u2os wild type cells  (DSMZ)

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