Long term observation of mitosis by live-cell microscopy is required for uncovering the role of Cohesin on compartmentalized nuclear architecture which is linked to nuclear functions. To perform long-term observation of mitosis devices are needed that have low phototoxic effects on living cells and enable high speed imaging Cohesin a ring like protein complex with its major subunits RAD 21 SMC 1 and SMC 3 is involved in sister chromatid entrapment to ensure proper chromosome segregation during mitosis, in double strand break repair and gene regulation Its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood.
By using the CSU-W1 confocal scanner unit for time lapse imaging entrance into mitosis, mitotic progression and exit can be examined (Figure 1 It was found that in control cells 80 of all recorded mitoses passed mitosis within 1 h and formed two daughter nuclei (Figure 1a) Whereas in cohesin depleted cells mitotic progress was delayed up to 14 h (median 4.5 h) The delayed mitotic passage was associated with the formation of abnormal, e g multipolar mitotic figures persisting over several hours with subsequent formation of one endomitotic multilobulated nucleus ( MLN)(Figure 1b).
Live cell microscopy demonstrating prolonged abnormal mitosis in cohesin depleted cells
Human colon cancer cells (HCT 116 RAD 21 mAID mClover cells (kindly provided by Kanemaki lab, Shizuoka, Japan)) were plated on poly L Lysine coated glass bottom 2 well imaging slides ibidi allowing to image control and auxin treated conditions in parallel For DNA staining cells were incubated in media containing 500 nM SiR DNA Spirochrome for 1 h before imaging Cohesin was depleted by auxin inducible degron ( The auxin inducible degron ( system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types.
Time lapse imaging of untreated control cells ( auxin)
The following figure shows selected points from time lapse imaging Σ t = 21 h, Δ t = 15 min) of untreated control cells auxin) with the accomplishment of mitosis (M1 with in 1h (time 02:45-03:45 and subsequent formation of two daughter nuclei A second mitosis (M2 of one daughter nucleus is shown at time 20:15. Scale bar 10 μ m.
Time lapse imaging of cohesin degraded cells (+auxin)
Time lapse imaging of cohesin degraded cells (+auxin) conducted in parallel to control cells demonstrate a prolonged mitotic stage.
Mitosis (M)( emerges at time 6:30 after auxin treatment, transition into one abnormal multilobulated daughter nucleus (MLN) is seen 14:45 h later (time 21:15) Mitosis (M) emerges 7h after auxin treatment (time 7:00), transition into a MLN is seen 4:45 h later (time 11:45). Scale bar 10 μ m.
- Confocal scanner unit CSU W 1 (pinhole 50 μ m)
- Laser 488 and 640 nm
- Microscope Nikon TiE microscope
- Camera Andor iXon 888 Ultra EMCCD camera
- Objective lens Nikon PlanApo 60 x/ 1.49 NA oil immersion objective
- Control software Nikon NIS Elements, ver 5.02.00
- Software Fiji software (ImageJ 1 51 j)
Images were recorded every 15 min for 21 h as z-stacks with two planes and a step size of 6 µm, unbinned and with a pixel size of 217 nm.
Long term observation of mitosis by live cell microscopy enables the analysis of nuclear architecture of cells in mitosis The CSU-W1 confocal scanner is a powerful tool for the analysis of the role of Cohesin on compartmentalized nuclear architecture
Data Cremer M., Brandstetter K., Maiser A, et al. Cohesin depleted cells rebuild functional nuclear compartments after endomitosis Nat Commun 11,6146 (2020)