Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5α/β

A newly discovered interaction between LL5s, laminins, and integrins reveals how the extracellular matrix directs microtubule polarity in epithelial tissues

Dissecting the Nanoscale Distributions and Functions of Microtubule-End-Binding Proteins EB1 and ch-TOG in Interphase HeLa Cells

<div><p>Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in <em>in vitro</em> systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other’s localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.</p> </div

EB1, but not ch-TOG, is recruited to CLASP-accumulating microtubule ends.

<p>(<b>A</b>) GFP-CLASP2γ was expressed in HeLa cells, and the cells were fixed and stained for endogenous EB1 and ch-TOG. The images were acquired by confocal microscopy. The GFP-CLASP2γ (green) and EB1 (red) signals are merged in the right panel. The insets show the boxed areas at 2× magnification. Note that only EB1, and not ch-TOG, was recruited to the GFP-CLASP2γ-accumulating microtubule ends. Scale bar, 20 µm. (<b>B–D</b>) mRFP1-CLASP2γ (white) was expressed in HeLa cell clones (1E10) expressing GFP-α-tubulin (green), and the cells were fixed and stained for endogenous ch-TOG (red). The images are merged in the right panel. The images were acquired by SIM. In the bottom panels, the boxed areas in the upper panels are magnified. Boxed areas in (B) and (C) are enlarged in (C) and (D), respectively. Note that ch-TOG distributes at the most distal portion of microtubule ends even after mRFP1-CLASP2γ overexpression. Scale bars, 10 µm (B, upper), 2 µm (B, bottom), 500 nm (C).</p

Impact of EB1 and/or ch-TOG depletion on microtubule dynamics in interphase HeLa cells.

<p>(<b>A–F</b>) Analysis of microtubule dynamics by time-lapse imaging of HeLa/GFP-α-tubulin clones (1E10) after transfection with the indicated siRNAs. Because ch-TOG depletion impairs mitosis and induces dell death, siRNA-treated cells were blocked in interphase 24 h after transfection by addition of thymidine (2.5 mM). Cells were imaged with a 0.5 s interval (Movie S5). In A–D, the first frames of the time-lapse movies (left), selected time-lapse images of the boxed areas (middle), and kymographs of the indicated microtubules (arrowheads) are shown (right). The time-lapse images are arranged with the microtubule distal ends upwards (the direction of the arrows in the left panels). Scale bars; 5 µm (left), 1 µm (middle and right). (<b>E</b>) Life-history plots of individual microtubules are shown. Plots for three different MTs are shown by the red, green and orange lines. In (<b>F</b>), the duration time of the “pause” state, in which microtubules exhibit no detectable growth/shortening or repeated growth/shortening within a limited distance (< 1 µm), was measured and plotted. The results are presented as means ± SEM (*, P < 0.01; n = 360 in 15 cells for mock, n = 204 in 15 cells for EB1 siRNA, n = 204 in 15 cells for ch-TOG siRNA, n = 179 in 15 cells for EB1 + ch-TOG siRNA). The pause duration time was increased additively following EB1 and ch-TOG double knockdown. (<b>G</b>, <b>H</b>) Western blotting (WB) analysis for acetylated tubulin, a marker of stable microtubules. In (G), a representative western blot of lysates from cells treated with the indicated siRNAs probed with the indicated antibodies is shown. The average densities of the acetylated tubulin bands obtained from five independent experiments are plotted in (H). The data were normalised and the density of the mock control was set as 1. The results are presented as means ± SEM (P = 0.061 for mock v.s. EB1 siRNA, P = 0.034 for mock v.s. ch-TOG siRNA, P = 0.006 for mock v.s. EB1+ch-TOG siRNA, P = 0.317 for EB1 siRNA v.s. ch-TOG siRNA, P = 0.042 for EB1 siRNA v.s. EB1+ch-TOG siRNA, P = 264 for ch-TOG siRNA v.s. EB1+ch-TOG siRNA; n = 7). For WB full scans, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442.s006" target="_blank">Figure S6A</a>.</p

EB1 and ch-TOG do not affect the binding of each other to microtubules.

<p>The effects of overexpression of mEB1-GFP or ch-TOG-GFP on the distributions of endogenous ch-TOG or EB1, respectively, were examined. (<b>A</b>, <b>B</b>) mEB1-GFP (green) was transiently expressed in HeLa cells, and the cells were fixed and stained for endogenous ch-TOG using a RhoX-conjugated secondary antibody (red). To analyse the effect of mEB1-GFP overexpression on the distribution of ch-TOG, cells expressing a large amount of mEB1-GFP that distributed throughout the entire microtubule lattice, in which microtubule ends were expected to be saturated with EB1, were selected and the images were acquired on a SIM microscope. The boxed areas in (A) are enlarged in (B). Microtubule ends positive for ch-TOG clusters are indicated by arrowheads. Scale bars, 10 µm (A), 2 µm (B). (<b>C</b>) Average FI profiles of overexpressed mEB1-GFP and ch-TOG were obtained by analysing multiple SIM images (n = 71, 4 cells in 4 images) and plotted. The data were normalised and the peak intensities were set to 1. The error bars are SEM. (<b>D</b>) ch-TOG-GFP was exogenously expressed in HeLa cells, and the cells were fixed and stained for ch-TOG and EB1. The images were acquired on a confocal microscope. The expression level of exogenous ch-TOG-GFP was determined by comparing the fluorescence intensity of ch-TOG staining in GFP-negative and positive cells. The cell indicated by the asterisks has an ∼8-fold higher expression level of ch-TOG than untransfected cells. The inset shows the boxed areas at 2.5× magnification. Scale bar: 20 µm. In (<b>E</b>), average FI profiles of EB1 in untransfected control cells and ch-TOG-GFP expressing cells (> 6-fold overexpression) were obtained by analysing multiple confocal images (n = 132, 7 cells in 3 images for control; n = 135, 5 cells in 3 images for ch-TOG-GFP expressing cells) and plotted without normalization. The comet-like distribution of EB1, as well as the fluorescence intensity of the EB1 comet, was not altered by ch-TOG-GFP overexpression. (<b>F</b>) A control experiment showing competitive binding of EB1 to microtubule ends. mEB1-RFP was transiently overexpressed at a level at which it distributes throughout entire microtubule lattices in HeLa/mEB1-GFP clones (2F10) stably expressing mEB1-GFP at ∼40% the level of endogenous EB1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442.s002" target="_blank">Figure S2D</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442.s006" target="_blank">S6E</a>). In RFP-negative cells, mEB1-GFP distributes in a typical comet shape. Overexpression of mEB1-RFP inhibited the accumulation of mEB1-GFP to microtubule ends and redistributed it throughout the entire microtubule lattice. The insets show the boxed areas at 2.5× magnification. Scale bar: 20 µm.</p

Evaluation of antibodies for EB1 and ch-TOG staining.

<p>To test the specificities of the antibodies and the effects of fluorophores (emission wavelength), ch-TOG (<b>A</b>) and EB1 (<b>E</b>) were labelled with two different secondary antibodies conjugated with Rhodamine Red-X (RhoX) or DyLight 649 (DL649), and visualised by SIM. The RhoX and DL649 channels are merged in the right panels in red and green, respectively. In (<b>B</b>) and (<b>F</b>), boxed areas in (A) and (E), respectively, are enlarged and merged in the right panels together with GFP-α-tubulin signals (white). In (B), both RhoX and DL649 signals are detected in the same clusters localised at microtubule ends (arrows), although their signal intensities are not necessarily equal probably owing to competing binding to the same antigen, while clusters positive only for RhoX (asterisks) or DL649 (arrowheads) are also observed. The colocalisation of RhoX and DL649 signals was analysed and the scatter plots of the pixel intensities for the RhoX and DL649 channels and their colocalisation parameters are shown, respectively, in (<b>C</b>) and (<b>D</b>) for ch-TOG and in (<b>G</b>) and (<b>H</b>) for EB1. The values indicating the colocalisation of the RhoX and DL649 channels show that 30–40% of pixels were not colocalised when analysed at the individual pixel level. However, considering that the same cluster often appears to be a different size when labelled with two different antibodies as shown in (B), the colocalisation percentage may be underestimated. (<b>I</b>) Using the ch-TOG-double labelling images including (A), ch-TOG-positive microtubule ends not overlapping with other microtubules were selected and the FI profiles were averaged (n = 52, 5 cells in 3 images). The error bars are SEM. (<b>J</b>) Using the EB1-double labelling images including (E), EB1-positive microtubule ends not overlapping with other microtubules were selected and the FI intensities were averaged (n = 52, 2 cells in 2 images). The error bars are SEM.</p

Effect of inhibiting microtubule dynamics on ch-TOG localisation.

<p>(<b>A, B</b>) ch-TOG-GFP (red) was expressed in HeLa cells expressing RFP-α-tubulin (green) and a low concentration of taxol (500 nM) was added. The living cells were observed by TIRF. The boxed area in (A) is enlarged in (B). Scale bars; 10 µm (A), 2 µm (B). (<b>C, D</b>) A HeLa cell clone expressing GFP-α-tubulin (1E10, green) was fixed and stained for endogenous ch-TOG using a secondary antibody conjugated with RhoX (red), and observed by SIM. The boxed area in (C) is enlarged in (D). Scale bars; 10 µm (A), 2 µm (B).</p

Behaviour of EB1 and ch-TOG in living HeLa cells observed by TIRF microscopy.

<p>(<b>A</b>) The distributions of mEB1-RFP and ch-TOG-GFP in a living HeLa cell. The merged images are shown in the right panel, where green indicates mEB1-RFP and red indicates ch-TOG-GFP. Scale bar, 10 µm. The boxed areas in the upper panels are enlarged in the bottom panels. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442.s008" target="_blank">Movie S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442.s009" target="_blank">S2</a>. Scale bar, 2 µm. (<b>B</b>) Kymographs showing the movements of ch-TOG-GFP and mEB1-RFP. The kymographs were generated from time-lapse sequences (33-sec long). Two example images are shown. The GFP (red) and RFP (green) signals are merged in the bottom panels. Scale bars, 2 µm. (<b>C</b>) Time-lapse series of part of a movie (Movie S3, S4) showing living HeLa cells expressing RFP-α-tubulin (green) and ch-TOG-GFP (red). Growing and shrinking microtubule ends, with bound ch-TOG-GFP, are indicated by the yellow arrows or arrowheads, respectively. The elapsed time is indicated in the figure as s:ms. Scale bar, 2 µm. (<b>D</b>) Kymographs showing the movements of ch-TOG-GFP and RFP-α-tubulin are presented similarly to in (B). Scale bar, 2 µm. In all figures, GFP and RFP are pseudo-coloured in red and green, respectively, for consistency with the other images. In the kymographs, the ch-TOG-GFP signals were always detected at a position slightly distal to the EB1 signals (B), although fast retrograde movement of ch-TOG-GFP was also observed (arrows in B). Simultaneous visualisation of ch-TOG-GFP and RFP-α-tubulin demonstrated the tracking of shortening microtubule ends by ch-TOG-GFP (arrows in D).</p

Overexpression of SLAIN2 enhanced the colocalisation of ch-TOG with EB1 comets.

<p>(<b>A</b>) Schema showing the domain structures of ch-TOG, SLAIN proteins, EB1 and CLASP proteins. Mutual binding sites are indicated by the arrows <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442-MimoriKiyosue4" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442-vanderVaart1" target="_blank">[24]</a>. (<b>B</b>) GFP-SLAIN2 (green) was transiently expressed in HeLa cells, and the cells were fixed and stained for endogenous EB1 (white) and ch-TOG (red). The images were acquired on a SIM microscope. The signals are merged in the right panel. Scale bar, 10 µm. In (<b>C</b>) and (<b>D</b>), magnified images of the boxed areas (1) and (2) in (B), respectively, are shown. Scale bars, 2 µm. (<b>E</b>) The line profiles along the EB1 comets marked with (i) and (ii) in (B) and (C) are plotted. Note that the fluorescence intensity of ch-TOG at the rear part of microtubules does not fall to zero, unlike in SLAIN2-untransfected cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone-0051442-g001" target="_blank">Figure 1E</a>). (<b>F</b>) Average FI profiles of EB1 and ch-TOG were obtained by analysing multiple SIM images (n = 79, 2 cells in 2 images) and plotted. The data were normalised and the peak intensities were set to 1. The error bars are SEM. Note that the ch-TOG distribution extends towards the rear of the microtubules and is co-localised with EB1 comets, while the strong ch-TOG peak is still present at the tips in front of the EB1 comets (arrows). GFP-SLAIN2 distributes on both ch-TOG clusters at the tips and EB1 comets. The difference in the ch-TOG distributions on the EB1 comets in the data sets shown in (F) and in Fig. 1F (as a control) was statistically significant (P < 0.01 at 25 nm rear of the ch-TOG peak, P < 0.001 at > 50 nm rear of the ch-TOG peak).</p

Model of the binding sites for EB1 and ch-TOG.

<p>EB1 and ch-TOG are displayed on a graphic of a growing microtubule structure. The shape of the tubulin/ch-TOG complex was adapted from ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442-Brouhard1" target="_blank">[3]</a>. XMAP215, which has a similar domain structure to ch-TOG, is a long molecule of ∼60 nm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442-Cassimeris2" target="_blank">[55]</a> that binds small tubulin oligomers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442-Slep2" target="_blank">[46]</a> or one free tubulin dimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442-Brouhard1" target="_blank">[3]</a> at its NH₂-terminus. The antibody against ch-TOG was generated using the COOH-terminal half of this molecule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442-Charrasse2" target="_blank">[28]</a>. EB1 binds to the closed B lattice of the microtubule wall <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442-Maurer2" target="_blank">[56]</a>. The microtubule tip probably contains protofilaments of different lengths. Based on our observation that the peak intensity of ch-TOG and EB1 comets was separated by ∼100 nm, the longer protofilaments for which growth is accelerated by ch-TOG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051442#pone.0051442-VanBuren1" target="_blank">[47]</a> are assumed to consist of ∼12 tubulin dimers. SLAIN proteins may recruit ch-TOG to EB1 comets to increase the local concentration of ch-TOG.</p