Girdin is a downstream effector of epidermal growth factor receptor (EGFR)-AKT and interacts with actin and microtubule. Increasing evidence confirmed that Girdin played an important role in cell migration. Here we report that Girdin also regulates cell division. Overexpression or suppression of Girdin leads to attenuated cell proliferation. Imaging of mitotic cells revealed that Girdin is located in the cell division apparatus such as centrosome and midbody. The sub-cellular localization of Girdin was dependent on the domains, which interacted with actin or microtubules. Overexpression of Girdin lead to increased centrosome splitting and amplification. In addition, data show that pAKT also locates in both the centrosome and midbody, indicating the regulating role of AKT in Girdin-mediated cell division. To elucidate the effect of Girdin on tumor growth in vivo, HeLa cells infected with retrovirus harboring either control or Girdin shRNAs were injected subcutaneously into the immunocompromised nude mice. Downregulation of Girdin by shRNA markedly inhibited the cell growth of subcutaneously transplanted tumors in nude mice. These data demonstrate that Girdin is important for efficient cell division. Taking our previous data into consideration, we speculate that Girdin regulates both cell division and cell migration through cytoskeletal molecules.
A central feature of animal cell division is the orchestration of cytoskeletal molecules such as actins and microtubules, which mediate cell shape transformation and mitotic spindle dynamics in a coordinated manner. When cells prepare to enter mitosis, they normally round up and suppress actin-filled protrusions. Control of the cytoskeleton is then given over to the microtubules, and the mitotic apparatus is generated at the beginning of centrosome-oriented spindles to separate chromosomes and subsequently gives rise to the central spindle (or midbody), forming a furrow at the cell equator. Ultimately, constriction at the midbody leads to abscission at the cell bridge resulting in the formation of two daughter cells.[2, 3] Recently it has become apparent that the spatiotemporal regulation of actin plays a key role during cell division.[4, 5] Thus, defining the mechanisms by which actins and microtubules act selectively or coordinately is crucial to understanding the basic processes of cell division.
Activation of intracellular signaling pathways by growth factors is one of the major causes of cell proliferation. Studies have demonstrated that epidermal growth factor (EGF) and its receptors (EGFRs) were key regulators of cell proliferation.[6, 7] Epidermal growth factor signaling has been shown to be an important mediator of cell proliferation in the adult mouse brain, rat aortic smooth muscle cell, as well as in human esophageal squamous epithelial cells.[8-10] Inhibition of EGFR leads to suppression of growth and motility of breast and pancreatic cancer cells.[11, 12] The PI3K-AKT pathway, which is downstream of EGFR activation, is involved in cell division. AKT activation stimulates proliferation through multiple downstream targets which impact cell-cycle regulation. For example, AKT phosphorylates the p27Kip1 cyclin-dependent kinase inhibitor at T157 and attenuates the cell-cycle inhibitory effects of p27. AKT-dependent phosphorylation of GSK3, TSC2 and subsequent activation of the mTOR complex 1 is also likely to drive cell proliferation through regulation of the stability and synthesis of proteins involved in cell-cycle entry.[15, 16] Interestingly, EGF-AKT activation leads to actin and microtubule remodeling.[17-20] However, it is not known whether AKT regulates cell proliferation through cytoskeleton.
Girdin is a newly identified protein that associates with both actin and microtubule.[21, 22] We and others have found that upon the stimulation with various types of growth factors, AKT phosphorylates Girdin at position Ser-1416 in the C-terminal domain, which plays an important role in remodeling the actin cytoskeleton during cell migration.[21, 23-25] Girdin also associates with Gαi protein and acts as a nonreceptor guanine nucleotide exchange factor (GEF) for Gαi proteins.[26, 27] The GEF motif of Girdin activates and sequesters Gαi, and then enhances AKT signaling through the Gβγ-PI3K pathway. Recently, Ghosh et al. reported that Gαi-Girdin complex bound EGFR and determined cell migration or proliferation through different signaling pathways. Similarly, Anai et al. found suppressed expression of endogenous Girdin by siRNA led to decreased DNA synthesis. And co-expression of AKT and Girdin generated cells that had DNA contents greater than normal G2/M cells. However, the detailed mechanisms are not clear so far.
In this article, we found Girdin and pAKT localized in centrosome and midbody. Overexpression of exogenous Girdin led to increased centrosome amplification and splitting. Mitotic failure was enhanced after Girdin was ove-expressed or suppressed. We also found that Girdin mediated the influence of EGF-AKT on cell division and regulated cell proliferation both in vitro and in vivo. These data demonstrated that Girdin was essential for cell division. Taking our previous data into consideration, we believed that Girdin regulated both cell mitosis and cell migration through cytoskeletal molecules.
Materials and Methods
All experiments involving animals were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee of Peking University Graduate School of Medicine.
Reagents and antibodies
Antibodies against Girdin, β-actin, α/γ-tubulin, siRNA targeting human Girdin and control non-targeting siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Epidermal growth factor, LY294002 and Alexa 488-conjugated phalloidin were obtained from Sigma (St. Louis, MO, USA). Anti-pAKT antibody was obtained from Cell Signaling (Danvers, MA, USA).
Wild-type, constitutively active, and dominant-negative human AKT1 constructs were generously provided by Liu Q (State Key Laboratory of Oncology in South China). The constructs for controls or Girdin shRNA-containing pNAMA Vectors, pGST-Girdin NT/CT and pEGFP-Girdin fragments were produced as described elsewhere.[23, 25]
Growth rates and cell viability
Cell growth rates were evaluated as reported previously. To estimate cell viability, cells were cultured in 24-well plates. Fifty milliliter of MTT solution was added to each well and incubated for 4 h at 37°C. Then, the medium was removed and 400 mL DMSO was added into each well. After shaking, DMSO from each well was transferred to a 96-well plate. Cell viability was determined by AD570 nm, and the results were expressed as the ratio of cell viability relative to the untreated control. Both assays were determined by three independent experiments.
Cells were plated on collagen I-coated glass base dishes, fixed, and stained with the indicated antibodies. Fluorescence was examined using a confocal laser-scanning microscope (Fluoview FV500; Olympus, Tokyo, Japan).
Centrosomal analysis and mitotic index
To stain centrosomes, cells were fixed and stained with anti-γ-tubulin and rhodamine-conjugated antibodies. They were then mounted and viewed with a Zeiss Photoscope III equipped with epifluorescence. In interphase cells, if the distance between duplicated centrosomes was longer than 2 μm, they were scored as centrosome splitting. Mitotic index was scored on the basis of chromosome condensation, disappearance of nuclear membrane, and spindle formation. To assess centrosome status and mitotic index, at least 1000 cells were scored per cover slip and for each condition at least three cover slips were scored. Mitotic index was indicated by the cell number per 1000 cells. Mitotic spindle type was investigated in more than 1000 mitotic cells and results were got from three independent experiments.
Tumor growth assay in nude mice
Tumor cells (3 × 106) were s.c. injected into 7-week-old female nude mice. Then the mice were kept in sterilized, filtered cages in a 12-h light/dark cycle under standardized environmental conditions throughout the experiments for 6 weeks. Growth of tumor was analyzed after dissection at the end of the experiments.
All data were analyzed with SPSS statistics software (Version 13.0; SPSS, Chicago, IL, USA). Results were shown as mean ± SD. Statistical analysis was performed using one-way anova or independent t-tests. A P-value less than 0.05 was considered statistically significant.
Girdin regulates cell proliferation
Girdin is an AKT substrate and it can induce AKT activation indirectly. In view of the fact that AKT is important for cell proliferation and survival, we want to determine whether Girdin affects cell growth. Endogenous Girdin was suppressed by siRNA, which was specific to Girdin, or exogenous Girdin was introduced into HeLa cells. The cells were collected 48 h after transfection and subjected to Western blotting. The efficiency of Girdin suppression or Girdin overexpression was shown in Figure 1(a). As shown in Figure 1(b), Girdin suppression and Girdin overexpression attenuate cell growth by 58.8 ± 11.3% and 29.7 ± 9.2%, respectively (P <0.05). These data suggested that disturbance of Girdin results in inhibition of cell proliferation. This conclusion was also supported by the results of MTT. As shown in Figure 1(c), inhibition or overexpression of Girdin leads to decreased cell viability. The percentage of cell viability decreased to 68.6 ± 5.9% and 84.2 ± 13.2%, respectively. To confirm that Girdin regulates cell proliferation, we also assessed the effects of Girdin disturbance on cell growth in COS7 cells. According to the results, identical conclusion was drawn (data was not shown).
Disturbance of Girdin leads to abnormal cell division
In addition to the depression of cell proliferation, the nuclear volume was obviously enlarged after Girdin was overexpressed or suppressed in HeLa cells, with the average volume increased by 19 ± 9.6% and 30 ± 17.2%, respectively (Fig. 2a). This observation was coordinate with the FACS result, showing an obviously increased 4 N population in Girdin overexpressed or suppressed cells (Fig. S1). Girdin suppression mainly caused mono-polar mitosis, while Girdin overexpression generated increased multi-polar mitosis and the representative mono-polar and multi-polar spindles are shown in Figure 2(b). Normal and aberrant spindles both existed in control and Girdin perturbed cells, the quantitative data regarding each type of spindle is shown in Figure 2(d). Next, we analyzed the effect of Girdin on cell mitosis directly, and found that Girdin suppression or overexpression inhibited cell mitosis significantly, as indicated by mitotic index (Fig. 2c): compared with control cells the mitotic index decreased by 45 ± 4.9% and 29 ± 7.2% in the Girdin suppressed and overexpressed groups, respectively (P <0.05). These data indicated that Girdin was essential for normal cell division.
Girdin locates in the centrosome and midbody
It was reported that Giα proteins could localize in centrosome and midbody, where it played an essential role for mammalian cell division. Acting as a Giα binding partner, Girdin shared some similarity with Giα in localization and function. In Hela cells, there was a strong Girdin accumulation in centrosome in any phase of cell cycle (Figs 2a, 3a), which was in accordance with the reports of Ghosh et al. Since centrosome was composed of centrioles and PCM (peri-centriole material), a centriole specific marker, centrin 2 was further used to confirm the precise location of Girdin in centrosome, and the result showed Girdin co-localized with centriole alone with cell cycle (Fig. S3a). Additionally, GFP-Girdin was introduced into Hela cells to avoid the possibility that Girdin's centrosomal staining could be non-specific (Fig. S3c). To examine which domain of Girdin is responsible for its centrosomal localization, GFP-NT (the N-terminal half), GFP-CT (the C-terminal half), GFP-M1 and GFP-M2 (the M1-terminal half and the M2-terminal half of the coiled-coil domain) were constructed. (Fig. S2b), and it was confirmed that the M1 domain was necessary for centrosomal localization of Girdin (Fig. S2c).
Interestingly, Girdin also localized in midbody, another cell division related apparatus. As shown in Figure 3(b,c), Girdin co-localized with the β-actin ring and α-tubulin at the place of midbody in early cytokinesis, and in late cytolinesis, a clear and precise midbody localization could been seen. Also, exogenous Girdin was introduced into cells to avoid the non-specific staining possibility of midbody (Fig. S3c). Further analysis showed that the NT and CT domains of Girdin, which could bind to microtubule (directly or through Dynamin) and actin filament respectively, were responsible for its midbody localization (Fig. S2c).
Then the centrosomal function of Girdin was investigated, and we used γ-tubulin and RFP-tagged centrin 2 to indicate centrosome statuses. In interphase cells, the appearance of more than two centrosomes was defined as centrosome amplification, and centrosome splitting indicated that two duplicated centrosomes were far away from each other, and the distance between them was longer than 2 μm (Fig. 3d and Fig. S3b). It was believed that splitted centrosomes could trigger additional centriole biosynthesis and lead to centrosome amplification, so, the word “generalized amplification” in our research was used to indicate overall centrosome splitting plus centrosome amplification. Compared with the control group, overexpression of Girdin led to increased generation of centrosome splitting and amplification dramatically (P <0.05) in HeLa and COS7 (data not shown) cells (Fig. 3e).
Girdin regulates cell division and centrosome through cytoskeleton
Girdin is a cytoskeleton-regulated protein. Since the cytoskeleton is essential for cell division, destruction of the association between Girdin and the cytoskeleton will lead to abnormal cell division. To examine our speculation, we introduced Girdin NT or Girdin CT domain into HeLa cells. Theoretically, the NT and CT domains of Girdin were speculated to seal the association between Girdin and microtubule or actin, respectively. The expression of Girdin NT and CT domains is shown in Figure 4(a). Expression of Girdin NT and CT domain led to decreased mitotic index and a corresponding increase of abnormal mitoses in HeLa cells (Fig. 4b,c). The representative bipolar, monopolar or multipolar mitotic cells are indicated in Figure 4(d) and the quantitative data regarding each type of spindle were also invest aged (Fig. 4c). Accordingly, the number of cells with mono-centriole increased in the Girdin NT and Girdin CT transfected cells (data not shown). These data strongly suggested that Girdin regulated cytoskeletal interaction was essential for cell division.
Girdin mediated the regulatory effects of EGF-AKT on cell division
As described above, Girdin regulated cell division by interacting with cytoskeleton. Since Girdin was a downstream molecule in the EGF-AKT signaling pathway, this raised the question as to whether Girdin mediated the effects of EGF-AKT on abnormal cell division. First, the staining of phosphorylated AKT (pAKT) was identified in HeLa cells. As shown in Figure 5(a,b), pAKT was found to localize in the centrosome and midbody, respectively. The results suggested that Girdin and pAKT were closely associated with the apparatus involved in mitotic cells.
Researchers have found that EGF stimulation or AKT activation can lead to centrosome splitting, centrosome amplification and abnormal mitosis. Similar to EGF and AKT, overexpression of Girdin induced centrosome splitting. However, the effects of Girdin on centrosome splitting could not be reversed by AKT inactivation (figure not shown). The addition of EGF enhanced cell mitosis by 50%, but this increased cell mitosis was attenuated in the Girdin-depleted cells. Compared with the Girdin inhibited cells, the mitotic index increased only about 20% in Girdin-inhibited + EGF cells (Fig. 5c). All of these results indicated that Girdin localized downstream of EGF-AKT signaling and mediated at least partial of the effects of EGF-AKT signaling on cell mitosis.
Girdin depletion delayed tumor growth in vivo
Finally, we designed the animal experiments for the exploration of the function of Girdin in vivo. HeLa cells were infected with a retrovirus harboring either control or Girdin shRNA, and several independent stable clones were isolated. Girdin was effectively depleted in Girdin shRNA-transfected cells (Fig. 6a). Compared with control cells, cell proliferation decreased to 41.1 ± 5.5% after Girdin was suppressed (Fig. 6b, P < 0.05). One clone was chosen for further in vivo tumor growth assays. Subcutaneous injection of HeLa cells into immunocompromised nude mice resulted in rapid growth of tumor cells. The growth ability of the cells injected in nude mice was statistically assessed by tumor weight. Large tumors were found in the mice injected with control cells, whereas small tumors were seen in the mice injected with Girdin-depleted cells (Fig. 6c). Compared with that of control tumors, the weight of Girdin depleted tumors decreased by 58.7 ± 6.5% (Fig. 6d, P < 0.05).
In this study, we performed a detailed analysis on the sub-cellular localization of Girdin during cell division. We also confirmed that Girdin played an important role in cell growth control and its probable mechanism. Enhanced or suppressed expression of Girdin in HeLa cells induced profound proliferation suppression. According to the previous reports, Girdin associated with actin filaments and microtubules by its C- and N-termini respectively.[22-24] Here we found that depletion and overexpression of Girdin, or introduction of Girdin N- and C-termini into HeLa cells led to abnormal cell mitosis, as indicated by large-nucleate and monopolar or multipolar mitoses. The mechanism, we speculate, involves Girdin-mediated actin-microtubules interaction, as well as centrosome and midbody regulation.
Centrosome and midbody are key components involved in the control of cell division.[4, 33] Studies have suggested that centrosome was also important for the completion of cytokinesis and cell cycle progression.[34-36] Since the behavior of centrosome is affected by cortical actin, and Girdin is important for the formation of cell cortex, it is possible that Girdin regulates centrosome through the cortical actin. Midbody is important for Cell division. Abscission of midbody leads to daughter cell separation.[33, 37] Although the function of midbody in cell division has not yet been clarified, it was suggested that microtubules and microtubule-associated proteins are required for the completion of cytokinesis.[4, 36, 38] Since Girdin recruits and associates with actin under the membrane, it is possible that Girdin plays a role in the formation of midbody. The recruitment of Girdin in midbody suggests that it is responsible, at least in part, for the abscission.
In addition, previously Simpson et al. found that Girdin was associated with Dynamin II. We also confirmed that Girdin interacts with dynamin II (data not shown). It was speculated that during cell division Dynamin is recruited to the midbody, where it plays an essential role in the membrane fusion and the final separation of dividing cells. So it is possible that Girdin acts as a linkage between Dynamin II and the membrane actin at the place of midbody, where it is important for membrane fusion and the concomitant cell separation.
Epidermal growth factor-PI3K-AKT signaling plays an important role in centrosome splitting and cell mitosis. Epidermal growth factor was found to induce centrosome splitting in HeLa cells 30 years ago. Recently, Wakefield et al. reported that the pAKT accumulates around centrosome throughout prophase and metaphase. Another group found that embryos containing reduced levels of active AKT show defects in centrosome splitting. Epidermal growth factor-PI3K-AKT signaling induced aberrant centrosomal behavior often parallels mitosis failure.[43-46] As our data in this study shows, overexpression of Girdin induces centrosome splitting while suppression of Girdin leads to abnormal mitosis. We suggest there are two possible mechanisms. First, Girdin regulates these cell processes through AKT since Girdin can induce AKT activation through G protein. Second, in view of the fact that Girdin associates with both actin filaments and microtubules, and noting that actin regulates centrosome directly, we suggest Girdin regulates centrosome splitting and mitosis through cytoskeleton. In our experiments, suppression of PI3K-AKT by LY294002 and dominant negative AKT did not affect Girdin overexpression induced centrosome splitting. This argues that the second of these two hypotheses is most likely.
In conclusion, in our experiments we found Girdin regulates cell division and plays an important role in cell growth control. The association of Girdin with the division apparatus might be another attractive means to control cell division and the concomitant cell growth. Our study shed light on the direct mechanism that Girdin controls cell division induced by EGF-AKT signaling activation.
This work was supported by the National Natural Science Foundation of China (30770830, 81072172); China Postdoctoral Science Foundation funded project (20100470170); and China Postdoctoral Science Foundation special funded project (201104043).