In a recent study published by the group of Prof. Luca L. Fava at CIBIO – University of Trento, it has been elucidated how centrosomes, the principal microtubule-organizing centres in human cells, can generate signals able to modulate cellular signalling and hence influence cell fate determination (Burigotto et al., 2021: EMBO J).
It is well known that centrosome amplification leads to genetic instability, a hallmark of cancer. To counteract this, cells have evolved mechanism which are able to sense supernumerary centrosomes and block the cell division cycle relying on the activation of the tumour suppressor p53 via the PIDDosome. The PIDDosome is a multiprotein complex composed of PIDD1, RAIDD and Caspase-2 which drives activation of Caspase-2, an endopeptidase originally implicated in apoptosis. In Burigotto et al, by combining super-resolution imaging, yeast-two-hybrid, and reverse genetics the authors demonstrate that PIDD1 is recruited to mature centrosomes by ANKRD26 protein, a component of the centriolar distal appendages, and its own localization is necessary for PIDDosome activation. In addition to that, they contribute to clarify that clustering of extra-centrosomes upon cytokinesis failure is crucial to avoid p53 activation via the PIDDosome in healthy cells.
Here we focus on a piece of data obtained with CrestOptics X-Light V2 spinning disk on fixed and living samples and shown in Figure 6 of the original publication (to read deeper into the study please click here for the whole publication). We present the PIDD1 localization throughout the cell cycle and, more specifically, during mitotic progression in unperturbed RPE1 cells (Figure A) and in cells failing cytokinesis (Figure B). Of note, in control cells endogenous PIDD1 dissociates from parental centrioles at mitotic entry and, during late mitotic stages, only one centriole for each spindle pole starts recruiting PIDD1 and showing PIDD1 positive staining (Figure A, yellow arrows). In fact, G1 cells display one PIDD1-positive centriole. In case of cytokinesis failure (e.g., after treatment with dihydrocytochalasin-B, DHCB), extra centrosomes generate clusters and PIDD1-positive centrioles are close to each other inside the cluster itself (Figure B).
To better appreciate centrosome cluster formation in its temporal and spatial dimension, the authors monitored by live imaging RPE1 cells stably expressing Centrin1-GFP in normal conditions and after treatment with DHCB, an inhibitor of actin polymerization (Figure C). They observed that centrosome clusters form about 3 hours after anaphase onset and then they remain stably associated.
Moreover, results shown in the Burigotto et al. paper demonstrate that the centrosome is not only involved in generating a cell cycle inhibitory response to mitotic errors playing a role in carcinogenesis, but also contributes to DNA damage response via a p53-PIDDosome pathway.
Figure A: PIDD1 localization along cell cycle and mitotic progression in control cells. Representative images of cells at the indicated cell cycle phases stably expressing Centrin1-GFP (CETN1-GFP, shown in green) and stained for centriole subdistal appendages (ODF2, shown in blue) and PIDD1 protein (shown in red). Enlarged images (2.5x) with DNA staining (Hoechst 33342) are shown. Images were acquired with a CrestOptics X-Light V2 spinning disk. Scale bar: 5 µm.
Figure B: PIDD1 localization upon cytokinesis failure. Representative images of cells stably expressing Centrin1-GFP (CETN1-GFP, shown in green) and treated either with DMSO (NT) or with DHCB for 24 hours. Enlarged images (2.5x) with DNA staining (Hoechst 33342) are shown. Images were acquired with a CrestOptics X-Light V2 spinning disk. Scale bar: 5 µm.
Figure C: Movies of RPE1 cells undergoing mitosis in control (i) and DHCB treatment conditions (ii). RPE1 cells stably expressing CETN1-GFP (shown in green) were subjected to time-lapse video microscopy in the presence of SiR-DNA (shown in blue). Time is expressed in minutes; t = 0 is one frame before anaphase onset. Movies were acquired with a CrestOptics X-Light V2 spinning disk. Scale bar: 5 µm.
All images and movies shown here were acquired on a spinning disc Eclipse Ti2 inverted microscope (Nikon Instruments Inc), equipped with Lumencor Spectra X Illuminator as LED light source, an X-Light V2 Spinning Disk (CrestOptics) and an Andor Zyla 4.2 PLUS sCMOS monochromatic camera using a Plan Apochromatic 100x 1.45 NA oil immersion objective. Images were deconvolved with Huygens Professional software (Scientific Volume Imaging, Hilversum, The Netherlands).
Movies were recorded every 4 minutes for the first hour and then every 10 minutes for up to 16 hours. Each field contained around 25 z-slices collected in 0.6 mm steps.
To read deeper into the Materials and Methods used by Burigotto and colleagues, please click here to read the whole publication.
Matteo Burigotto, Alessia Mattivi, Daniele Migliorati, Giovanni Magnani, Chiara Valentini, Michela Roccuzzo, Martin Offterdinger, Massimo Pizzato, Alexander Schmidt, Andreas Villunger, Stefano Maffini, Luca L Fava.
EMBO J (2021) 40: e104844
Re-use of images from this article is done under the terms and conditions of the Creative Commons Attribution (CC BY) license.
The application note has been prepared in collaboration with Dr. Matteo Burigotto and Prof. Luca L. Fava