To further analyse whether the three cellular proteins, periplakin, vitronectin and Kif14, have any role in the functioning of MCPyV ST, immunofluorescence studies were performed. This approach aimed to determine whether the subcellular localisation of the cellular proteins were altered upon MCPyV ST expression and/or the cellular protein co-localised with the ST protein. MCC13 cells were transfected with a control plasmid, pGFP, or a plasmid expressing an GFP-tagged ST construct (previously produced by Dr David Griffiths, Whitehouse laboratory). After 24 hours post transfection, the cells were fixed, permeabilised and incubated with primary antibodies specific for each target cellular protein and secondary antibodies conjugated with Alexa fluor 546. MCPyV ST expression was analysed by direct GFP fluorescence, while the nuclear DNA was stained with DAPI.
3.4.1. MCPyV ST and Periplakin.
Periplakin (PPL) is a member of the plakin family which have various functions in connecting cytoskeleton elements to form intercellular junction complexes (Sonnenberg and Liem, 2007). Most of the plakin family function as "molecular bridges" linking the intracellular cytoskeleton and cell-cell junctions. Gene mapping of the domains utilising a variety of periplakin deletion constructs (DiColandrea et al., 2000) showed that the periplakin N-terminus localizes at the plasma membrane in a punctate distribution, whereas the C-terminus associates with keratin filaments. Of particular interest in regard to MCPyV ST-induced tumourigenesis is that PPL knockdown resulted in reduced cellular movement and attachment activity of cells (Tonoike et al., 2011).
Several studies have investigated the subcellular localization of Periplakin. It has been shown to localize to desmosomes, the interdesmosomal plasma membrane and intermediate filaments between cells (DiColandrea et al., 2000). Moreover, in transient expression studies of primary human
keratinocytes, periplakin accumulated on the apical plasma membrane and at cell-cell contacts. Therefore, to examine the subcellular localisation of periplakin in control or GFP-ST expressing cells, the MCPyV negative MCC cell line, MCC13 was transfected with GFP or GFP-ST expression constructs and after 24 hours, cells were fixed, permeabilised and periplakin staining was analysed using a periplakin-specific antibody. GFP and GFP-ST localisation was visualised by direct fluorescence (Figure 3.3). GFP staining is observed diffuse throughout the cytoplasm and nucleus, where GFP-ST localises to both the nucleus and cytoplasm, with enhanced staining in the perinuclear region of the cell (Knight et al., 2015). Indirect immunofluorescence studies of periplakin in GFP-expressing control cells shows similar staining and localisation to previously described studies, where periplakin is present throughout regions of the cytoplasm. Enhanced staining was observed in the perinuclear region with some punctate staining discernibly visible which is reminiscent of vesticular staining. However, the exact localisation needs to be confirmed with colocalisation of characterised cellular marker proteins. A similar staining was observed for periplakin in GFP-ST expressing cells. A proportion of MCPyV ST appeared to colocalise with periplakin in the cytoplasm and also around the perinuclear region, where ST is evident. This may suggest a possible interaction and relocalisation of a small proportion of periplakin to this region upon ST expression, as an enhanced concentration of periplakin is observed in this region in GFP-ST expressing cells.
Figure 3.3: Immunofluorescence analysis of MCC13 cells transfected with GFP and GFP-ST expressing vectors.
MCC13 cells were transfected with GFP or GFP-ST expressing vectors. After 24 hours, the cells were fixed, permeabilised ,then incubated with anti-periplakin (1:100) overnight at 4°C. The secondary antibody Alexa fluor 488 goat anti rabbit used at 1:500 dilution for 1h at room temperature. Mounting medium VECTASHIELD contains 4’,6-diamidino-2- phenylindole (DAPI) and used to counterstain DNA (blue).
3.4.2. MCPyV ST and the motor protein kinesin family
(Kif14)
The kinesin superfamily of proteins (KIFs) generally function to transport membranous organelles and protein complexes in a microtubule- and ATP- dependent manner. KIFs have been associated with cancer progression, by disturbing the cell cycle. For example, downregulation of Kif14 expression has been shown to delay the transition of metaphase to anaphase in lung adenocarcinomas causing a binucleated status, which enhances tumour progression (Hung et al., 2013). Conversely, Kif14 overexpression inhibits anchorage-independent growth in vitro and also inhibits cancer cell migration, invasion and adhesion to the extracellular matrix. An adhesion molecule, cadherin 11 (CDH11), was recruited to the cellular membrane which has been implicated in the adhesive, migratory and invasive properties of the cell (Hung et al., 2013). In addition, Kif14 interacts with supervillin, a membrane protein involved in directing cellular motility (Smith et al., 2010). Considering the highly metastatic of the MCC,
GF P -ST G F P Dapi gfp PPL merge Dapi gfp st PPL merge Dapi gfp st PPL merge Dapi GFP PPL Merge
the further elucidation of a possible interaction of MCPyV ST and Kif14 was also examining initially by immunofluorescence studies. KIF14 is localized to the cytoplasm during interphase, and becomes tightly localized to the midbody and central spindle during cytokinesis (Carleton et al., 2006b; Gruneberg et al., 2006a). As above, the subcellular localisation of Kif14 was examined in GFP and GFP-ST expressing MCC13 cells, using a Kif14- specific antibody (Figure 3.4). Results show that in GFP-expressing control cells, Kif14 localised to the cytoplasm, mainly diffusely but some cells contained punctate structures. Expression of MCPyV ST was observed to colocalise with a diffuse proportion of Kif14 in the cytoplasm. More noticeable nuclear puncta were also observed in GFP-ST infected cells. However, these were also observed in a non-transfected cells in this sample. Although comparison between the GFP and GFP-ST showed possible alterations of Kif14 distribution, these were not conclusive. Further optimisation of the antibody for immunofluorescence studies was performed, but failed to show any further improvement.
Figure 3.4: Immunofluorescence analysis of MCC13 cancer cells transfected with GFP and GFP tagged ST antigen.
MCC13 cells were transfected with GFP or GFP-ST expressing vectors. After 24 hours, the cells were fixed, permeabilised and then incubated with anti-Kif14 (1:100) overnight at 4°C. The secondary antibody Alexa fluor 488 goat anti rabbit used at 1:500 dilution for 1h at room temperature. Mounting medium VECTASHIELD contains 4’,6-diamidino-2- phenylindole (DAPI) and used to counterstain DNA (blue).
Dapi gfp st Kif14 merge Dapi gfp Kif14 merge
Dapi gfp st Kif14 merge Dapi gfp Kif14 merge
Dapi GFP Kif14 Merge
GF P S T GF P
3.4.3. MCPyV ST and Vitronectin.
Vitronectin is an abundant and multi-functional glycoprotein found in serum and is also associated with the extracellular matrix. It has a defined activity in cell adhesion, promoting cell attachment, spreading, proliferation and differentiation of many normal and neoplastic cell types. As such it is a key protein in regulating cell homeostasis (Preissner and Seiffert, 1998) and tumour malignancy (Felding-Habermann and Cheresh, 1993). Vitronectin also possesses binding sites for membrane-bound integrins which function to anchor cells to the cellular matrix. These interactions are thought to be associated with vitronectin’s role in cell migration and signal transduction.
Immunofluorescence studies were therefore performed as above in transfected GFP and GFP-ST to determine if MCPyV ST colocalised or altered the subcellular localisation of vitronectin (Figure 3.5). Analysis of GFP-expressing cells appeared to show that vitronectin was present in the cytoplasm and the nucleus of GFP-expressing cells. This was rather surprising due to its known function as a glycoprotein. In contrast, vitronectin was observed in the cytoplasm and perhaps the extracellular matrix in MCPyV ST expressing cells. Due to the discrepancy of vitronectin staining in control cells further immunofluorescence studies are required to investigate any possible redistribution or colocalization with MCPyV ST.
Figure 3.5: Immunofluorescence analysis of MCC13 cells transfected with GFP and GFP tagged ST antigen.
MCC13 cells were transfected with GFP or GFP-ST expressing vectors. After 24 hours, the cells were fixed, permeabilised and then incubated with anti-vitronectin (1:100) overnight at 4°C. The secondary antibody Alexa fluor 488 goat anti rabbit used at 1:500 dilution for 1h at room temperature. Mounting medium VECTASHIELD contains 4’,6- diamidino-2-phenylindole (DAPI) and used to counterstain DNA (blue).