u2os wild type wt cells  (ATCC)

Journal: The Journal of Cell Biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: A subset of microtubules has an expanded lattice in cells. (A) Tomogram slice (thickness: 10 nm) showing two selected microtubule (MT) backbones in an untreated U2OS cell (red and pink). (B and C) Power spectra of the masked and transformed MT segments from the red MT (B) and pink MT (C) shown in A. (D) Overlay of the layer line plots of the power spectra of the MT segments from the compacted (red) and expanded (pink) MTs in A. Arrows indicate the location of the layer line peaks and their related lattice spacing. (E) Violin plot showing the distribution of lattice spacings in untreated U2OS cells ( N = 31, 12 tomograms, 7 cells), from microtubules assembled in vitro from GTP-bound soluble tubulin yielding dynamic microtubules ( N = 40, 6 tomograms), in the presence of Taxol ( N = 32, 3 tomograms), or from GMPCPP-bound soluble tubulin ( N = 33, 14 tomograms). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. (F) Simplified cartoon showing the long-range effect of a compacted or an expanded MT lattice. Scale bar: 100 nm (A).

Article Snippet: U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Zurich, Zürich, Switzerland).

Techniques: Transformation Assay, In Vitro

Journal: The Journal of Cell Biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Taxol treatment induces a hyperexpanded lattice within cells. (A) Tomogram slice showing a representative image of Taxol-treated microtubules in WT U2OS cells. Scale bar: 100 nm. (B) Violin plot showing the lattice spacing distribution in Taxol treated cells ( N = 30, 6 tomograms, 5 cells) and in untreated cells ( N = 31, 12 tomograms, 7 cells, same data as , included for comparison). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. Taxol distribution is significantly different from the untreated distribution (****P value <0.0001, unpaired t test based permutation test). (C) Microtubule average shows that Taxol-treated microtubules consist of 13 PFs. Central volume slices (28 nm thick) from top (left) and side (right) views. Scale bar: 5 nm.

Article Snippet: U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Zurich, Zürich, Switzerland).

Techniques: Comparison

Journal: The Journal of Cell Biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Correlation of FM to SEM data using an integrated cryo-FM. (A) Cartoon describing the FM-SEM correlation with FM data obtained after milling. Correlation is confirmed using extracellular beads. (B) FIB image of an intact U2OS cell (9° tilted side view). (C) Untilted SEM image of the same grid square as shown in B. The beads used to confirm FM-SEM correlation are indicated with white and black arrows in C and D, respectively. (D) Untilted SEM image of the polished lamella of the cell shown in C. (E) Scatterplot of correlation errors from leave-one-out calculations; each dataset has a unique color, grey circles mark the 1xSD and 2xSD boundaries (10 datasets, 51 beads), dx = difference in x, dy = difference in y. (F) Boxplot showing the distribution of scaling factors (mean = 0.584, standard deviation = 0.005, N = 10). (G) Scaled FM image of the extracellular beads used to guide FM-SEM overlay, beads used to confirm FM-SEM correlation are indicated with black arrows, similar to C and D. (H) Scaled FM image of fBSA-Au 5 beads used for subsequent FM-TEM correlation (see ). (I) Scaled FM image of the StableMARK signal. Scalebars: 10 µm (B–D and G–I).

Article Snippet: U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Zurich, Zürich, Switzerland).

Techniques: Standard Deviation

Journal: The Journal of cell biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices.

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Figure 1. A subset of microtubules has an expanded lattice in cells. (A) Tomogram slice (thickness: 10 nm) showing two selected microtubule (MT) backbones in an untreated U2OS cell (red and pink). (B and C) Power spectra of the masked and transformed MT segments from the red MT (B) and pink MT (C) shown in A. (D) Overlay of the layer line plots of the power spectra of the MT segments from the compacted (red) and expanded (pink) MTs in A. Arrows indicate the location of the layer line peaks and their related lattice spacing. (E) Violin plot showing the distribution of lattice spacings in untreated U2OS cells (N = 31, 12 tomograms, 7 cells), from microtubules assembled in vitro from GTP-bound soluble tubulin yielding dynamic microtubules (N = 40, 6 tomograms), in the presence of Taxol (N = 32, 3 tomograms), or from GMPCPP-bound soluble tubulin (N = 33, 14 tomograms). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. (F) Simplified cartoon showing the long-range effect of a compacted or an expanded MT lattice. Scale bar: 100 nm (A).

Article Snippet: Cell lines and cell culture U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Figure 4.

Techniques: Transformation Assay, In Vitro

Journal: The Journal of cell biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices.

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Figure 2. Taxol treatment induces a hyperexpanded lattice within cells. (A) Tomogram slice showing a representative image of Taxol-treated micro- tubules in WT U2OS cells. Scale bar: 100 nm. (B) Violin plot showing the lattice spacing distribution in Taxol treated cells (N = 30, 6 tomograms, 5 cells) and in untreated cells (N = 31, 12 tomograms, 7 cells, same data as Fig. 1 E, included for comparison). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. Taxol distribution is significantly different from the untreated distribution (****P value <0.0001, unpaired t test based permutation test). (C) Microtubule average shows that Taxol-treated microtubules consist of 13 PFs. Central volume slices (28 nm thick) from top (left) and side (right) views. Scale bar: 5 nm.

Article Snippet: Cell lines and cell culture U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Figure 4.

Techniques: Comparison

Journal: The Journal of cell biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices.

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Figure 3. Correlation of FM to SEM data using an integrated cryo-FM. (A) Cartoon describing the FM-SEM correlation with FM data obtained after milling. Correlation is confirmed using extracellular beads. (B) FIB image of an intact U2OS cell (9° tilted side view). (C) Untilted SEM image of the same grid square as shown in B. The beads used to confirm FM-SEM correlation are indicated with white and black arrows in C and D, respectively. (D) Untilted SEM image of the polished lamella of the cell shown in C. (E) Scatterplot of correlation errors from leave-one-out calculations; each dataset has a unique color, grey circles mark the 1xSD and 2xSD boundaries (10 datasets, 51 beads), dx = difference in x, dy = difference in y. (F) Boxplot showing the distribution of scaling factors (mean = 0.584, standard deviation = 0.005, N = 10). (G) Scaled FM image of the extracellular beads used to guide FM-SEM overlay, beads used to confirm FM-SEM correlation are indicated with black arrows, similar to C and D. (H) Scaled FM image of fBSA-Au5 beads used for subsequent FM-TEM correlation (see Fig. 4). (I) Scaled FM image of the StableMARK signal. Scalebars: 10 µm (B–D and G–I).

Article Snippet: Cell lines and cell culture U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Figure 4.

Techniques: Standard Deviation

Journal: Journal of Cell Science

Article Title: Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors

doi: 10.1242/jcs.234435

Figure Lengend Snippet: Distinct actin structures assemble in response to two stimuli: increased cytoplasmic calcium and mitochondrial depolarization. (A) Time-lapse image montage of ionomycin-induced (top) and CCCP-induced (bottom) actin polymerization for U2OS cells transfected with GFP–F-tractin (green) and mito–BFP (red). Imaging conducted at the basal cell surface. Ionomycin or CCCP added at time point 0. Scale bars: 10 μm (insets 5 μm). Corresponds to Movies 1 and 2. Movies 3 and 4 show similar time course in medial cell section. Orange arrows indicate actin assembly. (B) Comparison of ionomycin-induced and CCCP-induced actin polymerization time course for U2OS cells (15 s intervals); N=30 cells/60 ROIs for ionomycin (4 μM) treatment, 27 cells/27 ROIs for CCCP (20 μM) treatment. (C) CCCP-induced actin polymerization in U2OS-WT and U2OS-INF2-KO cells (14 s intervals); N=35 cells for WT, 35 cells for INF2-KO and 35 cells for WT cells stimulated with DMSO. (D) Effect of Arp2/3 complex inhibition on CCCP-induced actin polymerization (15 s intervals). U2OS cells were treated with either DMSO or 100 μM CK666 for 30 min and then stimulated with 20 μM CCCP; N=35 cells/35 ROIs for DMSO/CCCP, 41/41 for CK666/CCCP. (E) Effect of Arp2/3 complex inhibition on ionomycin-induced actin polymerization (15 s intervals). U2OS cells were treated with either DMSO or 100 μM CK666 for 30 min and then stimulated with DMSO or 4 μM ionomycin; N=23 cells/46 ROI for DMSO/ionomycin, 25/50 for CK666/ionomycin and 20/40 for DMSO/DMSO. All data (mean±s.e.m.) are from three experiments. Blue arrows denote drug addition.

Article Snippet: Wild-type U2OS (U2OS-WT) cells were procured from American Type Culture Collection (ATCC) and INF2-KO human osteosarcoma U2OS cells were prepared in our laboratory using CRISPR-Cas9 and are described elsewhere ( Chakrabarti et al., 2018 ).

Techniques: Transfection, Imaging, Comparison, Inhibition

Journal: Journal of Cell Science

Article Title: Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors

doi: 10.1242/jcs.234435

Figure Lengend Snippet: Actin polymerization accompanying transient depolarization of a subset of mitochondria in the absence of CCCP. (A) Time-lapse image montage of changes in mitochondrial polarization (TMRE, blue) and actin polymerization (GFP–F-tractin, green) in U2OS cells in the absence of uncoupler treatment. A mitochondrial matrix marker (mito–BFP, red) is also included. Top panels show a control cell (DMSO) and bottom panels show a CK666-treated cell (100 μM CK666 for 30 min before imaging and 50 μM during imaging). Time 0 denotes start of a depolarization event. Panels to the right of each time point denote zooms of boxed regions. Scale bars: 10 μm (insets 5 μm). Corresponds to Movies 5 and 6. (B) Number of spontaneous depolarization events associated with actin polymerization in either control (DMSO) or CK666-treated cells. Total numbers from three experiments (20 min imaging per cell, 1.2 s intervals): DMSO, 213 cells, 47 depolarization events (41 events accompanied by actin assembly); CK666, 217 cells, 81 depolarization events (8 events accompanied by actin assembly). (C) Percentage of depolarization events accompanied by actin polymerization for DMSO versus CK666 treatment, from same data as in B. Student's unpaired t-test, **P=0.0003. (D) Depolarization frequency (mean±s.e.m.) for DMSO versus CK666 treatment, from data set described in B. Student's unpaired t-test, *P=0.0484. (E) Scatter plot of depolarization duration, from data set described in B. Depolarization durations (mean±s.d.) are given for DMSO group (black line), 143.7 s±106.5 s and CK666 group (red line), 118.5 s±104.4 s. Gray and pink points represent depolarization events that occurred after 16 min of imaging time and failed to repolarize at the end of the 20 min imaging period. Student's unpaired t-test, P=0.1949; n.s., not significant.

Article Snippet: Wild-type U2OS (U2OS-WT) cells were procured from American Type Culture Collection (ATCC) and INF2-KO human osteosarcoma U2OS cells were prepared in our laboratory using CRISPR-Cas9 and are described elsewhere ( Chakrabarti et al., 2018 ).

Techniques: Marker, Control, Imaging

Journal: Journal of Cell Science

Article Title: Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors

doi: 10.1242/jcs.234435

Figure Lengend Snippet: Depolarization-induced mitochondrial shape changes in U2OS cells. (A) Examples of mitochondrial matrix dynamics after depolarization, leading to circularization. Three examples show matrix rearrangement and one example shows the mitochondrion curling back on itself to form the circle. U2OS cells were transfected with the mitochondrial matrix marker Mito–DsRed and treated with 20 μM CCCP at time point 0. Confocal imaging was conducted at the basal region of the cell at 4 s intervals, starting 10 frames before CCCP treatment. Corresponds to Movie 7. Orange arrows indicate mitochondrial circularization. (B) Example of mitochondrial circularization after transient depolarization in the absence of CCCP. Time indicates the time after depolarization. Time-lapse images taken at the basal region of the cell. (C) Quantification of depolarization time for depolarization events, with events resulting in mitochondrial circularization in green. Triangles indicate events in which no re-polarization was observed at the end of the imaging period (20 min). Red line indicates depolarization duration (mean±s.d.) of 310.2 s±308.1 s. Total numbers from two experiments, 144 cells and 41 depolarization events. (D) Dynamics of the OMM and mitochondrial matrix upon depolarization. U2OS cell transfected with Mito–DsRed (red) and Tom20–GFP (green) was treated with 20 μM CCCP at time point 0. Airyscan images (basal region) were acquired at 4 s intervals starting 10 frames before CCCP treatment. Corresponds to Movie 8. (E) Maximum intensity projections of glutaraldehyde-fixed U2OS cells (transfected with mito–GFP, green) after treatment with either DMSO (top) or 20 μM CCCP (bottom) for 20 min. Cells were stained with anti-Tom20 (OMM, red) and DAPI (nucleus, blue). Z-stacks were taken at step size of 0.4 μm. Zoomed images show representative examples of mitochondrial circularization after CCCP treatment. Scale bars: 2 μm in A; 2.5 μm in B,D; 10 μm in E (insets, 2 μm).

Article Snippet: Wild-type U2OS (U2OS-WT) cells were procured from American Type Culture Collection (ATCC) and INF2-KO human osteosarcoma U2OS cells were prepared in our laboratory using CRISPR-Cas9 and are described elsewhere ( Chakrabarti et al., 2018 ).

Techniques: Transfection, Marker, Imaging, Staining

Journal: Journal of Cell Science

Article Title: Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors

doi: 10.1242/jcs.234435

Figure Lengend Snippet: Effect of LatA and Arp2/3 complex inhibition on depolarization-induced mitochondrial shape changes. (A) Effect of LatA on CCCP-induced actin polymerization. U2OS cells were treated with combinations of LatA (500 nM) and CCCP (20 μM) at time 0 (blue arrow). Confocal images (medial section) were acquired at 15 s intervals starting four frames before treatment. Data (mean±s.e.m.) are from three experiments; N=23 cells/23 ROIs for LatA/DMSO, 38/38 for LatA/CCCP and 31/31 for DMSO/CCCP. (B) Effect of LatA on CCCP-induced mitochondrial matrix circularization (‘centroids’). U2OS cells were transfected with mito–BFP and GFP–F-tractin. CCCP (20 μM) was added at time point 0, simultaneously with LatA addition (500 nM). Data (mean±s.e.m.) are from three experiments; N=22 cells/4354 μm2 total mitochondrial area for DMSO/CCCP control cells; 18 cells/3823 μm2 for LatA/CCCP. Mean±s.e.m. P-value for time points 4–18 min post-treatment was 0.0115±0.0119 for DMSO versus LatA. (C) Time-course montage of LatA-treated cells under control conditions (DMSO treatment, top) or CCCP treatment (20 μM, bottom). U2OS cells transfected with F-tractin (not shown) and mito–BFP (green) were treated with 500 nM LatA and CCCP or DMSO simultaneously at 0 min. Confocal images (basal section) acquired at 15 s intervals starting four frames before treatment. Orange arrows denote centroids (circular mitochondrial matrix). (D) Time-course montage of CCCP-induced mitochondrial matrix circularization in the absence (top) or presence (bottom) of CK666 pretreatment. U2OS cells transfected with GFP–F-tractin (not shown) and mito–BFP (green) were treated with DMSO or CK666 (100 μM) for 30 min before stimulation with 20 μM CCCP at time point 0 (blue arrow). Confocal images (basal section) were acquired at 15 s intervals starting four frames before CCCP treatment. Orange arrows denote centroids (circular mitochondrial matrix). Corresponds to Movie 9. (E) Change in mitochondrial matrix circularization (defined as centroids per total mitochondrial area in the region of interest) from time-courses as described in A. Data (mean±s.e.m.) from three experiments. The three conditions tested were CK666 pretreatment followed by CCCP stimulation (N=46 cells/8539 μm2 total mitochondrial area), DMSO pretreatment, followed by CCCP stimulation (58 cells/11473 total mitochondrial area) and CK666 pretreatment followed by DMSO stimulation (36 cells/6655 total mitochondrial area). Mean±s.e.m. P-value for time points 4–18 min post-treatment was 0.00141±0.00217 for CK666/CCCP versus DMSO/CCCP. Scale bars: 10 μm in C; 10 μm in D (insets 2.5 μm).

Article Snippet: Wild-type U2OS (U2OS-WT) cells were procured from American Type Culture Collection (ATCC) and INF2-KO human osteosarcoma U2OS cells were prepared in our laboratory using CRISPR-Cas9 and are described elsewhere ( Chakrabarti et al., 2018 ).

Techniques: Inhibition, Transfection, Control

Journal: Journal of Cell Science

Article Title: Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors

doi: 10.1242/jcs.234435

Figure Lengend Snippet: Role of OMA1 in depolarization-induced mitochondrial shape change. (A) Western blots of OPA1 and OMA1 in control, OMA1-KD and Yme1-KD U2OS cells before and after treatment with 20 μM CCCP for 30 min. The positions of the five OPA1 forms (L1, L2, S1–S3) are indicated. GAPDH was INF2 loading control. (B) Western blots of OPA1 in control and OMA1-KD U2OS cells during time course of CCCP treatment (20 μM). Tubulin was loading control. (C) Western blot of OPA1 in control and CK666-treated U2OS-WT cells during the time course of CCCP treatment (20 μM). GAPDH was loading control. (D) CCCP-induced actin polymerization for control and OMA1-KD U2OS cells, transfected with GFP–F-tractin and mito–BFP, and stimulated with 20 μM CCCP (blue arrow). Data (mean±s.e.m.) from three experiments; N=40 cells/40 ROIs for scrambled control, 28/28 for OMA1-KD. (E) Change in mitochondrial matrix circularization (‘centroids’) over time for control and OMA1-KD U2OS cells transfected with mito–BFP and GFP–F-tractin. CCCP (20 μM) added at time point 0. Data (mean±s.e.m.) from three experiments; N=36 cells/4733 μm2 total mitochondrial area for control cells; 53 cells/9519 μm2 for OMA1-KD cells. Mean±s.e.m. P-value for time points 4–20 min post-treatment was 0.000185±0.000171 for control versus OMA1-KD. (F) Change in mitochondrial matrix circularization upon CCCP treatment for control-KD and OMA1-KD U2OS cells in the presence or absence of CK666. Data (mean±s.e.m.) from three experiments; N=87 cells/11,778.29 μm2 total mitochondrial area for control-KD/CK666 cells; 28 cells/4010.9 μm2 for control-KD cells; 81 cells/10,691.47 μm2 for OMA1-KD/CK666 cells; 28 cells/3126.79 μm2 for OMA1-KD cells. Mean±s.e.m. P-value for time points 4–20 min post-treatment was 0.00590±0.0103 for control KD versus control KD/CK666, and 0.256±0.229 for OMA1-KD versus OMA1-KD/CK666. (G) Time-course montage of CCCP-induced mitochondrial shape change (orange arrows) in control (green) and OMA1-KD U2OS cells (red), imaged in the same field. Control cells (scrambled siRNA) were transfected with mito–GFP whereas OMA1-KD cells were transfected with Mito–DsRed. The two cell populations were trypsinized, mixed and plated 24 h before imaging. Confocal images (basal cell section) were acquired at 15 s intervals starting four frames before CCCP treatment. CCCP (20 μM) was added at time point 0. Corresponds to Movie 11. Scale bar: 10 μm in G (insets 2.5 μm).

Article Snippet: Wild-type U2OS (U2OS-WT) cells were procured from American Type Culture Collection (ATCC) and INF2-KO human osteosarcoma U2OS cells were prepared in our laboratory using CRISPR-Cas9 and are described elsewhere ( Chakrabarti et al., 2018 ).

Techniques: Western Blot, Control, Transfection, Imaging