The effect of the actin cytoskeleton on mitosis after culture on 2D square patterned substrates.

<p>(A) HeLa cells (YFP-paxillin) were cultured on 2D square patterns for 18 hours and imaged during interphase for actin (red) and paxillin (green). HeLa cells (RFP-tubulin/GFP-H2B) were assessed for the angle of the mitotic spindle in the xy plane at metaphase after culture on 2D square patterns and treatment with (B) media or (C) latrunculin A. A high proportion of the cells cultured on square patterns aligned the mitotic spindle along the diagonal of the cell, which was reduced by the addition of latrunculin A. (D) Conversely the addition of latrunculin A had little effect on the average orientation of the mitotic spindle perpendicular to the substrate plane. Values represent spindle angle in degrees ± SEM. (E) Furthermore, the addition of latrunculin A had little effect on the distribution of the spindle orientation. (F) Cells treated with latrunculin A (light grey bars) took significantly less time to progress from late prometaphase to metaphase and to complete mitosis than untreated cells (black bars). All values represent time in minutes ± SEM. Key: *** p<0.001; NB  =  nuclear envelope breakdown, S =  spindle formation, LPM  =  late prometaphase, M =  metaphase, A =  anaphase.</p

The effect of cell shape on the cells' progression through mitosis.

<p>HeLa cells (RFP-tubulin/GFP-H2B) were synchronized and cultured on 2D homogenously coated substrates (black bars) or within square (light grey bars) or circular (dark grey bars) microwells and assessed for the time required to progress through each stage in mitosis. Cells cultured in circular microwells took longer to progress through mitosis and specifically to reach late prometaphase, than cells cultured in square microwells or on 2D substrates. All values represent time in minutes ± SEM. Key: * p<0.05, ** p<0.01; NB  =  nuclear envelope breakdown, LPM  =  late prometaphase, M =  metaphase, A =  anaphase, C =  cytokinesis.</p

Control of 3D cell shape using a microwell cell culture platform.

<p>(A–B) HeLa cells (RFP-tubulin/GFP-H2B) were synchronized and cultured in square (A) or circular (B) microwells for 18 hours and imaged for nucleus (green) and cell outline (Vybrant DiD cell labeling solution, red). Images show the central xy slice of the cell (top) and the corresponding xz slice of the same cell (bottom); bars: 10 µm.</p

Analysis of mitotic timings in single cells cultured on different cell culture platforms.

<p>Analysis of mitotic timings in single cells cultured on different cell culture platforms.</p

The role of the actin cytoskeleton in the shape dependent differences in mitosis.

<p>HeLa (YFP-paxillin) cells were cultured for 18 hours in either (A) square microwells or (B) circular microwells and imaged during interphase for actin (red) and paxillin (green); bars: 10 µm. HeLa (GFP-H2B/RFP-tubulin) cells were assessed for the parallel orientation of the mitotic spindle at metaphase after culture in (C) square or (D) circular microwells and treatment with latrunculin A for 1 hour prior to mitosis. Cells cultured in circular microwells showed a random orientation of the mitotic spindle, whilst cells cultured in square microwells predominately aligned the spindle along the long axis of the cell. (E) The inhibition of actin polymerization in HeLa (GFP-H2B/RFP-tubulin) cells lead to increased tilting in cells cultured in the square microwells. Values represent spindle angle in degrees ± SEM. (F) Perturbation of actin polymerization in cells cultured in square microwells also increased the distribution of spindle orientation, but had little effect on cells cultured in circular microwells. (G) The perturbation of the actin cytoskeleton negated the differences in mitotic timings observed between cells cultured in square microwells (light grey bars) and cells cultured in circular microwells (dark grey bars). All values represent time in minutes ± SEM. NB  =  nuclear envelope breakdown, S =  spindle formation, LPM  =  late prometaphase, M =  metaphase, A =  anaphase.</p

FRET interactions between CENP-O class proteins.

<p>The FRET pair EGFP-mCherry is used. “F” indicates that for these fusions FRET was detected also by FLIM. ++: strong FRET, +: weak FRET, −: no FRET.</p

CENP-O loading to kinetochores measured by the SNAP-tag technology.

<p>(A) Top: schematic representation of the performed experiment. Below: representative images of cells showing TMR-star fluorescence for SNAP-CENP-O in G2, M-phase and the following G1. Cell cycle phases G2 (CENP-F staining of the whole nucleus) and mitosis (specific kinetochore binding of CENP-F) are clearly identified. (B) The same experiment as in (A) was performed with U2OS cells stably expressing PCNA-GFP. SNAP-CENP-O fluorescence appears at kinetochores in late S-phase as judged from cellular PCNA distributions. (C) Top: schematic representation of the performed experiment. Below: representative images of cells expressing SNAP-CENP-O showing no fluorescence at kinetochores during G1. CENP-O is thus loaded to kinetochores in S-phase.</p

Acceptor-bleaching FRET of the protein pairs EGFP-CENP-U/CENP-R-mCherry (FRET signal) and EGFP-CENP-Q/CENP-P-mCherry (no FRET signal).

<p>Typical HEp-2 cell nuclei are displayed in (A) and (D) showing centromere location of all four CEN proteins. Two of these locations, spot 1 and spot 2 in each of the two graphs, were selected for fluorescence intensity analysis before and after acceptor bleaching (see enlargements below). Spot 1 served as control and showed no detectable intensity change. At spot 2, the acceptor fluorophore mCherry was bleached (compare pre-bleach and post-bleach in (A) and (D)). In (B) and (E), the time course of the fluorescence intensity of the donor and the acceptor of both FRET pairs are shown. The acceptor intensities in the ROI (“region of interest”; open squares) were averaged and normalized to the mean intensity measured at the three time points before bleaching. The donor intensities in the ROI were averaged and normalized to the intensity measured at the time point after bleaching. Bleaching of the acceptor resulted in a fluorescence intensity increase of the donor (black dots) for EGFP-CENP-U (B) indicating the presence of FRET (see arrow), but no fluorescence intensity increase for EGFP-CENP-Q (E) indicating the absence of FRET. (C) and (F): Donor fluorescence intensity variation observed during acceptor bleaching normalized to the intensity measured at the first time point after bleaching. Control: spot 1 (acceptor not bleached) yielding E_var (grey bars), FRET measurement: spot 2 (acceptor bleached) yielding E_FRET (black bars). For protein pairs indicated, number of observed single cases (grouped into E_var or E_FRET value ranges of 4%) displayed versus values of E_var or E_FRET. (C) EGFP-CENP-U (donor) and CENP-R-mCherry (acceptor): distribution of E_FRET (40 bleached kinetochores) is clearly distinct from the distribution of E_var (39 non-bleached kinetochores) indicating FRET. (F): EGFP-CENP-Q (donor) and CENP-P-mCherry (acceptor): distribution of E_FRET (52 bleached kinetochores) superimposes the distribution of E_var (51 non-bleached kinetochores) indicating no FRET.</p

Fluorescence recovery after photobleaching of EGFP-CENP-P in mid S-phase.

<p>Normalised mean fluorescence values of 55 kinetochores taken in time steps of 30 min over 4 hours. Recovery levels off, indicative of an about 40% immobile fraction.</p

Cell cycle-dependent FRET between CENP-Q C- (grey bars) and N-termini (white bars).

<p>In late S-phase and G2, significant FRET is observed (p<0.001). In G1, early and mid S-phase, no FRET is observed (p>0.005).</p