We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores suggesting that macropinocytosis can supply nutrients other than amino acids. Non-transformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically.

We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution.

Membrane wetting by biomolecular condensates recently emerged as a key phenomenon in cell biology, playing an important role in a diverse range of processes across different organisms. However, an understanding of the molecular mechanisms behind condensate formation and interaction with lipid membranes is still missing. To study this, we exploited the properties of the dyes ACDAN and LAURDAN as nano-environmental sensors in combination with phasor analysis of hyperspectral and lifetime imaging microscopy. Using glycinin as a model condensate-forming protein and giant vesicles as model membranes, we obtained vital information on the process of condensate formation and membrane wetting. Our results reveal that glycinin condensates display differences in water dynamics when changing the salinity of the medium as a consequence of rearrangements in the secondary structure of the protein. Remarkably, analysis of membrane-condensates interaction with protein as well as polymer condensates indicated a correlation between increased wetting affinity and enhanced lipid packing. This is demonstrated by a decrease in the dipolar relaxation of water across all membrane-condensate systems, suggesting a general mechanism to tune membrane packing by condensate wetting.