Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

<div><p>Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.</p></div

Sonication efficiently disrupted cells and released AVs that retained their integrity.

<p>(<b>A</b>) Forward scatter (FSC) vs. side scatter (SSC) plots of flow cytometric analysis of PC3 cell homogenates after a 2-day treatment with 1 µM GDC-0941 and 10 µM CQ, stained with AO and subjected to 0 (left) or 3 (right) 1s pulses of sonication. The position of the beads (7 µm) used as a standard for size threshold to distinguish cells from subcellular structures is circled in black. The distinct low FSC and SSC population circled in red, containing cell debris and organelles, is named the “subcellular” population, and used as such throughout this report. The high FCS and SSC population of intact cells is circled in blue. (<b>B</b>) Quantitative analysis of the number of total and AO⁺ subcellular events or cells in the corresponding populations defined in (<b>A</b>) as a function of the number of 1s sonication pulses. Error bars represent standard errors of mean (SEM), n = 7. Representative data from 2 independent experiments are shown. (<b>C,D</b>) Microscopy images of PC3 cells stained with AO and Hoechst 33342 and sonicated with 3 x 1s pulses showing the bottom focus plane with released vacuoles in focus (<b>C</b>) and the mid-cell range focus plane with an unbroken cell in focus (<b>D</b>). RGB images obtained in ex FITC/em Cy5 and ex DAPI/em DAPI channels are merged using Adobe Photoshop. Inset: 7 µm beads in the green (em FITC/ex FITC) channel under microscope at the same magnification. Examples of AVs are indicated with white arrows. Scale bars: 20 µm. (<b>E</b>) Microscopy image of PC3 cells stably expressing mCherry-eGFP-LC3B co-treated with 1 µM GDC-0941 and 10 µM CQ for 2 days and sonicated with 3 x 1s pulses. Two unbroken cells in the upper left corner filled with AVs are out of focus. Mostly “yellow” (“red” and “green” dual positive) AVs on the bottom of the plate are in focus. RGB images from red (mCherry) and green (eGFP) channels are merged using Adobe Photoshop. Examples of AVs are indicated with white arrows. Scale bar: 20 µm.</p

TEM analysis of subcellular structures in both intact and 3 x 1s sonicated homogenates of PC3 cells.

<p>(<b>A–C</b>) PC3 cells treated with DMSO showed only occasional autophagosome and autolysosome structures in both intact cells and homogenates. (<b>D–F</b>) PC3 cells treated with 2 µM GDC-0941 for 24 hours showed a slightly increased number of autophagosome and autolysosome structures in both intact cells and homogenates compared to (<b>A</b>). (<b>G–I</b>) PC3 cells treated with 10 µM CQ contained increased number of AVs mostly of autolysosomal nature in both intact cells and homogenates. (<b>J–L</b>) PC3 cells treated with both GDC-0941 and CQ are packed with AVs mostly of autolysosomal nature in both intact cells and homogenates. Arrows indicate representative AV structures in each population; scales bars: 2 µm (A,D,G,J), 0.2 µm (B,C) & 0.5 µm (E,F,H,I,K,L).</p

mCherry-eGFP-LC3B, eGFP-p62, and Acridine Orange are co-localized with fluorescently-labeled chloroquine in AVs detected by OFACS.

<p><b>(A)</b> Representative microscopy images of PC3 cells stably expressing mCherry-eGFP-LC3B and treated with 1 µM GDC-0941 +/− 9 µM CQ and 1 µM LynxTag-CQ-blue for 24 hours. Images are shown in each fluorescence channel separately (top panels) or merged (bottom panels). Scale bar: 20 µm. <b>(B)</b> OFACS quantification of the experiment in <b>(A)</b> showing the normalized numbers of events with the indicated labels: total normalized number of “subcellular” events (black bars), CQblue⁺ (blue bars), eGFP⁺ (green bars) and dual positive (CQblue⁺eGFP⁺) (cyan bars). *, P<0.05 vs. DMSO, CQ or GDC-0941 only groups. <b>(C)</b> Representative microscopy images of PC3 cells transiently transfected with eGFP-p62 for 48 hours and treated with GDC-0941 (1 µM) +/− 9 µM CQ and 1 µM LynxTag-CQ-blue for 24 hours. Images are shown in green or blue channels separately or merged. Scale bar: 10 µm. <b>(D)</b> OFACS quantification of the experiment in <b>(C) </b>showing the normalized number of CQblue⁺eGFP⁺ events under each treatment. <b>(E)</b> “Red” (PerCP) vs. “blue” (DAPI) channel plots of PC3 cells treated with 1 µM GDC-0941 +/− 5uM LynxTag-CQ-blue for 24 hours, stained with AO, sonicated and analyzed by OFACS. Error bars represent SEM (n = 4). Representative data from 2 independent experiments are shown.</p

Pharmacologically induced AVs can be individually detected by OFACS from sonicated PC3 cells expressing mCherry-eGFP-LC3B.

<p><b>(A)</b> mCherry (ex PE/em Texas Red, “red” channel) vs. eGFP (FITC “green” channel) plots showing accumulation of the mCherry⁺eGFP⁺ organelle population in PC3 cells co-treated for 2 days with 1 µM GDC-0941 and 10 µM CQ vs. each drug alone or no drug. Control PC3 cells expressing free mCherry (top) or eGFP-LC3B (bottom) treated with GDC-0941 and CQ analyzed the same way are shown on the right as single channel controls. Numbers in upper right quadrants represent the percentage of mCherry⁺eGFP⁺ events of the total subcellular population. <b>(B) </b>Normalized number of mCherry⁺eGFP⁺ events in cells treated with the indicated agents and the effects of Atg5 or Atg7 siRNA on this population. *, P<0.05 vs. DMSO, CQ or GDC-0941/GDC-0068 alone with non-target siRNA; **, P<0.05 vs. non-target siRNA in the same treatment group. <b>(C,D)</b> Normalized number of mCherry⁺eGFP⁺ AVs per cell <b>(C)</b> or total GFP intensity of the mCherry⁺GFP⁺ AVs <b>(D)</b> in PC3 cells co-treated with GDC-0941/GDC-0068 and protease inhibitors or CQ as indicated. *, P<0.05 both vs. DMSO group treated with the same lysosomal inhibitor and vs. GDC-0941 or GDC-0068 alone. PC3 cells stably expressing the mCherry-eGFP-LC3B marker were treated with 1 µM of GDC-0941 <b>(A–D)</b>, or 5 µM of GDC-0068 <b>(B-D)</b> +/− 10 µM CQ or the indicated concentrations of protease inhibitors for 1 day, sonicated and analyzed by OFACS. Error bars represent SEM (n = 4). Representative data from one of three independent experiments are shown.</p

Pharmacologically induced acidic vacuoles can be individually detected by OFACS from sonicated cells stained with AO.

<p><b>(A)</b> PC3 cells treated with 1 µM GDC-0941 and 10 µM CQ for 2 days analyzed by OFACS after staining with AO. FITC (green) vs. PerCP (red) channels of the organelle population are plotted. Percentage of the gated AO⁺ population is shown for each plot. <b>(B)</b> Quantification of the number of AO⁺ events as gated in <b>(A)</b> normalized to the number of cells used for sonication with each treatment. *, P<0.05. <b>(C)</b> Quantitative analysis by OFACS showing the normalized number of AO⁺ events under indicated treatments. PC3 cells transfected with Atg5 or Atg7 siRNA for 2 days were treated with 1 µM GDC-0941 or 5 µM GDC-0068 +/− 10 µM CQ for an additional day. *, P<0.05 vs. DMSO, CQ or GDC-0941/GDC-0068 alone with non-target siRNA; **, P<0.05 vs. non-target siRNA in the same treatment group. <b>(D,E)</b> Quantification of AO⁺ events obtained by OFACS analysis of PC3 cells treated with GDC-0941 or GDC-0068 +/− CQ or protease inhibitors for 2 days, showing the normalized number of AO⁺ events <b>(D)</b> and the total “red” signal intensity of AO⁺ events <b>(E)</b>, derived by multiplying the number of AO⁺ organelles by the mean value of the “red” AO signal. *, P<0.05 both vs. DMSO group treated with the same lysosomal inhibitor and vs. GDC-0941 or GDC-0068 alone. Error bars represent SEM (n = 3). P values are determined by Student's t-test. Representative data from one of three independent experiments are shown. All quantifications are normalized to the number of cells used for the sonication under each condition.</p

Inhibition of autophagy by 3-MA at an early stage and by BafA1 at a later stage can be distinguished by individual AV analysis with OFACS.

<p>Untransfected PC3 cells <b>(A,B)</b> or PC3 cells expressing mCherry-eGFP-LC3B <b>(C)</b> treated with 1 µM GDC-0941 or 5 µM GDC-0068 +/− 10 µM CQ for 24 hours with the addition of: nothing (upper panels), 0–10 mM 3-MA (middle panels) or 0–100 nM Bafilomycin A1 (BafA1; bottom panels). AVs were analyzed by OFACS after staining with AO <b>(A,B)</b> or unstained <b>(C)</b>. Y axes represent the total “red” channel intensity of AO⁺ organelles normalized to cell number <b>(A,B)</b> or the percentage of mCherry⁺eGFP⁺ events of the total subcellular events <b>(C)</b>. Note the different scales of the y-axes in<b> (A)</b> vs.<b> (B).</b> Error bars represent standard deviations (SD), n = 3.</p

Three-dimensional distribution of VZV nucleocapsids in cell nuclei without PML cages.

<p>Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial section TEM (F and G). A) BSE-SEM images of four representative sections (s20, s30, s40, s50) from a series of 50 consecutive 100 nm sections are shown. A nuclear indentation is outlined and marked with a blue arrow. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s001" target="_blank">Video S1</a>. (B) 3D model of the shape of the VZV infected nucleus (grey). Upper panel (front view): the cross section plane and a deep invagination (blue arrow) of the nucleus are visible. The middle panel (side view) and bottom panel (rear view) reveal the irregular shape of the nucleus with numerous indentations. (C) View of the same nucleus at different angles in transparent mode. Color code: transparent grey, boundary of the nucleus; transparent blue, electron dense heterochromatin; brown, nucleolus; red spheres (mature capsids, C-type) and yellow spheres (immature capsids, A and B-type). A total of 4,223 capsids were identified and visualized. (D) Higher magnification view; color code as in C, but nuclear envelope not shown. The dense heterochromatin (solid dark blue) hides nucleocapsids that are located deeper in the nuclear volume. (E) Same view as in D, but with transparent heterochromatin: the distribution of capsids throughout the nucleus is revealed. See also Video S2. (F and G) Two different nuclei that were reconstructed from serial sections imaged by TEM. The color code is the same as above. Insets show representative images from the TEM series. The 3D models show the distribution of 425 (F) and 1,340 capsids (G), respectively. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s003" target="_blank">Video S3</a>. All scale bars are 5 µm.</p

Large volume-reconstruction of a VZV infected cell nucleus with four PML cages.

<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) Five representative BSE-SEM images (s5, s20, s30, s47, s70) from a series of 82 consecutive sections through a VZV nucleus with four PML cages (1–4, black arrows) with sequestered VZV capsids. A valley-like indentation of the nucleus (blue outline and arrow) and the endoplasmic reticulum (ER) are marked. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s006" target="_blank">Video S6</a>. (B–D and E–G, respectively) show 3D models of the nucleus in two angles (100 degrees rotation to the left). (B and E) Shape of the nucleus based on tracing its outer boundary (grey). (C and F) The shape of the nucleus (transparent grey) is overlaid with the dense heterochromatin (transparent blue). PML cages 1–4 (solid green) and VZV capsids that escaped sequestration (red spheres) are visible in the interior of the nucleus. (D and G) Same view as above, but the nuclear envelope and the PML domains are completely transparent. This reveals the location of all VZV capsids (5,597) identified in this nucleus; red spheres represent free capsids (70) and yellow spheres represent sequestered capsids (5,527). Scale bars are 5 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s007" target="_blank">Video S7</a>. (H and I) 3D models of PML cages (transparent green) from the same nucleus at higher magnification that reveal the dense packaging of capsids (yellow) and the close association of PML cages with the electron dense heterochromatin (blue). Red spheres represent free capsids. Scale bars are 2 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s008" target="_blank">Video S8</a>.</p

Electron tomography of PML cages reveals the cross-linking of VZV capsids by an electron-dense meshwork.

<p>Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and then high pressure frozen, freeze-substituted and embedded in epoxy-resin. 80 nm sections (A–E) or 300 nm sections (F–L) were investigated by dual-axis electron tomography. (A) A representative tomographic slice shows the periphery of the nucleus with the electron dense heterochromatin (blue bottom area) and part of the PML cage (light green area) containing numerous VZV capsids. A light electron-dense fibrous meshwork (grey) is visible within the PML-domain. These fibers are directly associated with capsids (arrows) and can cross-link them. (B) The area in the black square in A is shown at higher magnification in inverted mode, e.g. electron dense structures appear bright. Arrows depict fibrous material associated with VZV capsids. (C) Same image as in B but with traces for 3D reconstruction shown: capsids (yellow) were traced manually; electron dense meshwork (green) was traced automatically by thresholding. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s010" target="_blank">Video S10</a>. (D and E) show 3D models of the VZV capsids (yellow) associated with the electron-dense meshwork (green). Scale bars are 200 nm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002740#ppat.1002740.s011" target="_blank">Video S11</a>. (F) Volume view with inverted contrast of a reconstruction from a dual-axis tomogram of a 300 nm section. The arrangement of VZV capsids within a PML cage is visible. (G) Same reconstruction as in F but in orthoslice mode that reveals the arrangement of capsids in the interior of the reconstructed volume. (H) Volume view of a part of the tomographic reconstruction that was then traced and segmented (I) to reveal the position of capsids and the electron dense meshwork in a 3D model. (I) Traces on one representative digital tomographic slice: immature capsids (yellow), mature capsids (red), electron dense fibers and meshwork (green). (J) 3D model shows the packaging of capsids (protein meshwork is green/transparent for unobscured view of capsids). (K) 3D model shows capsids with associated electron-dense meshwork (green) at higher magnification. (L) Representative tomographic slice images that show protein fibers (green arrows) associated with VZV capsids. Scale bars are 200 nm (A–D and F–J) and 100 nm (E, K and L).</p