what has been observed in Xenopus extracts (Abrieu et al., 2001). Finally, both CENP-F (Fig. 27E) and CENP-B (Fig. 27F) could still localize to kinetochores efficiently, indicating that siRNA of hMps1 did not disrupt the structural assembly of kinetochores (Fig. 27F).
Figure 27: Depletion of hMps1 by siRNA does not interfere with kinetochore binding of Hec1, hBub1, hBubR1, CENP-E, CENP-F and CENP-B.
(A-F) HeLa S3 cells were mock-transfected (upper panels) or transfected with hMps1 siRNA duplex (lower panels) for 48 hours. Prometaphase cells were analysed by indirect immunofluorescence microscopy. Cells were fixed and permeabilized simultaneously with formaldehyde/Triton-X-100, and stained with appropriate antibodies as indicated. In Figures (A-C) and (E), hMps1 staining is shown in the left rows, and staining with corresponding antibodies is shown in the right rows. In Figures (D) and (F) we could not stain for hMps1 because of the identical source of the primary antibodies (mouse).
Instead, DNA staining by DAPI is shown (right rows). Bar = 10µm.
Taken together, these data reveal that elimination of hMps1 interferes with kinetochore association of a specific subset of checkpoint proteins. In particular,
kinase, whereas Hec1, the hBub1/hBubR1 protein kinases and the centromere proteins CENP-E, CENP-F and CENP-B do not depend on hMps1. These observations suggest that all these proteins function either upstream of hMps1 or in a parallel, hMps1-independent branch of a checkpoint pathway that regulates kinetochore localization of checkpoint components in human cells.