Transcription factor EB regulates phosphatidylinositol-3-phosphate levels that control lysosome positioning in the bladder cancer model.

Lysosomes orchestrate degradation and recycling of exogenous and endogenous material thus controlling cellular homeostasis. Little is known how this organelle changes during cancer. Here we investigate the intracellular landscape of lysosomes in a cellular model of bladder cancer. Employing standardized cell culture on micropatterns we identify a phenotype of peripheral lysosome positioning prevailing in bladder cancer cell lines but not normal urothelium. We show that lysosome positioning is controlled by phosphatidylinositol-3-phosphate (PtdIns3P) levels on endomembranes which recruit FYVE-domain containing proteins for lysosomal dispersion. We identify transcription factor EB (TFEB) as an upstream regulator of PtdIns3P production by VPS34 that is activated in aggressive bladder cancer cells with peripheral lysosomes. This conceptually clarifies the dual role of TFEB as regulator of endosomal maturation and autophagy, two distinct processes controlled by PtdIns3P. Altogether, our findings uncover peripheral lysosome positioning, resulting from PtdIns3P production downstream of TFEB activation, as a potential biomarker for bladder cancer.

Analysis of Organelle Positioning Using Patterned Microdevices.

The consequences of alterations in the distribution of intracellular organelles, observed in many diseases, are often not clear. Intracellular organelles alter their morphology and positioning to regulate cell homeostasis and function. We outline how organelle positioning can be studied employing a density-based analysis of 3D images applied to cells that show similar cellular geometries. Quantification is facilitated by the use of single cells seeded on micropatterned substrates that provide cues for controlled cell spreading. This minimal system mimics the reproducible distribution of organelles typically observed in tissues, simplifying image analysis and minimizing the number of cells required for the observation of robust phenotypes. Here we provide guidelines for how the majority of organelles can be efficiently analyzed in cells seeded on adhesive micropatterns. We exemplify how alterations in the positioning of different organelles as a result of the perturbation of the cytoskeleton or associated motor proteins can be efficiently quantified. © 2018 by John Wiley & Sons, Inc.

Determining the Intracellular Organization of Organelles.

Many studies have found alterations in the positioning and morphology of intracellular organelles under different experimental conditions. Although the precise quantification of these changes is challenging, it is strongly facilitated in single cells that are seeded on micropatterned substrates. Indeed, the controlled microenvironment of the cell leads to a reproducible distribution of organelles, simplifying image analysis and minimizing the number of cells required for robust phenotypes. Here, we outline how alterations in the intracellular organization of lysosomes and mitochondria, as a result of different growth conditions, can be efficiently quantified in cells seeded on adhesive micropatterns.