Journal: bioRxiv

Article Title: Microtubules tune mechanosensitive cell responses

doi: 10.1101/2020.07.22.205203

Figure Lengend Snippet: A . Schematic and western blots showing the tools used to manipulate the levels of microtubule acetylation in this study. 2 distinct sets of siRNAs targeting αTAT1 (siαTAT1-1 and siαTAT1-2) reduce microtubule acetylation and Tubacin, an inhibitor of HDAC6 (deacetylase), increases microtubule acetylation in astrocytes plates on hydrogels or glass. Representative blots from N = 3 independent experiments are shown. In the main figures, only results obtained with siαTAT1-2, which induces the strongest decrease in acetyl tubulin, are shown. Although less pronounced, siαTAT1-1 produced similar effects as siαTAT1-2, in experiments corresponding to , , and . B . Epifluorescence images of astrocytes transfected with indicated siRNAs and plated on crossbow-shaped micropatterned hydrogels of different rigidities, stained for acetylated tubulin and α-tubulin. C . Epifluorescence images showing detyrosinated tubulin and α-tubulin staining of astrocytes sparsely plated on hydrogels of different rigidities. Graph shows the intensity ratio of detyrosinated tubulin over total tubulin in each condition; N = 2 independent experiments; ns – not significant. D . Western blot showing the levels of β 1 integrin, acetylated tubulin and GAPDH in astrocytes transfected with control siCtl or siβ 1 integrin. E . Western blot showing the levels of acetylated tubulin and GAPDH in DMSO Ctl or Y-27632 treated (2 h) astrocytes, 6h after wound healing of the monolayer. F . Volcano plot analysis identifying interactors of αTAT1 in HEK cells. Binding partners were obtained by using quantitative label-free mass spectrometry analysis performed from four replicates. Shown are the fold changes (GFP-αTAT1/GFP-Ctl) of the quantified proteins with thresholds of 3 or more peptides, minimum absolute fold change of 1.5 (green lines) and maximum adjusted p-value of 0.05 (red line). Enriched protein interactors related to GO:0005925 focal adhesion (Ratio = 1.98 and p = 7.89 × 10 −5 ) are shown (red). External plots show proteins with peptides identified only in one sample type (left in GFP-Ctl and right in GFP-αTAT1). The ratio of talin is indicated in the table. G . Epifluorescence images showing GFP-αTAT1 localisation on microtubules in astrocytes (as previously described in ). Scale bar (B, C, G): 10 μm.

Article Snippet: Movies were acquired with a Zeiss Axiovert 200M or a Nikon Eclipse Ti-E epifluorescence inverted microscope with cells maintained at 5% CO 2 and 37°C.

Techniques: Western Blot, Histone Deacetylase Assay, Produced, Transfection, Staining, Binding Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: Microtubules tune mechanosensitive cell responses

doi: 10.1101/2020.07.22.205203

Figure Lengend Snippet: A . Inverted-contrast epifluorescence images of astrocytes plated on polyacrylamide gels of different rigidities (1.26 kPa, 48 kPa), stained with acetylated tubulin and α-tubulin. Graph shows the ratio of the intensities of acetylated tubulin over total tubulin intensity of each cell; n > 67 cells, N = 4 independent experiments; one-way ANOVA followed by Tukey’s multiple comparison’s test. B . Astrocytes were treated with RGD control or RGD peptides (upper panels) prior to wounding or transfected with siCtl or siβ 1 integrin (lower panels), allowed to migrate for 8 h. Images show migrating astrocytes stained with acetylated tubulin, α-tubulin and DAPI. Graph shows the ratio of the intensities of acetylated tubulin over total tubulin of each cell; n ≥ 100 cells, N = 3 independent experiments; one-way ANOVA followed by Tukey’s multiple comparison’s test. C . Inverted-contrast epifluorescence images of astrocytes, treated with DMSO (Ctl) or ROCK inhibitor Y-27632 for 2 h, and stained with acetylated tubulin and α-tubulin. Graph shows the ratio of the intensities of acetylated tubulin over total tubulin intensity of each cell; n ≥ 80 cells, N = 3 independent experiments; Student’s t-test. D . Inverted-contrast epifluorescence images of migrating astrocytes transfected with siCtl or siαTAT1 and treated with or without Tubacin prior to wounding. Representative images from N > 3 independent experiments are shown. E . Table shows the mass spectrometry data obtained on Talin-1 following the analysis of αTAT1 interactors. F . Immunoprecipitations using anti-GFP nanobodies were performed with lysates from astrocytes transfected with GFP-Ctl or GFP-αTAT1. Samples were analysed by immunoblotting using talin and GFP antibodies. Representative blot from N = 3 independent experiments is shown. G . TIRF images of GFP-αTAT1 and epifluorescence images of mCherry-vinculin expressing astrocytes before and after nocodazole or Y27 treatment. Insets represent regions of the cell where αTAT1 and vinculin colocalise. Representative images from N = 3 independent experiments. H . Pulldowns using GST-αTAT1 resin were performed with lysates from astrocytes treated with tubacin, nocodazole or Y27632, and analysed by red Ponceau staining and immunoblotting using talin. Representative blots from N = 3 independent experiments are shown. Scale bar (A-D): 10 μm, (G): 20 μm; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Movies were acquired with a Zeiss Axiovert 200M or a Nikon Eclipse Ti-E epifluorescence inverted microscope with cells maintained at 5% CO 2 and 37°C.

Techniques: Staining, Transfection, Mass Spectrometry, Western Blot, Expressing

Journal: bioRxiv

Article Title: Microtubules tune mechanosensitive cell responses

doi: 10.1101/2020.07.22.205203

Figure Lengend Snippet: A . Inverted epifluorescence images of astrocytes plated on polyacrylamide gels of different rigidities (1.26 kPa and 48 kPa), stained with α-tubulin and talin. Images shown correspond to the same cells depicted in . Talin images were segmented by adjusting the threshold to detect FAs. Histogram shows the mean ± SEM of FA density (number of FAs/μm 2 ) in different regions of the cells plated on substrate of indicated rigidities; n = 40 for 1.26 kPa, 51 for 2 kPa, 94 for 9 kPa, 77 for 48 kPa, N = 3 independent experiments; two-way ANOVA followed by Tukey’s multiple comparison’s test. B . Inverted epifluorescence images of astrocytes transfected with siCtl and siαTAT1, plated on different substrate rigidities and stained with paxillin. C . Inverted epifluorescence images of astrocytes treated with with Niltubacin and tubacin, plated on different substrate rigidities and stained with paxillin. D . Histogram shows the mean ± SEM of FA density (number of FAs/μm 2 ) in the central region (16μm-cell center) of cells depicted in B and C; n = 60 for 1.26 kPa siCtl, 36 for 1.26 kPa siαTAT1, 54 for 48 kPa siLuc and 47 for 48 kPa siαTAT1; n = 59 for 1.26 kPa Niltubacin, 55 for 1.26 kPa tubacin, 46 for 48 kPa Niltubacin and 37 for 48 kPa tubacin; N = 3 independent experiments; one-way ANOVA followed by Tukey’s multiple comparison’s test. E . Schematic showing the effects of microtubule acetylation on the distribution of FAs in cells plated on polyacrylamide gels of different rigidities. Scale bar (A-C): 10 μm; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Movies were acquired with a Zeiss Axiovert 200M or a Nikon Eclipse Ti-E epifluorescence inverted microscope with cells maintained at 5% CO 2 and 37°C.

Techniques: Inverted Epifluorescence, Staining, Transfection

Journal: bioRxiv

Article Title: Microtubules tune mechanosensitive cell responses

doi: 10.1101/2020.07.22.205203

Figure Lengend Snippet: A . Schematic representation of the different cell regions used to quantify FA density . B . Histograms show mean ± SEM of FA density (number of FAs/μm 2 ) in different regions of astrocytes transfected with siCtl or siαTAT1 or treated with Niltubacin or Tubacin, and plated on 1.26kPa or 48 kPa substrates; N = 3 independent experiments. C . Inverted epifluorescence images of siCtl or siαTAT1-transfected astrocytes plated on crossbow-shaped micropatterned polyacrylamide gels of 40 kPa or 2 kPa, stained with paxillin and tubulin. Representative images from N = 3 independent experiments are shown. Scale bar: 10 μm D . Graph shows the percentage of siCtl or siαTAT1-transfected astrocytes with transverse interjunctional actin arcs; n ≥ 159 cells, N = 3 independent experiments; Student’s t-test; ****p<0.0001.

Article Snippet: Movies were acquired with a Zeiss Axiovert 200M or a Nikon Eclipse Ti-E epifluorescence inverted microscope with cells maintained at 5% CO 2 and 37°C.

Techniques: Transfection, Inverted Epifluorescence, Staining

Journal: bioRxiv

Article Title: Microtubules tune mechanosensitive cell responses

doi: 10.1101/2020.07.22.205203

Figure Lengend Snippet: A-C Inverted epifluorescence images of astrocytes transfected with siCtl or siαTAT1 and stained with ( A ) phalloidin, pMLC and paxillin; ( B ) myosin IIa, acetylated tubulin and α-tubulin; and ( C ) vimentin and α-tubulin. Images are representative of N = 3 independent experiments. Scale bar (A-C): 20 μm. D . Ultrastructural organization of focal adhesion-associated cytoskeleton in siCtl or siαTAT1 depleted cells. (1) Platinum replica electron microscopy (PREM) survey view of the cytoplasmic surface of the leading edge in siCtl unroofed cells. Boxed regions correspond to focal adhesions. Extracellular space is pseudo-coloured in purple. (i, ii) High magnification views corresponding to the boxed regions in panel 1. White arrows indicate microtubules and yellow arrowheads denote intermediate filaments. (iii) Zoom-in region corresponding to the boxed region in marked in region ii. Scale bar: 1 µm. White arrows indicate microtubules and yellow arrowheads denote intermediate filaments. Scale bar: 2 µm and 1 µm. (2, 3) PREM survey view of the cytoplasmic surface of the leading edge in αTAT1-depleted cells. Extracellular space is pseudo-coloured in purple. Scale bars: 10 µm, 1 µm (inset). (iv, v) High magnification views corresponding to the boxed regions in panel 2 and 3 respectively. Scale bar: 1 µm.

Article Snippet: Movies were acquired with a Zeiss Axiovert 200M or a Nikon Eclipse Ti-E epifluorescence inverted microscope with cells maintained at 5% CO 2 and 37°C.

Techniques: Inverted Epifluorescence, Transfection, Staining, Electron Microscopy

Journal: bioRxiv

Article Title: Microtubules tune mechanosensitive cell responses

doi: 10.1101/2020.07.22.205203

Figure Lengend Snippet: A . Stress-field maps of astrocytes transfected with siCtl, siαTAT1, siαTAT1 + GFP-αTAT1, treated with Niltubacin or Tubacin, and plated on crossbow-shaped micropatterned polyacrylamide gels of 40 kPa or 2 kPa. B, C . Corresponding stored energies (in Joules, J) of cells in each of the above mentioned conditions. Values represent mean ± SEM stored energies of cells within the range of 0 to 5 × 10 −13 J; n ≥ 142 cells for siCtl and siαTAT1, n ≥ 71 cells for siCtl + GFP-αTAT1 and siαTAT1 + GFP-αTAT1, n ≥ 137 cells for Niltubacin and tubacin, N = 3 independent experiments; One-way ANOVA followed by Tukey’s multiple comparison’s test (graph B) or Student’s t-test (graph C). D . GST-Rhotekin pulldowns were performed using siCtl or siαTAT1-transfected astrocytes. Red Ponceau and anti-RhoA western blot analysis of lysates. Representative blot from N = 3 independent experiments is shown. E . Inverted epifluorescence images of migrating astrocytes transfected with siCtl, siαTAT1 and siαTAT1 treated with tubacin, stained with acetylated tubulin, α-tubulin, GEF-H1. Graph represents mean ± SEM of the percentage of GEF-H1 colocalised with microtubules; n ≥ 73 cells; N = 3 independent experiments; One-way ANOVA followed by Tukey’s multiple comparison’s test or Student’s t-test. Scale bar (A): 10 μm, (E) 20 μm; **p<0.01,b ***p<0.001, ns – not significant.

Article Snippet: Movies were acquired with a Zeiss Axiovert 200M or a Nikon Eclipse Ti-E epifluorescence inverted microscope with cells maintained at 5% CO 2 and 37°C.

Techniques: Transfection, Western Blot, Inverted Epifluorescence, Staining