AKAP95 bind to Cx43 and transfer it into nucleus during G1 phase in A549 cells.
Treated with Lovastatin, Mimosine and Thymidine respectively, cells were arrested at P, M and R stages [27–30]. Flow cytometry (FCM) was used to detect the cell cycle (Fig. 1A). After treated with Lovastatin and Mimosine respectively, proportion of cells in G1 phase increased while proportion of cells in G2 phase decreased. No significant changes were found of percentage of cells in S phase compared with the control group. Percentage of cells in G1 phase of Lovastatin group was higher than Mimosine group. In Thymidine group, whose cells were mainly arrested between late G1 and early S phase, proportion of cells in S phase increased while proportion of cells in G1 phase decreased. No significant change was found of percentage of cells in G2 phase compared with the control group (Fig. 1Aa). Figure 1.Ab shows the bar chart of the statistical results of Fig. 1Aa. These data show that cells were arrested in P, M, and R stage efficiently.
We further detected expression of AKAP95 and Cx43 in cancer and normal lung cells at different stages of G1 phase (Fig. 1B). In A549 cells, both AKAP95 and Cx43 had low level expression during P and M stages but highly expressed in R stage (Fig. 1Ba). Expressions of both AKAP95 and Cx43 in these three stages were significant respectively (p < 0.05), and both of their expression increased gradually form P to R stage and peaked at R stage. In Beas-2B cells, AKAP95 expressed much lower than that in A549 cells or Cx43 in Bease-2B cells (Fig. 1Bb). Both AKAP95 and Cx43 in Beas-2B cells highly expressed form M stage, which was earlier than that in A549 cells, and kept highly expression.
Since the expression of AKAP95 and Cx43 in G1 phase showed a consistent increasing trend, we detected the localization of these two proteins in different stages by CLSM. The blue fluorescence showed DNA of nucleus and green and red fluorescence showed AKAP95 and Cx43 respectively. In our images, AKAP95 mainly expressed in nucleus during G1 phase (Fig. 2Ab,h,n). In addition, some strong fluorescent patches could be found in nucleus, suggesting that some AKAP95 aggregated and form protein aggregates. Cx43 expressed both in nucleus and cytoplasm during G1 phase (Fig. 2Ac,i,o). Due to superposition of blue and red fluorescence, purple color could be observed in Merge images of DAPI and Cx43 (Fig. 2Ae, k, q) and yellow color could be observed in Merge images of AKAP95 and Cx43 (Fig. 2Af, l, r), suggesting that some Cx43 might bind to DNA and AKAP95 in nucleus.
By TEM assay, we further confirmed that AKAP95 could bind to Cx43 both in cytoplasm and nucleus and could transport it into nucleus through nuclear pore. In Fig. 2Ba, green triangle pointed to AKAP95 (15 nm label) and red triangle pointed to Cx43 (10 nm label). Taking the scale on the picture as the standard, the distance of the two labels was less than 15 nm, suggesting that AKAP95 can bind to Cx43 in cytoplasm (It is generally considered that two proteins bind to each other when the distance of gold labels of them is less than 15 nm in TEM images [31,32]). Figure 2Bb showed that AKAP95-Cx43 complex just entered nucleus through nuclear pore. Blue arrow refers to the nuclear membrane. The nuclear pore is between the two arrows on the right of the image (the interval marked by the red line).
Dynamic process of nuclear transport of Cx43.
We had further detected the process of nuclear transport of Cx43 in cells arrested at different stages of G1 phase by TEM assay.
In TEM assay, different concentrations of gold labeled IgG may affect the results. When the dilution ratio of Gold labeled IgG was 1:100, a few gold particles showed nonspecific adhesion causing false positive while when the dilution ratio was 1:100, though false positive could be avoided, some target proteins could not be labeled and displayed caused by low concentration of gold labeled IgG. In order to verify whether there was false positive interference under our experimental conditions, Rabbit anti- and Mouse anti-GAPDH were used to incubated sections before incubation with Gold labeled IgG (15 nm and 10 nm) in Positive Control while PB buffer was used instead of specific GAPDH antibodies in Negative Control group. Results showed that single gold labels could be found in Positive control (Fig. 3Aa) and distance of different labels was significantly more than 15 nm (Fig. 3Ab). Scales on each TEM images were chosen to be standard and distance of 10 pairs of 15 nm and 10 nm gold labels in each group were detected. Average distance of different gold labels of Control (Positive Control) group was more than 100 nm and no nonspecific aggregation had been detected while it was less than 10 nm in arrested cells groups, suggesting that our positive results were reliable. Only extremely few gold labels were found in Negative Control groups, and no nonspecific aggregation were found (Fig. 3B), suggesting that the false positive interference could be ignored under our experimental condition.
In arrested A549 cells, we found that binding of AKAP95 and Cx43 could be detected during whole G1 phase and binding of the them could be detected in nucleus at three stages of G1 phase (Fig. 4A). Interestingly, we found that AKAP95 could bind to Cx43 in two forms. In addition to bind to each other and form AKAP95-Cx43 complexes, we had detected aggregations of gold labels with different diameters, suggesting that some AKAP95 and Cx43 proteins could aggregate and form bigger protein aggregates. AKAP95-Cx43 complexes/aggregates existed in both nucleus and cytoplasm, suggesting that both the complexed and aggregates of AKAP95 and Cx43 had the ability of entering nucleus. In nucleus, no single AKAP95 nor Cx43 was found, suggesting that Cx43 kept binding to AKAP95 after transported into nucleus. In Beas-2B cells, we had found AKAP95-Cx43 complexes/aggregates both in nucleus and cytoplasm as well (Fig. 4B). However, owing to expression level of AKAP95 was lower than that of Cx43 in Beas-2B cells, we had found some single 10 nm gold labels (Cx43) which could not enter the nucleus, suggesting that Cx43 could not enter the nucleus without transport of AKAP95.
To clarify the process of nuclear transport of Cx43, we further photographed more TEM images of various locations of AKAP94-Cx43 complexes/aggregates in A549 cells at different stages of G1 phase and organized a series of typical images that could represent every stages of the process (Fig. 5). After expression, AKAP95 could bind to Cx43 in cytoplasm and form AKAP95-Cx43 complex, and some AKAP95 and Cx43 could form aggregates as well during the stage (Fig. 5a). In formed AKAP95-Cx43 complex/aggregates, AKAP95 then targeted nuclear membrane, driving the complex/aggregates to move towards the nuclear membrane (Fig. 5b). After located on the nuclear membrane (Fig. 5c), AKAP95 further targeted nuclear matrix and transported Cx43 into nucleus (Fig. 5d). In nucleus, AKAP95 and Cx43 kept binding and further functioned in the form of AKAP95-Cx43 complex/aggregates (Fig. 5e, f).
Cyclin E1 was involved in nuclear transport of Cx43 during G1 phase.
AKAP95 and Cx43 could bind to cyclin D1 and competitively bind to cyclin E1/E2 respectively, regulating the G1/S conversion [7,33–35]. Our previous study had shown that cyclin D1 and cyclin E1 were correlated with AKAP95 respectively while were not correlated with Cx43 [34]. To further discuss if cyclin D1 and cyclin E1, which were important regulators of G1/S transition, were involved in nuclear transport process of Cx43, we analyzed these proteins in arrested A549 cells.
Our results showed that cyclin D1 highly expressed at P and S stages while lowly expressed at R stage (Fig. 6Aa, the third row). Expressions of cyclin E1 were low at P and M stages while peaked at R stage (Fig. 5Aa, the fourth row). Statistical results were shown in Fig. 6Ab. According to the data, expression of cyclin E1 during G1 phase changed similarly to that of AKAP95 and Cx43 while cyclin D1 showed the opposite trend (Fig. 5Ab). Specific AKAP95 and Cx43 antibodies were used in Co-IP assay to detect protein bindings in arrested cells. Both cyclin D1 and cyclin E1 could be detected in coprecipitation in Co-IP: AKAP95 group (Fig. 5B), suggesting that AKAP95 could bind to cyclin D1 and cyclin E1 during whole G1 phase. Similar results were found in Co-IP: Cx43 group (Fig. 5C), suggesting that Cx43 could bind to cyclin D1 and cyclin E1 during G1 phase as well. These results suggested that AKAP95, Cx43, cyclin D1, and cyclin E1 could bind to each other and formed at least four complexes, including AKAP95-cyclin D1, AKAP95-cyclin E1, Cx43-cyclin D1, and Cx43-cyclin E1 during G1 phase. However, owing to the difference of expression level of the four proteins, we considered that AKAP95-cyclin D1 and Cx43-cyclin D1 mainly existed at P and M stage while AKAP95-cyclin E1 and Cx43-cyclin E1 were formed mainly at R stage of G1 phase.
In CLSM images, no significant nuclear expression of cyclin D1 was found during whole G1 phase (Fig. 7A). Cyclin E1 mainly expressed at R stage and obvious nuclear expression could be detected at this stage. In addition, strong fluorescent plaques of cyclin E1, similar with AKAP95, could be found in cells at R stage. During the other two stages, cyclin E1 mainly located in cytoplasm (Fig. 7B). These images suggested that even both cyclin D1 and cyclin E1 could bind to AKAP95 and Cx43 respectively, cyclin E1 might take participate in nuclear transport of Cx43 while cyclin D1 not.
We further carried out TEM assay to seek direct evidences at subcellular level. Results showed that gold labels that marked cyclin D1 only existed in cytoplasm. Some cyclin D1 could located on nuclear membrane, but could not enter nucleus (Fig. 7C), suggesting that cyclin D1 did not entered nucleus with AKAP95 and might dissociate from AKAP95 before it entered the nucleus at the latest. On the contrary, cyclin E1 could be detected both in nucleus and cytoplasm in P, M, and R stage cells. Significant aggregations of gold labels could also be found, especially in R stage cells (Fig. 7D), suggesting that cyclin E1 entered nucleus with AKAP95 mainly at R stage and might be involved in aggregations of AKAP95 and Cx43.
When AKAP95 and cyclin D1 were simultaneously labeled in cells, AKAP95-cyclin D1 complexes located in cytoplasm during whole G1 phase. Single 10 nm gold labels (AKAP95) could be found in nucleus while 15 nm labels (cyclin D1) could not. In addition, no significant protein aggregation was found (Fig. 8Aa,b,c). Images of Cx43/cyclin D1 group were similar and no labels of cyclin D1 were found in nucleus (Fig. 8Ad,e,f). In R stage cells, we didn’t find Cx43-cyclin D1 complex this time. We considered that highly expressed Cx43 might promotes degradation cyclin D1, which lowly expressed at R stage, after their binding, thus we didn’t catch some images. It was completely different when cyclin E1 was labeled together with AKAP95 and Cx43 respectively. Both AKAP-cyclin E1 complexes and Cx43-cyclin E1 complexes could entered nucleus and protein aggregation could be found during G1 phase (Fig. 8B). These results had supported our conjecture that cyclin E1 was involved in aggregations of AKAP95 and Cx43 while cyclin D1 was not.