Actin Cytoskeleton Controls Movement Of Intracellular Organelles In Epithelia

Abstract

Background: Intracellular organelles move along microtubules and filamentous actin (F-actin) using molecular motors. Previously we reported that movement of secretory granules was hindered by Ca2+-dependent formation of a dense cytoplasmic F-actin meshwork in pancreatic duct epithelial cells (PDEC). Aim: Here we addressed whether other organelles such as mitochondria and lysosomes also are controlled by the same mechanism. Methods: Mitochondria and lysosomes were labeled with fluorescent MitoTracker Red and LysoTracker Red, respectively. Their movements were monitored every 1.2 s with high resolution fluorescence microscopy and measured by comparing subsequent images. Cells were stimulated with UTP to activate an endogenous P2Y2 G-protein coupled receptor to increase intracellular Ca2+. Results: Mitochondria and lysosomes moving actively at rest stopped rapidly (τ ∼ 2.6 s) after Ca2+ rise. This organelle ‘freezing’ was accompanied by the formation of F-actin in the whole cytoplasm as stained with phalloidin-Alexa 488. EGFP-actin binding domain 2 (ABD2) expressed in PDEC, indicated a rapid formation of cytoplasmic F-actin (τ ∼ 7.8 s). In addition, the freezing of the organelles was blocked by latrunculin B, an inhibitor of F-actin formation. Conclusion: Ca2+-dependent formation of a fine F-actin meshwork reduces the movement of intracellular organelles physically in PDEC. The freezing mechanism controls exocytosis of secretory granules and may affect local ATP supply by controlling mitochondrial distribution in PDEC.Supported by UW Royalty Research Fund and an NIH grant (GM083913).