The RAS association domain family 1A (RASSF1A) gene is silenced by DNA methylation in over 50% of all solid tumors of different histological types. However, the biochemical function of the RASSF1A protein is unknown. We show that RASSF1A colocalizes with microtubules in interphase and decorates spindles and centrosomes during mitosis. RASSF1A has a strong cytoprotective activity against the microtubule-destabilizing drug nocodazole, and against cold-treatment in vivo. Conversely, loss of RASSF1 in RASSF1−/− mouse embryonic fibroblasts renders the cells more sensitive to nocodazole-induced depolymerization of microtubules. The domain required for both microtubule association and stabilization was mapped to a 169 amino-acid fragment that contains the RAS association domain. Overexpression of RASSF1A induces mitotic arrest at metaphase with aberrant mitotic cells reminiscent of such produced by the microtubule-stabilizing drug paclitaxel (taxol), including monopolar spindles, or complete lack of a mitotic spindle. Altered microtubule stability in cells lacking RASSF1A is likely to affect spindle assembly and chromosome attachment, processes that need to be carefully controlled to protect cells from genomic instability and transformation. In addition, knowledge of the microtubule-targeting function of RASSF1 may aid in the development of new anticancer drugs.
Recently, we have cloned and characterized the RAS association domain family 1 (RASSF1) gene (Dammann et al., 2000). The RASSF1 gene is located at 3p21.3 within an area of common heterozygous and homozygous deletions, which occur frequently in a variety of human solid tumors (Kok et al., 1997; Sekido et al., 1998; Lerman and Minna, 2000). None of the eight genes in the common homozygous deletion area at 3p21.3 is mutated at a significant frequency in cancer (Lerman and Minna, 2000). However, one of the isoforms of the RASSF1 gene, RASSF1A, is inactivated by DNA methylation of its promoter CpG island at a high frequency, as observed originally in lung cancers (Dammann et al., 2000; Burbee et al., 2001). RASSF1A is the most frequently methylated gene so far described in human cancer (Pfeifer et al., 2002; Dammann et al., 2003). RASSF1A methylation occurs in a broad spectrum of carcinomas and other solid tumors (Dammann et al., 2000, 2001a,2001b; Agathanggelou et al., 2001; Burbee et al., 2001; Byun et al., 2001; Dreijerink et al., 2001; Lee et al., 2001; Lo et al., 2001; Maruyama et al., 2001; Morrissey et al., 2001; Kuzmin et al., 2002; Liu et al., 2002; Lusher et al., 2002; Schagdarsurengin et al., 2002; Toyooka et al., 2002; van Engeland et al., 2002; Wagner et al., 2002; Zhang et al., 2002; Chan et al., 2003; Dammann et al., 2003; Honorio et al., 2003; Schagdarsurengin et al., 2003; Spugnardi et al., 2003).
Re-expression of RASSF1A reduces the growth of human cancer cells supporting a role for RASSF1 as a tumor suppressor gene (Dammann et al., 2000; Burbee et al., 2001; Dreijerink et al., 2001; Kuzmin et al., 2002). The biochemical function of the RASSF1A protein is unknown. The homology of RASSF1A with the mammalian RAS effector NORE1 suggested that the RASSF1A gene product may function in signal transduction pathways involving RAS-like proteins. Recent data indicate that RASSF1A itself binds to RAS only weakly, and that binding to RAS may require heterodimerization of RASSF1A and NORE1 (Ortiz-Vega et al., 2002). There is evidence for an association of both NORE1 and RASSF1A with the proapoptotic kinase MST1 and that this interaction is involved in apoptosis (Khokhlatchev et al., 2002). Other investigations have uncovered a role of RASSF1A in suppression of cyclin D accumulation and cell cycle progression (Shivakumar et al., 2002).
Here, we present evidence that RASSF1A is a microtubule binding protein that can stabilize microtubules from depolymerization in vivo. RASSF1A localizes to the mitotic apparatus and controls mitotic progression.
RASSF1A associates with microtubules in both interphase and mitosis
To analyse the function of RASSF1A, we investigated the subcellular localization of this protein in COS-7 cells. Using EGFP-tagged RASSF1A, we observed that the RASSF1A signal formed a meshwork-like pattern in the cytoplasm with brighter signals surrounding the nucleus (Figure 1A). When EGFP-RASSF1A was introduced into other mammalian cell lines, including A549, LNCaP, HeLa, and NIH-3T3, the same pattern was observed (data not shown). When we transfected EGFP-RASSF1A into COS-7 cells and costained with an anti-α-tubulin antibody, colocalization of RASSF1A with microtubules was observed in the interphase cells (Figure 1a–a″). However, no significant colocalization of RASSF1A with F-actin was observed (data not shown). To further demonstrate the association of RASSF1A with microtubules, we treated EGFP-RASSF1A-transfected COS-7 cells with nocodazole, a microtubule depolymerizer, and with taxol, a microtubule stabilizer. Upon disruption of microtubules by nocodazole (2 μ M, 20 h), the fibrous structure of EGFP-RASSF1A disappeared. Instead, the RASSF1A signal was diffused throughout the cytoplasm with about 15–25% entering the nucleus (Figure 1b–b″). Conversely, RASSF1A colocalized with bundled microtubules in cytoplasmic protrusions when taxol was used (Figure 1c–c″). These results suggest that the localization of RASSF1A depends on the integrity of the microtubule network. Next, we performed an in vivo GST pull-down assay. β-tubulin and γ-tubulin could only be detected in the pull-down product from GST-RASSF1A-transfected 293T cells, but not in the pull-down product from pEGB (GST)-transfected cells (Figure 1d), suggesting that RASSF1A can associate with β-tubulin and γ-tubulin physically. Since γ-tubulin is a centrosome-specific protein, this is consistent with our results showing that RASSF1A intensely stained the microtubule-organizing center (MTOC) in interphase cells (Figure 1a–a″).
To further explore the association of RASSF1A with microtubules, we studied cells throughout mitosis (Figure 2). In prophase and prometaphase, RASSF1A localized to spindle poles and attached spindles. When cells progressed into metaphase and anaphase, RASSF1A staining accumulated to spindle poles and only slightly decorated the interpolar spindles. In telophase, RASSF1A intensely stained both the spindle poles and the interzone region. During cytokinesis, the cytoplasmic microtubules reappear, and RASSF1A staining was distributed along the astral microtubules and concentrated at the spindle poles. Strikingly, the midbody region, where the microtubules overlap and the cleavage furrow forms, was also intensely stained by RASSF1A. These results suggest that RASSFIA may play a role during mitosis and cytokinesis.
RASSF1A stabilizes microtubules in vivo
We treated COS-7 cells transfected with EGFP-RASSF1A with nocodazole, a tubulin-depolymerizing drug. When untransfected or EGFP-transfected COS-7 cells were incubated with 20 μ M nocodazole for 60 min, their microtubular array was completely destructed (Figure 3b–b″). In contrast, the microtubule structure was largely intact in over 75% of the COS-7 cells with forced expression of EGFP-RASSF1A (Figure 3a–a″). To further explore the microtubule stabilizing effect of RASSF1A, we held COS-7 cells on ice for 30 min, conditions that resulted in loss of microtubules in all the untransfected or EGFP-transfected cells (Figure 3d–d″). Surprisingly, the microtubules still remained intact in ice-treated COS-7 cells that were transfected with EGFP-RASSF1A (Figure 3c–c″). To support these data, we generated RASSF1−/− mouse embryonic fibroblasts (MEFs) (Figure 4). The sensitivity of these cells to nocodazole was analysed. Consistent with the protective effect seen in the transfection studies, loss of RASSF1 rendered the MEFs more sensitive to nocodazole treatment. When different concentrations of nocodazole were used for 30 min, we found that microtubules began to partially depolymerize at 1 μ M but microtubule fibers were still detectable at 6 μ M in wild type cells (Figure 5). In RASSF1−/− MEFs, microtubules began to disrupt at 0.5 μ M nocodazole and microtubular fibers could hardly be seen at 2 μ M nocodazole (Figure 5).
Compared with cytoplasmic microtubules, mitotic spindles are more dynamic structures. Overexpression of RASSF1A cannot protect spindles from depolymerization by cold treatment for 30 min. Interestingly, the centrosome spots were still visible in these cells and RASSF1A staining coincided with these spots (Figure 6a–a′″, b–b′″). Since RASSF1A can interact with γ-tubulin (Figure 1d) and RASSF1A concentrates at the spindle pole during mitosis (Figure 2), the data strongly indicate that RASSF1A can interact with the centrosome.
When EGFP-RASSF1A-transfected COS-7 cells were incubated with 2 μ M nocodazole for 20 h, over 80% of the cells lose the integrity of microtubules. When a 5 min time period was allowed for the cells to recover after removal of nocodazole, the microtubular array began to nucleate from the newly forming centrosome (Figure 6c′). Strikingly, 90% of RASSF1A staining was solely concentrated at this star-like structure (Figure 6c–c′″), suggesting that RASSF1A may promote microtubule polymerization in vivo.
Microtubule-binding and -stabilizing domain mapping of RASSF1A
The RASSF1A cDNA encodes a protein of 340 amino acids with a calculated MW of 38.8 kDa. The C-terminus of RASSF1A contains a putative Ras-association domain (R194 to S288), which shows high homology to the murine Ras-effector protein Nore1 (Vavvas et al., 1998) and may mediate interaction with Ras oncoproteins. The N-terminus of RASSF1A has high homology to a cystein-rich diacyl-glycerol/phorbol ester-binding domain (Newton, 1995). RASSF1A also has a putative ATM kinase phosphorylation consensus motif (Kim et al., 1999) spanning amino acids W125 to K138. To determine the domain(s) that is (are) required for RASSF1A to associate with and to stabilize microtubules, we generated a series of deletion mutants of RASSF1A tagged with EGFP (Figure 7a). The subcellular distribution of these mutants was analysed by fluorescence microscopy. Only two mutants, RASSF1A 120-340, and RASSF1A 1-288, could bind to microtubule-like structures in the cytoplasm (Figure 7b), and they colocalized with microtubules in the interphase cells (data not shown). Unlike full-length RASSF1A and RASSF1A 120–340, a nuclear accumulation of RASSF1A 1–288 is predominant in COS-7 cells. This suggests that the C-terminal 52 amino acids play a role in retaining full-length RASSF1A in the cytosol. It is less likely that this region or this region combined with an adjacent region contain a nuclear export signal (NES), because no typical NES was detected. When this region alone (RASSF1A 289–340) or this region plus an adjacent region (RASSF1A 194–340) was cloned into an EGFP vector, the signal largely localized to the nucleus (Figure 7b). Like RASSF1A full-length protein, both mutants can localize to the spindle apparatus during mitosis (data not shown). Similarly, these two mutants were also capable of protecting microtubules from disruption by nocodazole (Figure 7c, d) and by cold treatment (data not shown). The other mutants did not significantly colocalize with microtubules and were incapable of protecting microtubules from depolymerization (data not shown). These results indicate that the minimal overlapping domain required for association with and stabilization of microtubules is a 169 amino-acid fragment from D120 to S288, which includes the intact RAS-association domain (RA) and putative ATM phosphorylation site of RASSF1A. This sequence shares no significant homology with any other known microtubule-binding protein, defining a novel microtubule interacting domain. Although we have done no detailed study with RASSF1C, the other major isoform expressed from the RASSF1 locus, the presence of this domain in RASSF1C would suggest that this protein may bind to microtubules as well. In addition, these sequences are highly conserved in the two closest RASSF1 homologues, NORE1 and RASSF3 (Tommasi et al., 2002), implying a conserved function of RASSF1-related proteins.
RASSF1A regulates mitotic progression
To determine whether RASSF1A has any effect on mitosis, EGFP-tagged RASSF1A and its truncated mutant constructs were transfected into COS-7 cells. Cells were double-stained with anti-α-tubulin antibody and 4,6-diamidino-2-phenylindole (DAPI), and mitotic cells were scored. Strikingly, overexpression of RASSF1A resulted in over 50% of mitoses with only monopolar spindles (Figure 8a–a′″, c). In these cells, RASSF1A colocalized with the monopolar spindles. Typically these cells have highly condensed ball-shape chromatin (Figure 8a″). Similarly, ectopic expression of RASSF1A 120–340 also led to 46% mitoses with monopolar spindles. In contrast, only 5 and 12% monopolar mitoses were observed in untransfected and EGFP-transfected COS-7 cells, respectively (Figure 8c). In RASSF1A 1–288-transfected cells, 18% of all mitoses were monopolar, but 51% of all mitoses were without any spindle structure (Figure 8b–b′″, d). In these spindle-less mitoses, the chromosomes were also highly condensed and the only tubulin-containing structures were some punctate aggregates of tubulin throughout the cell. RASSF1A 1–288 also distributed without any definitive spindle structure in these mitotic cells (Figure 8b–b′″). In all, 10% of mitoses were found to be without spindle in RASSF1A 120–340-transfected cells. However, less than 2% of spindle-less mitoses were found in cells untransfected or transfected with EGFP, full-length RASSF1A, RASSF1A 1–193, RASSF1A 1–220, RASSF1A 194–340, or RASSF1A 152-340, respectively (Figure 8D). Since a large percentage of abnormal mitoses appeared in cells overexpressing RASSF1A or two of its truncated mutants that can bind to microtubules, we calculated the ratio of cells in anaphase to cells in metaphase. In untransfected COS-7 cells, this ratio was 0.16. A significant increase in this ratio may indicate an anaphase arrest, while a significant decrease in this ratio may suggest a metaphase arrest. As shown in Figure 8e, this ratio dramatically dropped to 0.016, 0.015, and 0.002 in RASSF1A-, RASSF1A 120-340-, and RASSF1A 1–288- transfected cells, respectively, suggesting a metaphase arrest in these cells.
We have shown here that the RASSF1A protein localizes to the microtubule network. This result was somewhat unexpected since RASSF1 is endowed with a RAS association domain, a domain found in proteins that can bind to activated (GTP bound) RAS family members (Ponting and Benjamin, 1996). However, previous studies have shown that RASSF1A is able to bind to activated RAS only indirectly through its closest homologue and heterodimerization partner NORE1 (Ortiz-Vega et al., 2002). The intracellular localization of NORE1 has not yet been reported. Activated k-RAS proteins are normally localized at the plasma membrane although an interaction of k-RAS with the microtubule network has been described (Chen et al., 2000). An intact RAS association domain is required for RASSF1A to bind to microtubules (Figure 7). It remains to be determined if (indirect) k-RAS interaction and the proapoptotic effect of RASSF1A (Khokhlatchev et al., 2002) take place in the setting of microtubule association of RASSF1A. Interestingly, a putative Ras effector in Schizosaccharomyces pombe, Scd1, was reported to localize to mitotic spindles (Li et al., 2000; Segal and Clarke, 2001). Loss of Scd1 resulted in hypersensitivity to microtubule depolymerizing drugs, while overexpression of Scd1 induced mitoses with abnormal spindles and missegregated chromosomes. Our report is the first one to describe microtubule and spindle association for a putative mammalian RAS effector. When RASSF1A was introduced into a K-ras transformed cell line, its cellular localization was not significantly changed compared to that in the paired untransformed cell line (data not shown), suggesting that microtubule targeting of RASSF1A is independent of active k-RAS.
Recently, several other tumor suppressor gene products have been reported to associate with the microtubule network. The adenomatous polyposis coli (APC) tumor suppressor protein localizes to and stabilizes microtubules (Zumbrunn et al., 2001). The von Hippel–Lindau tumor suppressor protein (pVHL) also is a microtubule-associated protein that can protect microtubules from depolymerization (Hergovich et al., 2003). BRCA1, a tumor suppressor for breast cancer, has been shown to associate with centrosomes (Hsu and White, 1998) and mitotic spindles (Lotti et al., 2002). It remains to be seen if microtubule association and stabilization by several important tumor suppressor proteins can be functionally linked to a common pathway that needs to be disabled in tumorigenesis.
We have shown here that expression of RASSF1A and several of its deletion mutants arrests cells in mitosis and promotes the formation of aberrant spindle structures. The microtubule stabilizing effect of RASSF1A in interphase cells and the effects of its overexpression on mitotic cells are surprisingly similar to those produced by the commonly used cancer chemotherapy drug paclitaxel (taxol). Taxol produces mitotic arrest by stabilization of microtubules leading to a loss of microtubule dynamics (Jordan et al., 1993). The effects of taxol on the mitotic spindle are dose-dependent (Jordan et al., 1996). At the lowest doses, a bipolar spindle is still formed but some of the chromosomes fail to align along the metaphase plate (Jordan et al., 1996). At higher concentrations of taxol, monopolar spindles, with congression of the chromosomes into a ball-shaped structure, has been observed (Jordan et al., 1993; Jordan et al., 1996). We see exactly the same effect with overexpression of full-length RASSF1A (Figure 8). At the highest concentrations of taxol or other microtubule-targeting drugs, the drugs promote destruction and loss of the mitotic spindle (Jordan et al., 1992), a phenomenon observed with the C-terminal RASSF1 deletion mutant (RASSF1A 1–288) in our studies (Figure 8d). The mechanistic basis of the taxol-like effect of RASSF1A is not known, but we speculate that RASSF1A overexpression reduces microtubule dynamics, thus leading to a defect in centrosome separation and bipolar spindle formation (monopolar or spindle-less mitoses). Knowledge of the microtubule-targeting function of RASSF1 may be exploited for the development of new anticancer drugs. In addition, it may be important to determine if patients with tumors harboring RASSF1 deficiencies may respond differently to treatment regimens involving spindle poisons.
Most solid tumors are characterized by frequent epigenetic inactivation of the RASSF1A gene (Dammann et al., 2000, 2001a,2001b; Agathanggelou et al., 2001; Burbee et al., 2001; Byun et al., 2001; Dreijerink et al., 2001; Lee et al., 2001; Lo et al., 2001; Maruyama et al., 2001; Morrissey et al., 2001; Kuzmin et al., 2002; Liu et al., 2002; Lusher et al., 2002; Schagdarsurengin et al., 2002; Toyooka et al., 2002; van Engeland et al., 2002; Wagner et al., 2002; Zhang et al., 2002; Chan et al., 2003; Dammann et al., 2003; Honorio et al., 2003; Schagdarsurengin et al., 2003; Spugnardi et al., 2003). We have prepared RASSF1 knockout mice in order to investigate the role of this gene in tumorigenesis. Homozygous knockout mice are viable and fertile. They are currently being observed for spontaneous and induced tumor formation. Fibroblasts were prepared from day 13.5 embryos. These RASSF1−/− fibroblasts are only 60–70% the size of wild-type MEFs (data not shown) and are much more sensitive than fibroblasts from wild-type littermates towards microtubule destruction by nocodazole (Figure 5), suggesting cytoskeletal defects. This confirms that the stabilizing effect seen in the cell line studies is not due to expression of the protein at nonphysiological levels.
Loss of RASSF1A expression by promoter methylation is a very common phenomenon observed in almost every type of cancer so far described (Dammann et al., 2003). One notable exception is cervical carcinoma for which an inverse association between RASSF1A methylation and presence of high-risk papillomavirus (HPV16/HPV18) sequences has been seen (Kuzmin et al., 2003). Human papillomavirus oncoproteins E6 and E7 promote mitotic infidelity by promoting centrosome duplication errors, aberrant mitoses, and aneuploidy (Duensing and Münger, 2002). Aberrations in chromosome numbers (aneuploidy, polyploidy) are a common feature of most solid tumors for which RASSF1A inactivation has been reported, suggesting that loss of RASSF1A and papillomavirus infection may engage the same tumorigenic pathway. Thus, one hypothesis to be tested would be that lack of RASSF1A affects mitotic spindle function. In preliminary analysis of a limited number of metaphase spreads of RASSF1 knockout fibroblasts, we have not detected gross chromosomal aberrations. However, one requirement for such mitotic aberrations to result in viable daughter cells is that the mitotic spindle checkpoint, which monitors unaligned chromosomes (Rieder et al., 1995), also needs to be defective and/or that the apoptotic mechanism that eliminates such defective cells is abrogated. This could be accomplished, for example, by mutation or epigenetic inactivation of checkpoint genes such as BUB1, BUB3, MAD2, or others (Cahill et al., 1998; Michel et al., 2001) or by overexpression of the serine–threonine kinase AURORA-A (Anand et al., 2003). We are currently testing this hypothesis by analysing tumor samples for simultaneous aberrations in RASSF1A and in known spindle checkpoint genes.
Materials and methods
Cell culture and transfection
COS-7, 293T, HeLa, A549, LNCaP, and NIH3T3 cells were obtained from the ATCC and were cultured in high-glucose DMEM with 10% fetal bovine serum. Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
Knockout mice and embryonic fibroblasts
To construct RASSF1 knockout mice, we sequenced 18 kb of the mouse 129/SvImJ RASSF1 locus (Genbank Accession number AF333027). All RASSF1 exons are highly conserved between mouse and human with >90% identity at the amino-acid level. We generated mouse PCR products by high-fidelity PCR from genomic 129/SvImJ DNA. Appropriate fragments flanking exon 1α and exon 6 of mouse RASSF1 (3.9 and 5.3 kb in length, respectively) were cloned into the pKSneolox targeting vector and were confirmed by DNA sequencing. The vector was linearized and introduced into 129/SvImJ ES cells by electroporation. Drug-resistant ES cell clones were isolated and Southern analysis was used to identify recombinant clones. After production of chimeras and germline transmission, the RASSF1-targeted mice were bred to homozygosity (129S1 × B6J background). Absence of RASSF1 transcripts in the mouse tissues and embryonic fibroblasts from RASSF1−/− mice was confirmed by RT–PCR (Figure 4c). We used previously reported methods (Abbondanzo et al., 1993) to isolate and culture primary MEFs derived from 13.5 day embryos.
To generate EGFP- or GST-tagged RASSF1A, the cDNA encoding human RASSF1A was subcloned into EGFP-C2, EGFP-N2 (Clontech, Palo Alto, CA, USA), or pEGB (kindly provided by Dr J Avruch; Massachusetts General Hospital) vectors. Each experiment concerning EGFP-RASSF1A was carried out using both EGFP-C2- and EGFP-N2-tagged RASSF1A. Similar results were obtained with C-terminal- and N-terminal-tagged constructs. The truncated mutant RASSF1A sequences were generated by a single-step PCR using EGFP-RASSF1A as template and cloned into EGFP-C2 or EGFP-N2.
At 24 h after transfection, COS-7 cells were trypsinized and seeded at 60% confluence into six-well plates containing coverslips coated with poly-L-lysine (Sigma; St Louis, MO, USA). Cells were allowed to grow for an additional 24 h. The cells were washed with PBS, fixed with formaldehyde, and permeabilized with 0.1% Triton X-100 for 5 min. The slides were treated with 1% BSA in PBS for 30 min. For tubulin staining, the cells were incubated with a mouse monoclonal anti-α-tubulin antibody (Molecular Probes; Eugene, OR, USA) for 1 h at room temperature. After washing with PBS, Alexa Fluor 568-conjugated anti-mouse secondary antibody (Molecular Probes) was used and DNA was counterstained with DAPI (Sigma). Images were obtained with an Olympus IX81 automated inverted microscope equipped with a Spot RT Slider high-resolution cooled CCD color camera (Olympus; Melville, NY, USA) or with a Zeiss LSM310 laser scanning confocal microscope with Zeiss MC100 35 mm camera. Color images were processed using Photoshop 7.0 (Adobe Systems; San Jose, CA, USA) or ImagePro Plus (Media Cybernetics; Silver Spring, MD, USA).
In vivo GST pull-down assay
At 60–72 h after transfection with GST-RASSF1A or pEBG (GST) vector alone, 293T cells were harvested and lysed in ice-cold cytoskeleton lysis buffer (0.5% Nonidet P-40, 10 mM PIPES, pH 7.0, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1 mM DTT, 1 mM Na3VO4) with protease inhibitor cocktail (Roche; Indianapolis, IN, USA). The lysates were incubated with glutathione sepharose 4B (Amersham; Piscataway, NJ, USA) for 45 min at room temperature. After centrifugation and washing with PBS, the pellet was collected and the bound proteins were eluted with SDS loading buffer by heating to 95°C for 5 min. The eluted proteins were fractionated on a 10% SDS–polyacrylamide gel and transferred to a PVDF membrane. The blots were probed with monoclonal antibodies against GST (Amersham), β-tubulin, or γ-tubulin (Santa Cruz Biotechnologies; Santa Cruz, CA, USA) followed by appropriate secondary antibodies conjugated with horseradish peroxidase. The signals were detected using ECL-Plus (Amersham).
Microtubule stability and repolymerization analysis
To evaluate microtubule stability, coverslip cultures of COS-7 cells transfected with the indicated constructs were incubated with 20 μ M nocodazole (Sigma) for 1 h. The coverslips were washed twice briefly with cold PBS and then were double-stained with anti-α-tubulin antibody (Molecular Probes) and DAPI (Sigma) as described above. For MEFs, the cells were seeded onto lysine-coated eight-chamber cover slides and were allowed to grow for 24 h. Nocodazole was used to treat the cells at concentrations of 0.5, 1, 2, 4, 6, and 8 μ M for 30 min. The cover slides were washed twice with cold PBS and were stained with anti-α-tubulin as described above. At each concentration of nocodazole, 100–150 cells were scored for microtubule integrity. The micrographs shown are representative of >75% of the cells in terms of microtubule integrity at each concentration of nocodazole. To study repolymerization of microtubules, coverslip cultures of COS-7 cells transfected with EGFP-RASSF1A were incubated with 2 μ M nocodazole for 20 h. Cells were incubated at 37°C for 5 min after removal of nocodazole and then were processed for immunofluorescence microscopy as described above.
COS-7 cells transfected with EGFP-RASSF1A or its truncated mutant constructs were double-stained with anti-α-tubulin antibody (Molecular Probes) and DAPI (Sigma). Mitotic cells were scored and counted under a fluorescence microscope, based on both spindle structure and chromosome configuration. Mitotic cells with highly condensed chromosomes but without spindle or with a monopolar spindle were categorized as metaphase according to the convention in the literature. For each transfection, at least 300 mitotic cells were enumerated. COS-7 cells, either untransfected or transfected with EGFP only, were analysed in parallel as controls.
mouse embryonic fibroblast
RAS association domain family 1A
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We thank R Barber, M Lee, and P Salvaterra for assistance with microscopy, S Bates for culturing cells, W Tsark and F Silva for ES cell electroporation and blastocyst injection, L Brown and C Spalla for FACS analysis, S-G Jin for plasmid construction, and J Shively, CJ Chen, and CL Jiang for instructive discussions. This work was supported by NIH Grant CA88873 (to GPP) and by BMBF Grant FKZ01ZZ0104 (to RD).
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