AMPK subcellular localisation during mitosis

3.2.1.1 Subcellular localisation of AMPKα subunits during mitosis

It has been reported that the AMPKα2 subunit localises to the midbody in HUVECs and that phospho-T172 AMPKα similarly localises to the midbody and MTOC in HeLa cells and the A431 cancer cell line (Vazquez-Martin et al., 2009; Pinter et al., 2012;). The localisation of AMPKα subunits during mitosis was therefore investigated in primary human cells (HUVECs) and HeLa cells. Using antibodies raised to amino acids 344-358 of AMPKα1, immunoreactivity was observed at the MTOCs in HeLa cells (Figure 3-1) and HUVECs (Figure 3-2), whereas no staining was observed at the midbody. Conversely, when stained with an antibodies raised to amino acids 351-356 of AMPKα2, immunoreactivity was observed at the midbody with no immunoreactivity at the MTOCs in HeLa cells (Figure 3-1) and HUVECs (Figure 3-2). In both cell lines, AMPKα2 localised to two discreet areas on either side of the midbody (highlighted in Figure 3-2D). AMPKα1 and AMPKα2 immunoreactivity was also observed in the cytoplasm of mitotic cells. Further immunofluorescence experiments were undertaken using antibodies raised to distinct epitopes (amino acids 357-524 of AMPKα1 or amino acids 29-510 of AMPKα2), however no immunoreactivity was observed in HUVECS (data not shown).

To avoid specificity issues of antibodies, HeLa cells were transfected with plasmids encoding genes for either AMPKα1-GFP, AMPKα2-GFP or GFP alone. Cells were fixed with PFA and GFP localisation assessed by fluorescence confocal microscopy. Despite successful induction of AMPKα1/2-GFP expression (Figure 3- 3D), the majority of GFP-positive cells were senescent. Only three GFP positive cells were observed which were connected by an intracellular bridge, and in all three cases GFP was apparent at the midbody (Figure 3-3).

79 Figure 3-1. Subcellular localisation of AMPKα subunits during mitosis in HeLa cells

HeLa cells were fixed with 4% (w/v) PFA and stained with anti-tubulin (Abcam, ab6160), anti- AMPKα1 (epitope raised against amino acids 344-358 of AMPKα1) (A and B) or anti-AMPKα2 (epitope raised against 351-356 of AMPKα2) antibodies (C and D). DNA was stained with RedDot2. Cells undergoing metaphase are shown in A and C, cells undergoing telophase are shown in B and D. Scale bar 10 μm. Representative images are shown from three independent experiments in each case.

80 Figure 3-2. Subcellular localisation of AMPKα subunits during mitosis in HUVECs

HUVECs were fixed with 4% (w/v) PFA and stained with anti-tubulin (Abcam, ab6160), anti- AMPKα1 (epitope raised against amino acids 344-358 of AMPKα1) (A and B) or anti-AMPKα2 (epitope raised against 351-356 of AMPKα2) antibodies (C and D). DNA was stained with RedDot2. Cells undergoing metaphase are shown in A and C, cells undergoing telophase are shown in B and D. Scale bar 10 μm. Representative images are shown from three independent experiments in each case.

81 Figure 3-3. Subcellular localisation of AMPKα-GFP in HeLa cells

HeLa cells were transfected with plasmids containing the genes for GFP, AMPKα1-GFP or AMPKα2-GFP. (A-C) Representative images of transfected cells stained with anti-tubulin (Abcam, ab6160) antibodies and for DNA with RedDot2 from two independent experiments. Scale bar 10 μm. (D) HeLa cells were transfected with the plasmids described. Lysates were prepared 24 h following transfection and resolved by SDS-PAGE followed by immunoblotting using anti-GFP antibodies. N=1.

82 3.2.1.2 Subcellular localisation of phospho-T172 AMPK during mitosis

Using two different anti-phospho-T172 AMPK antibodies, immunoreactivity was observed at the MTOCs when cells were in metaphase and at the midbody during telophase in both HeLa cells (Figure 3-4) and HUVECs (Figure 3-5).

Immunoreactivity of phospho-T172 AMPK was also observed in the cytoplasm of mitotic cells using both antibodies. Anti-phospho-T172 AMPK immunoreactivity was also detected at the spindle midzone of metaphase cells (Figure 3-4 and Figure 3-5).

To determine if the observed anti-phospho-T172 AMPK immunoreactivity was specific, immunoreactivity of the antibodies was examined in AMPK α1/α2 double knockout (AMPK -/-) MEFs. The wild type and AMPK -/- MEFs rapidly progress through the cell cycle, as a result M-phase cells were rarely observed when examined by microscopy (data not shown). To examine mitotic cells, MEFs were synchronised using a thymidine block followed by nocodazole arrest. Anti- phospho-T172 AMPK (sc-101630) immunoreactivity was observed at the midbody in both wild type and surprisingly in AMPK -/- MEFs (Figure 3-6). Comparative levels of cytosolic and nuclear immunoreactivity was observed between the cell types (Figure 3-6).

83 Figure 3-4. Subcellular localisation of phospho-T172 AMPK during mitosis in HeLa cells HeLa cells were grown on glass cover slips, fixed with 4% (w/v) PFA and stained with anti-tubulin (Abcam ab6160), and either anti-phospho-T172 AMPK from Cell Signalling Technologies (CST) #2535 (A and B) or Santa Cruz Inc. sc-101630 (C and D). DNA was stained with RedDot2. Cells undergoing metaphase are shown in A and C. Cells undergoing telophase are shown in B and D. Scale bar 10 μm. Representative images from three independent experiments are shown.

84 Figure 3-5. Subcellular localisation of phospho-T172 AMPK during mitosis in HUVECs HUVECs were grown on glass cover slips, fixed with 4% (w/v) PFA and stained with anti-tubulin (Abcam ab6160) and either anti-phospho-T172 AMPK from Cell Signalling Technologies (CST) #2535 (A and B) or Santa Cruz Inc. sc-101630 (C and D). DNA was stained with RedDot2. Cells undergoing metaphase are shown in A and C. Cells undergoing telophase are shown in B and D. Scale bar 10 μm. Representative images from three independent experiments are shown.

85 Figure 3-6 Subcellular localisation of phospho-T172 AMPK during mitosis in wild type and AMPK -/- MEFs

(A) Wild type and AMPK -/- MEFs were synchronised using a thymidine and nocodazole block. Synchronised MEFs were seeded onto 8-well Ibidi chambers and incubated until cells entered telophase. Cells were fixed using 4% (w/v) PFA and stained with anti-tubulin (Abcam, ab6160) and anti-phospho-T172 AMPK (Santa Cruz Inc. sc-101630) antibodies. Scale bar represents 10 μm. Representative images from two independent experiments are shown. (B) Unsynchronised wild type and AMPK -/- MEFs were incubated with 100 μM A769662 for 30 min before lysates were prepared. Lysates were resolved by SDS-PAGE followed by immunoblotting with the antibodies indicated. Arrow indicates a non-specific band of a higher molecular mass than AMPK.

86 3.2.1.3 Subcellular localisation of phospho-S79 ACC

It has been reported that phospho-S79 ACC, a site phosphorylated exclusively by AMPK, exhibits a subcellular localisation which mirrors that of phospho-T172 AMPK during mitosis in A431 cells. (Ha et al., 1994; Vazquez-Martin et al., 2013). However, during the course of this investigation, phospho-S79 ACC was not found to localise to the MTOC or the midbody in HUVECs (Figure 3-7). Immunoreactivity was observed in the cytoplasm of HUVECs. Additionally, phospho-S79 ACC

immunoreactivity did not increase in response to A769662 (100 μM, 30 min) when examined by immunofluorescence microscopy, although A769662 stimulation caused a clear increase in phospho-S79 ACC signal intensity when assessed by immunoblotting (Figure 3-7).

87 Figure 3-7. Subcellular localisation of Phospho-S79 ACC during mitosis in HUVECs

(A) HUVECs were seeded onto glass coverslips and stained with anti-tubulin (Abcam, ab6160) and anti-phospho-S79 ACC (Cell Signalling technology ®, #11818) antibodies and for DNA with

RedDot2. Scale bar 10 μm. Representative images from three independent experiments are shown. (B-E) HUVECs were incubated with DMSO vehicle control or A769662 (100 μM, 30 min). (B/C) Cells were stained for anti-tubulin, anti-phospho-S79 ACC antibodies and DNA. (D) Cells were lysed and resolved by SDS-PAGE followed by immunoblotting with the antibodies indicated. (E) Mean fluorescence intensity of phospho-S79 ACC staining was measured in 20 cells using image J software. N=1.

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