Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells - PubMed (original) (raw)
Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells
Yukako Nishimura et al. PLoS One. 2012.
Abstract
Polarized microtubule (MT) growth in the leading edge is critical to directed cell migration, and is mediated by Rac1 GTPase. To find downstream targets of Rac1 that affect MT assembly dynamics, we performed an RNAi screen of 23 MT binding and regulatory factors and identified RNAi treatments that suppressed changes in MT dynamics induced by constitutively activated Rac1. By analyzing fluorescent EB3 dynamics with automated tracking, we found that RNAi treatments targeting p150(glued), APC2, spastin, EB1, Op18, or MARK2 blocked Rac1-mediated MT growth in lamellipodia. MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study. We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner. In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration. Thus, automated image analysis of MT assembly dynamics identified MARK2 as a target regulated downstream of Rac1 that promotes oriented MT growth in the leading edge to mediate directed cell migration.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Rac1 promotes pioneer MTs in U2-OS cells.
(A) Immunolocalization of microtubules (MTs) and fluorescent phalloidin staining of F-actin in mock-transfected cells (control) or cells expressing CA-Rac1 or DN-Rac1. Contrast inverted, left, center. Zoom of boxed region, second column. Insets, BFP-Rac1. Bar, 10 µm. (B) Workflow of plusTipTracker software for detecting mKO-EB3 comets, tracking them, and classifying MT growth excursions. (C) Top: Proportion of MT growth excursions in each subpopulation in non-targeting control vector, CA-Rac1 or DN-Rac1 expressing cells. Bottom: Color key showing MT growth speed and growth excursion lifetime ranges for subpopulations. Box-plots of speed (D) and lifetime (E) of MT growth excursions, conditions as in C. (*, p<0.001, Kolmogorov-Smirnov, *, p<0.05, Students). (F) Top: mKO-EB3 tracks from 2 min time-lapse movies (frame rate = 3 s) colored according to the key in C overlaid on images of mKO-EB3 (inverted contrast), conditions described in A. Bottom: zoom of boxed region. Bars, 10 µm. (G) Percentage of MT growth tracks within 5 µm from the leading edge whose angle relative to the edge is between 0–45° (blue) or between 45–90° (green), conditions as in C.
Figure 2. Effects of MT regulatory protein depletion on MT growth dynamics in U2-OS cells.
Top: Results of analysis of time-lapse movies of mKO-EB3 with PlusTipTracker software. Proportion of MT growth excursions in each subpopulation in control shRNA vector-transfected cells (WT) or cells treated with RNAis targeting the protein noted (kd). shRNA vectors were used for RNAi targeting of EB1, CLASP2, dynamitin, DCX, MAP1A, MAP1B, MAP2, MAP4, MARK1, MARK2 and MARK3. siRNA oligos were used for RNAi targeting of APC, APC2, ACF7, XMAP215, Op18, p150_glued_, CLIP115, CLIP170, STOP, MAP1S, Spastin and Katanin p60 (see Methods). Bottom: Color key showing MT growth speed and growth excursion lifetime ranges for each subpopulation.
Figure 3. RNAi screen for proteins whose depletion blocks CA-Rac1 effects on MT growth and orientation.
(A) Top: Proportion of MT growth excursions in each subpopulation in control shRNA vector-transfected cells (control), cells expressing CA-Rac1, CA-Rac1 and additionally treated with RNAis targeting the protein noted (kd), DN-Rac1. shRNA vectors were used for RNAi targeting of EB1, CLASP2, dynamitin, DCX, MAP1A, MAP1B, MAP2, MAP4, MARK1, MARK2 and MARK3. siRNA oligos were used for RNAi targeting of APC, APC2, ACF7, XMAP215, Op18, p150_glued_, CLIP115, CLIP170, STOP, MAP1S, Spastin and Katanin p60 (see Methods). Dashed line 1: proteins which pass the first criteria (red tracks<50%); solid line 2: proteins which pass the second criteria (40%<red+green tracks <60%). Bottom: Color key showing MT growth speed and growth excursion lifetime ranges for subpopulations. (B) mKO-EB3 tracks from 2 min time-lapse movies (frame rate = 3 s) colored according to the key in A overlaid on images of mKO-EB3 (inverted contrast) in cells treated with RNAis targeting the noted proteins with (bottom) or without (top) CA-Rac1 expression. Bar, 10 µm. (C) Percentage of MT growth tracks within 5 µm from the leading edge whose angle relative to the cell edge is between 0–45° (blue) or between 45–90° (green), conditions as in B.
Figure 4. MARK2 regulates MT growth dynamics and orientation downstream of Rac1.
(A) Western blot of lysates of U2-OS cells transfected with control shRNA (lane 1), MARK2-shRNA (lane 2), MARK2-shRNA and shRNA-resistant GFP-MARK2, (lane 3), non-targeting siRNA pool (lane 4) or MARK2 siRNA (lane 5). GAPDH and DM1A were used as a loading control. (B) Immunostaining of MTs (inverted contrast) in cells expressing BFP-CA-Rac1 (Rac1) and GFP-shRNA targeting MARK2 (shRNA), or rescued with GFP-MARK2 expression. Center column: zoom of boxed region. (C) Top: Proportion of MT growth excursions in subpopulations, conditions as in B. Control represents control shRNA vector transfected cells. Bottom: Color key showing MT growth speed and growth excursion lifetime ranges for subpopulations. Box-plots of speed (D) and lifetime (E) of MT growth excursions, conditions as in B. (*p<0.001, Kolmogorov-Smirnov, *p<0.05, Students). (F) Top: mKO-EB3 tracks from 2 min time-lapse movies (frame rate = 3 s) colored according to the key in C overlaid on images of mKO-EB3 (inverted contrast), conditions as in B. Bottom: Zoom of boxed regions. (G) Percentage of MT growth tracks within 5 µm from the leading edge whose angle relative to the cell edge is between 0–45° (blue) or between 45–90° (green), conditions as in B. Bars, 10 µm.
Figure 5. MARK2 regulates MT growth lifetime.
(A) Immunolocalization of MTs (inverted contrast) in cells expressing control shRNA-GFP (top), MARK2 shRNA-GFP (middle), or MARK2 shRNA-GFP together with RNAi-resistant GFP-MARK2 (bottom). Cells co-expressing MARK2 shRNA-GFP and GFP-MARK2 could be recognized by the targeting of GFP-MARK2 to the centrosome (arrowhead). (B) Top: Proportion of MT growth excursions in each subpopulation in cells under the conditions described in A. Bottom: Color key showing MT growth speed and growth excursion lifetime ranges for subpopulations. Box-plots of speed (C) and lifetime (D) of MT growth excursions, conditions as in A. (*, p<0.001, Kolmogorov-Smirnov, *, p<0.05, Students) (E) Top: mKO-EB3 tracks from 2 min time-lapse movies (frame rate = 3 s) colored according to the key in B overlaid on images of mKO-EB3 (inverted contrast), conditions as in A. Bottom: zoom of boxed regions. (F) Percentage of MT growth tracks within 5 µm from the leading edge whose angle relative to the cell edge is between 0–45° (blue) or between 45–90° (green), conditions as in A. Bars, 10 µm.
Figure 6. MARK2 is required for leading edge MT dynamics of directed migrating cells.
(A) Above: Proportion of MT growth excursions in each subpopulation in control shRNA vector-transfected whole cells (W), expression of CA-Rac1, MARK2 shRNA treatment or from within 5 µm from the leading edge of wound edge cells with MARK2 shRNA (MARK2 RNAi (LE)) or control shRNA (control (LE)) treatment. Below: Color key showing MT growth speed and growth excursion lifetime ranges for subpopulations. Box-plots of speed (B) and lifetime (C) of MT growth excursions, conditions as in A. (*, p<0.001, Kolmogorov-Smirnov, *, p<0.05, Students). (D) mKO-EB3 tracks from 2 min time-lapse movies (frame rate = 3 s) of cells at the edge of a scratch-wound, colored according to the key in A overlaid on images of mKO-EB3 (inverted contrast) with MARK2 shRNA (MARK2 RNAi) or control shRNA (control RNAi) treatment. Only MT growth tracks that come within 5 µm of the leading edge are shown. Bar, 10 µm. (E) Percentage of MT growth tracks within 5 µm from the leading edge whose angle relative to the cell edge is between 0–45° (blue) or between 45–90° (green), conditions as in A.
Figure 7. Accumulation of MARK2 in the leading edge depends on Rac1 activity.
(A) Fluorescence images of GFP-MARK2 (upper left), mCherry-C1 (upper right) and phase image (lower left) in a non-polarized cell. Line scans (lower right) show the average normalized (to maximal) fluorescent intensity of three different cells expressing GFP-MARK2 (red) and mCherry-C1 (blue) along lines places similarly as those shown in the upper panel. Arrow represents cell protrusion. (B) Fluorescence image of GFP-MARK2 (upper left), and phase image (upper right) in migrating cell at a wound edge. Arrow: cell protrusions, Asterisks: cell-cell junctions. Line scans (lower panel) show average normalized (to maximal) fluorescent intensity of lines in three different cells expressing GFP-MARK2 along protrusion area (Lines 1) or non-protrusion area (Line 2). (C) Fluorescence images of GFP-MARK2 (upper left and upper right) in cell expressing BFP-CA-Rac1 (inset upper left) or BFP-DN-Rac1 (inset upper right). Insets show the BFP fluorescence images. Line scans indicate average normalized (to maximal) fluorescent intensity of three different cells expressing GFP-MARK2 along lines placed similarly to those in in CA-Rac1 (left) or DN-Rac1 (right). Bars, 10 µm.
Figure 8. MARK2 is required for cell polarization and directional migration.
(A) Immunostaining of MTs (left and right) and γ-tubulin (merge in center) in wound edge cells in non-targeting siRNA pool (top, control RNAi) and MARK2 siRNA treatment (bottom, MARK2 RNAi). Dotted circles, centrosome; bar, 10 µm. (B) Percentage of cells in the wound edge with centrosomes in front of the nucleus. In B, D, F *p<0.05, Student’s t-test. (C) Wound-healing assay in non-targeting siRNA pool (control RNAi; left) or MARK2 siRNA (MARK2 RNAi; right), time after wounding shown. Dashed line, position of wound edge at t = 0. Bar, 50 µm. (D) Average migration velocity of non-targeting siRNA pool (control) and MARK2 siRNA-treated (MARK2 RNAi) cells. (E) Rose plots of the position of nuclei over 5 hr (each cell track colored differently) for non-targeting siRNA pool-(above, control RNAi) or MARK2 siRNA-treated cells (below, MARK2 RNAi) at the edge of a wound. Arrows: open region of wound. (F) Distance from origin (position at time = 0) divided by total distance travelled over 5 hr for non-targeting siRNA pool and MARK2 siRNA treated cells.
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