Data for: SUN2 regulates mitotic progression in response to extracellular matrix rigidity
Guilluy, Christophe; Belaadi, Nejma (2022), Data for: SUN2 regulates mitotic progression in response to extracellular matrix rigidity, Dryad, Dataset, https://doi.org/10.5061/dryad.ns1rn8pvv
How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here we examined how ECM rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrate. While we observed no effect of ECM rigidity on rounding or post-mitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the LINC complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation. Whereas when cells adhere on soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.
Raw data corresponding to Figure 3A and 3B (quantifications are provided as Data and Videos as Supplemental Information)
Quantification of the duration of mitosis phases in HeLa Kyoto EGFP-LaminA/H2B-mCherry cells transfected with control siRNA or siRNA targetting SUN2. Mitosis duration was calculated as the time from the first image showing the Nuclear Envelope Breakdown (NEBD) to the first image showing nuclear envelope reformation. Prometaphase duration was calculated as the time from the first image showing NEBD to the last image showing all chromosomes congression at the metaphase plate. Metaphase was calculated as the time from chromosomes congression at the metaphase plate to the image just before chromosomes segregation. Anaphase was calculated as the time from the first image showing chromosomes segregation to the first image showing nuclear envelope reformation. Data were collected from 3 independent experiments (biological replicates). Table provides: biological replicate (n=1 to 3), condition name (control or SUN2 siRNA), first frame number and last frame number for each phase, number of frames for each phase and duration (min) of each phase, calculated as number of frames * 2.5 (2.5=frame rate, as one frame every 2.5 min)
Live imaging was performed using spinning disk confocal microscope Andromeda TILL-FEI fitted with a Plan-Apochromat 20x/0.75, WD 610 objective and coupled to a EMCCD camera iXon 897. The microscope was equipped with a temperature- and CO2-controlled incubator box (37°C and 5% CO2) and images were taken every 2.5 minutes for 15 hours minimum. Acquisition was performed with LA, FEI software and analysis with OA, FEI software and with ImageJ. 4 avi videos: 2 control siRNA and 2 SUN2 siRNA videos are provided as Supplemental Information.
Raw data corresponding to Figure 2B (western blot as Supplemental Information).
A7R5 were cultured on 1 or 50 kPa matrix for 6h, 24h and 5 days. Cells were lysed in Laemli and processed for western blot (SUN2 and GAPDH) to compare SUN2 expression. Images of uncropped gels for 2 replicates are provided (Western_Blot_1 and Western_Blot_2) for both SUN2 and GAPDH as Supplemental Information.
H2020 European Research Council