(A) Coomassie- and silver-stained full-length CENP-E (340 kD) purified to near homogeneity from baculovirus-induced insect cells

(B) Electron micrographs of individual CENP-E molecules. Two motor heads are clearly visible, as indicated by yellow arrows. (C) Length distribution of CENP-E. Contour lengths of molecules were measured, and the mean length of CENP-E was 230 ± 25 nm (mean ± SD; = 20). (D) Coiled-coil prediction of CENP-E. Coiled-coil scores were generated using Protean software (DNAstar) and are graphed below the amino acid scale bar. The number 1.3 is the default value indicating the minimum score for known coiled coils, and resulting predicted coiled-coil domains are shown as solid orange rectangles.<p><b>Copyright information:</b></p><p>Taken from "CENP-E combines a slow, processive motor and a flexible coiled coil to produce an essential motile kinetochore tether"</p><p></p><p>The Journal of Cell Biology 2008;181(3):411-419.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364708.</p><p></p

(A) Using its slow processive motor activity and a weak diffusive binding mode to microtubules, CENP-E walks toward the plus ends of kinetochore microtubules or diffuses along the lattice without dissociating for extended periods

(B) The 230-nm-long coiled coil of CENP-E functions as a safety catch for disassembling microtubules detached from the core kinetochore attachment components, thereby stabilizing the microtubule and enabling rescue. (C) CENP-E is likely to be a part of the kinetochore slip clutch that is engaged on fluxing kinetochore microtubules with its slow plus end–directed motility (). CENP-E bound to the microtubule surface may affect kinetochore microtubule plus ends, thereby promoting growth and allowing recapture. (D) Unlike other shorter and more rigidly structured kinetochore capture components, multiple CENP-E molecules are likely to work together by allowing the simultaneous attachment at many different microtubule orientations relative to the kinetochore axis without forcing each other into unproductive conformations. (E) The highly flexible extended coiled coil of CENP-E mediates the initial capture of microtubules by searching a large volume in cells. (F) Its slow, processive motility powers monooriented chromosomes to congress using an adjacent kinetochore fiber ().<p><b>Copyright information:</b></p><p>Taken from "CENP-E combines a slow, processive motor and a flexible coiled coil to produce an essential motile kinetochore tether"</p><p></p><p>The Journal of Cell Biology 2008;181(3):411-419.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364708.</p><p></p

(A) Coomassie-stained full-length CENP-E–GFP (366 kD) purified from baculovirus-induced insect cells

(B) Microtubule gliding assay with full-length CENP-E. CENP-E–GFP proteins were tethered to a GFP antibody-coated surface of a flow chamber, and polarity-marked microtubules were subsequently introduced. Minus ends of the microtubules are brightly marked. Colored arrowheads indicate the starting positions of three microtubules, and colored dots indicate the minus ends. The mean gliding velocity was 30 ± 7.6 nm/s (mean ± SD; = 112). (C) Purified full-length CENP-E–GFP was added into the extract before spindle assembly. (D) Added CENP-E–GFP was localized to the kinetochore in extract spindle. A frame from a time-lapse video of a metaphase spindle in the extracts is shown. Red, X-rhodamine tubulin; green, CENP-E–GFP. Bars: (B) 2 μm; (D) 10 μm.<p><b>Copyright information:</b></p><p>Taken from "CENP-E combines a slow, processive motor and a flexible coiled coil to produce an essential motile kinetochore tether"</p><p></p><p>The Journal of Cell Biology 2008;181(3):411-419.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364708.</p><p></p

Rac1 promotes pioneer MTs in U2-OS cells.

<p>(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.</p

MARK2 regulates MT growth lifetime.

<p>(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.</p

MARK2 is required for cell polarization and directional migration.

<p>(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.</p

MARK2 is required for leading edge MT dynamics of directed migrating cells.

<p>(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.</p

RNAi screen for proteins whose depletion blocks CA-Rac1 effects on MT growth and orientation.

<p>(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<i>^glued</i>, 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%</p

MARK2 regulates MT growth dynamics and orientation downstream of Rac1.

<p>(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.</p

Effects of MT regulatory protein depletion on MT growth dynamics in U2-OS cells.

<p>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<i>^glued</i>, 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.</p