VITO-1 Localizes prominently in the nucleus of undifferentiated C2C12 cells

C2C12 cell line is a subclone of the mouse myoblast cell line established by D. Yaffe and O. Saxel. The C2C12 cell line differentiates rapidly, forming contractile myotubes and producing characteristic muscle proteins. Care should be taken not to allow the cells to become confluent as this will deplete the myoblastic population in the culture.

Diffentiation and myotube formation of C2C12 cells is stimulated when the medium is supplemented with 2% horse serum instead of FCS.

Northern blot analysis using RNA from C2C12 cells had revealed the presence of m-VITO-1 RNA in proliferating C2C12 cell indicating expression of the gene in dividing myoblasts that have not yet undergone terminal differentiation (Mielcarek, Gunther et al.

2002). The expression level in C2C12 myoblasts was lower compared to differentiated C2C12 myotubes.

To investigate the localization of VITO-1 in C2C12, cells were cultured on 6 well fibronectin coated plates and transfected with 0.8 µg of VITO-1 pEGFPc2 using FuGene 6 reagent. After transfection, cells were incubated with serum free medium without antibiotics for 8 hrs after which the medium was replaced with DMEM containing 10%

FCS and penicillin/streptomycin). Cells were fixed with methanol-acetone for 10 min and the nucleus was stained with Dapi.

Figure 7. Expression of VITO-1 (green) in undifferentiated C2C12 muscle cell line.

VITO1-GFP was transfected into C2C12 cells using Fugene 6 and the distribution pattern was monitored at different time points. (A) After 12 hrs, VITO1 show a puntacted distribution all over the cytoplasm in most of the cells. (C) After 24 hrs, VITO1 is distributed both in the cytoplasm as well as the nucleus. (E) After 48 hrs, VITO1 is associated prominently in the nucleus which supports its role as a transcriptional co-activator.

After 12 hrs of transfection, VITO-1-GFP was found predominantly in the cytoplasm in a punctuated pattern in most of the cells although some cells still show a weak nuclear localization (Fig. 7A). In 24 hrs, one can observe the distribution of VITO-1 both in the nucleus as well as in the cytoplasm (Fig. 7C). After 48 hrs, VITO-1 completely translocates into the nucleus (Fig. 7E). VITO-1 shows an endogenous expression in undifferentiated C2C12 myoblasts and the localization of VITO-1 in the nucleus could support its possible role as a co-activator where it might interact with TEFs or other transcriptional factors that are involved in the terminal differentiation of muscle cells and control gene regulation.

3.1.4 Expression of VITO-1 in differentiated C2C12 myotubes

Proliferating C2C12 cells can be triggered to differentiate into myotubes by culturing them in low growth factor medium when they are confluent. To monitor the expression of VITO-1 in differentiated myotubes, a GFP tagged fusion plasmid (VITO-1-pEGFP-c2) was used. C2C12 (mouse myoblast cell line) cells were grown on 6 well plates in DMEM containing 10% FCS and Penicillin/Streptamycin (P/S) antibiotics. When the cells reached 60% confluence, they were transfected with 2 µgs of the plasmid VITO-1-GFP using FuGENE 6 reagent and incubated for 48 hrs. When the cells reached ~ 100%

confluence, the medium was replaced with DMEM containing 2% Horse serum. Cells were allowed to differentiate for 4 – 5 days until the formation of multinucleated mature myotubes. Later the cells were fixed with 4% PFA and the nuclei were stained with DAPI. The cover slips containing the fixed cells were visualized using a fluorescence

Figure 8. Over expression of VITO-1 in matured myotubes of C2C12 cells. The top four panels show the expression of VITO-1-GFP in C2C12 cells after differentiation into myotubes. Nuclei were stained using Dapi (bottom panels).

VITO-1-GFP was predominantly expressed in differentiated myotubes (Fig. 8 top panels) indicating that cells expressing VITO-1 showed an increased property to form myotubes compared to their neighboring untransfected cells which did not form the mature myotubes. These findings show that VITO-1 might play an important role in association with transcription factors to facilitate terminal differentiation of muscle cells.

3.1.5 Expression of mutant VITO-1 constructs in C2C12 cells shows the requirement of SID domain to translocate to the nucleus.

VITO-1 mediates interaction with TEFs and other proteins through its scalloped interaction domain (SID) [(Gunther, Mielcarek et al. 2004)]. We hypothesized that,

evidence of the importance of SID domain, a mutant construct lacking the SID domain was fused to IRES-GFP (VITO-1 Δ SID-GFP). This construct was transfected into C2C12 cells plated in 6 well chambers with 1 µ g of plasmid DNA using the FuGene 6 reagent and were fixed using 4% PFA. After 48 hrs of transfection, full length VITO-1 was mainly expressed in the nucleus (Fig. 9A). VITO-1 lacking the SID domain did not translocate into the nucleus even after 48 hrs and displayed a prominent cytoplasmic localization (Fig. 9B). The corrected total cell fluorescence (CTCF) was quantitated using Image J software.

(A) Full length VITO-1-GFP localized mainly in the nucleus after 48 hrs of transfection.

(B) VITO-1 mutant lacking the SID domain (VITO-1∆ SID-GFP) is distributed throughout the cells with prominent punctated pattern. (Histograms represent total cell fluorescence quantitated using Image J software)

The results demonstrate the SID domain of VITO-1 is instrumental for an efficient translocation into nuclei where it may act as a transcriptional co-activator to regulate expression of target genes.

3.2 Over-expression of TEF3 together with VITO-1, results in a complete recruitment of VITO-1 into the nucleus in various cell types

The expression of VITO-1 was analyzed in various cell types like C2C12, HEK293 and 10T1/2 in the presence of TEF3. VITO-1 – GFP was co-transfected with TEF3-pCDNA5 in an equimolar ratio using the FuGene 6 reagent. After 48 hrs of transfection, cells were fixed with 4% PFA and nucleus was stained by DAPI. VITO-1 when transfected alone in these cell lines did not completely localize in the nucleus. After 24 hrs of co-transfection, VITO-1 still remained in the cytoplasm in most of the cells and did not show much difference with cells transfected with 1 alone. But interestingly after 48 hrs, VITO-1 was completed translocated into the nucleus in all cell types including HEK 293 cells (Fig. 10). It should be noted that VITO-1 transfected alone never showed a nuclear expression (Fig. 5). This is probably due to the association of VITO1 with the transcription factor TEF3 during translocation into the nucleus. The endogenous expression of TEF transcription factor seems not sufficient to drag over-expressed VITO-1 into the nucleus.

Figure 10. Full length VITO-1 translocates completely into the nucleus in the presence of TEF3 in different cell types. 10T1/2, C2C12 and HEK 293 cells were trasnfected with VITO-1-GFP and TEF3. In the presence of TEF3, VITO-1 completely translocated to the nucleus in all three cell types. Nuclei were stained with Dapi.

The next important question was whether VITO-1 lacking the SID domain translocates into the nucleus in the presence of TEF3. To investigate this, VITO-1 Δ SID-GFP was co-transfected in C2C12 cells with TEF3 in an equimolar concentration with a total DNA

Figure 11. Subcellular localization of VITO-1ΔSID-GFP in the presence of TEF3.

VITO-1ΔSID-GFP is distributed all through the cell and not restricted to nuclear compartment alone.

Cells were fixed and stained as stated previously and visualized in the microscope. It should be noted that after 48 hrs of transfection, cells expressing VITO-1 Δ SID-GFP did not completely translocate into the nucleus even in the presence of TEF3 (Fig. 11). This shows the requirement of the SID domain to activate TEF3 and its downstream target genes. These findings underline the importance of the SID domain in mediating interaction of VITO-1 with other proteins such as TEF3.

3.3 Screening for novel interacting partners of VITO-1