Figure 7.

Redistribution of myosin II during nutrient starvation. (A) The fat body of spaghetti-squash (sqh^AX3); sqh–GFP larva under fed or starved conditions were dissected and subjected to immunofluorescence analysis. Sqh–GFP and phospho-myosin regulatory light chain (MRLC) were enriched in cell–cell junction under nutrient-rich (fed) condition. Under starvation conditions, Sqh–GFP and phospho-MRLC were localized to both cell–cell junction and perinuclear region. Actin was stained with TRITC-labelled phalloidin (red) and nucleus was stained with DAPI (blue). Bar, 20 μm. (B) Starvation induced redistribution of myosin II in MCF7/GFP–LC3 cells. Both phospho-MRLC and MRLC were localized in peripheral region of cells in nutrient-rich medium (DMEM), whereas they redistributed to the perinuclear region and co-localized with GFP–LC3 under starvation conditions (Earle's balanced salt solution; EBSS). Bar, 10 μm. (C) MCF7/GFP–LC3 cells cultured in nutrient-rich (DMEM) or starved (EBSS) conditions were homogenized and subjected to centrifugation, and the resulting post-nuclear supernatant (PNS) was fractionated by high-speed centrifugation into membrane pellet and cytosol. Proteins were resolved by SDS–PAGE and immunoblotted with anti-TGN46 antibody as a control for membrane-association proteins, anti-caspase-3 as a control for cytosolic proteins. The levels of phospho-MRLC and MRLC in each fraction were quantified using ImageJ and plotted relative to their amounts in PNS (n=3). Each value represents mean±s.e. of three experiments. ^*P<0.05, ^**P<0.01.