Accumulation of an Antidepressant in Vesiculogenic Membranes of Yeast Cells Triggers Autophagy

Many antidepressants are cationic amphipaths, which spontaneously accumulate in natural or reconstituted membranes in the absence of their specific protein targets. However, the clinical relevance of cellular membrane accumulation by antidepressants in the human brain is unknown and hotly debated. Here we take a novel, evolutionarily informed approach to studying the effects of the selective-serotonin reuptake inhibitor sertraline/Zoloft® on cell physiology in the model eukaryote Saccharomyces cerevisiae (budding yeast), which lacks a serotonin transporter entirely. We biochemically and pharmacologically characterized cellular uptake and subcellular distribution of radiolabeled sertraline, and in parallel performed a quantitative ultrastructural analysis of organellar membrane homeostasis in untreated vs. sertraline-treated cells. These experiments have revealed that sertraline enters yeast cells and then reshapes vesiculogenic membranes by a complex process. Internalization of the neutral species proceeds by simple diffusion, is accelerated by proton motive forces generated by the vacuolar H+-ATPase, but is counteracted by energy-dependent xenobiotic efflux pumps. At equilibrium, a small fraction (10–15%) of reprotonated sertraline is soluble while the bulk (90–85%) partitions into organellar membranes by adsorption to interfacial anionic sites or by intercalation into the hydrophobic phase of the bilayer. Asymmetric accumulation of sertraline in vesiculogenic membranes leads to local membrane curvature stresses that trigger an adaptive autophagic response. In mutants with altered clathrin function, this adaptive response is associated with increased lipid droplet formation. Our data not only support the notion of a serotonin transporter-independent component of antidepressant function, but also enable a conceptual framework for characterizing the physiological states associated with chronic but not acute antidepressant administration in a model eukaryote

Clathrin mutant ultrastructure

<p>Electron micrographs of chc1 (clathrin heavy chain) mutant yeast cells.</p

The vacuole life cycle of yeast series

<p>Transmission electron microscopy is a powerful technique that allows us to peer deep inside cells. Dynamic cellular processes, like the fission and fusion of internal compartments, are frozen in time by an aldehyde-containing fixative, allowing us to get a glimpse of a population of near 100% genetic clones.</p> <p>The cell in this gallery is a wildtype strain (BY4716) of Saccharomyces cerevisiae, or budding yeast. As is evidenced in the high magnification zooms, the formation of multilamellar compartments is a normal event in the lives of vacuoles and related trafficking organelles.</p

Crowd4Discovery ventral medial primary neuronal culture experiment 1

<p>In this pilot experiment, Danny (@badomens) has treated fixed cultured ventral midbrain neurons with a pH-sensitive dye called pHrodo, which accumulates and "glows" red inside acidic compartments of the endocytic pathway. Danny is comparing untreated neurons ("steady-state") vs 24-hr amphetamine-treated neurons ("400µM").</p> <p> </p> <p>He also treated these cells with an antibody that recognizes a protein called LC3, which marks intracellular structures called autophagosomes, which deliver cellular bits to the recycling centers of cells. This LC3-specific antibody was visualized with a second, fluorescent antibody that "glows" green. Yellow indicates co-localization of endocytic compartments with autophagosomes. </p> <p><br></p> <p>This experiment is now being done using a confocal microscope to thoroughly assess colocalization of the two labels.</p

Wildtype non-spherical yeast vacuoles (replicate 2)

<p> </p> <p>Individual vacuoles from untreated wildtype yeast cells at steady state. These vacuoles were classified as non-spherical.</p> <p><br></p> <p> </p> <p>These images are supplementary data for my lab's PLoS ONE paper, which is linked below.</p> <p><br></p> <p> </p> <p>Similar data from my lab is available for viewing on The Cell: An Image Library, which is a repository maintained by the American Society for Cell Biology (ASCB).</p> <p> </p

Tranmission electron microscopy of a swa2 mutant in yeast

<p>Electron micrographs of <em>swa2</em> mutant cells.</p> <p> </p

Wildtype yeast autophagosomes (replicate 2)

<p> </p> <p>Individual autophagosomes from untreated wildtype yeast cells at steady state.</p> <p><br></p> <p> </p> <p>These images are supplementary data for my lab's PLoS ONE paper, which is linked below.</p> <p> </p> <p><br></p> <p>Similar data from my lab is available for viewing on The Cell: An Image Library, which is a repository maintained by the American Society for Cell Biology (ASCB).</p> <p> </p

Wildtype spherical yeast vacuoles (replicate 1)

<p> </p> <p>Individual vacuoles from untreated wildtype yeast cells at steady state. These vacuoles were classified as spherical.</p> <p><br></p> <p> </p> <p>These images are supplementary data for my lab's PLoS ONE paper, which is linked below.</p> <p><br></p> <p>Similar data from my lab is available for viewing on The Cell: An Image Library, which is a repository maintained by the American Society for Cell Biology (ASCB).</p

Crowd4Discovery striatal primary neuronal culture experiment 1

<p>Primary striatal neuronal culture troubleshooting experiment.</p> <p>As described in the C4D Spring update (link below), these cells were stained with a variety of markers to assess autophagy induction as a positive control in response to rapamycin treatment, a known inducer of autophagy.</p> <p>These cells were untreated.</p

Crowd4Discovery protocols and experimental summaries

<p>Fileset containing C4D protocols, experimental summaries, and other notes that help in the interpretation and analysis of our data.</p> <p> </p> <p>Documents in this fileset will be regularly updated, and new files will be added as the project progresses.</p