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Journal of Cell Science Travelling Fellowship Project Report. Does the Structure of the Pericentriolar Material Control Checkpoi

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Journal of Cell Science Travelling Fellowship Project Report. Does the Structure of the Pericentriolar Material Control Checkpoi Journal of Cell Science Travelling Fellowship project report. Does the structure of the pericentriolar material control checkpoint responses to DNA damage? Alicja K. Antonczak1, Laurence Pelletier2, Ciaran Morrison1 1Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland; 2Samuel Lunenfeld Research Institute, University of Toronto, Canada Fellowship duration: 13 to 27 October 2012 Host laboratory: Samuel Lunenfeld Research Institute, University of Toronto, Canada Research Objectives Centrosomes are the principal microtubule organising centres of animal cells and consist of a pair of centrioles (mother and daughter) surrounded by pericentriolar material (PCM). Pericentrin (Pcnt; also known as kendrin) is a large coiled coil protein that anchors many PCM proteins. Importantly, it associates with γ-tubulin to form a complex that controls spindle organisation and mitotic entry. Whereas Pcnt depletion leads to mitotic delay, Pcnt overexpression leads to centrosomal abnormalities, including PCM expansion and the formation of multiple centrioles. The PCM is often referred to as an amorphous mass, although recent data have indicated an underlying three-dimensional scaffold-like structure. The cell cycle requires a tight spatiotemporal control on the regulation of its key components, such as the checkpoint kinases and the CDKs. Therefore, we hypothesize that the PCM is an ordered structure that responds to checkpoint signals by changing the spatial relationship between key cell cycle regulators. Our proposed project was to characterize structural changes in the PCM after DNA damage using structured illumination microscopy (SIM) in the host laboratory of Dr. Laurence Pelletier at the Samuel Lunenfeld Research Institute in Toronto (Canada). We decided to use Pcnt as a model for visualizing PCM in chicken DT40 cell line. As there are no commercial antibodies against chicken pericentrin, we have used gene targeting in the hyper-recombinogenic DT40 cell line to target the 3’ end of the Pcnt locus with a sequence that encodes superfolder-GFP (sfGFP), which we used because it folds more efficiently than the eGFP variant. The advantage of using Pcnt gene targeting is that we should be able to see all the tagged molecules. We have successfully generated cell lines that express only sfGFP-tagged pericentrin (Pcnt- sfGFP+/+). Results During my work in Toronto, I obtained high-resolution images of the PCM as the cells progress through the cell cycle. The cells were co-stained with antibody against centriolar protein, Cep135, to orient the centrioles inside the PCM structure. As predicted, the PCM forms a ring structure around centriols (Figure 1). This confirmed that the GFP tagged pericentrin retains its centrosomal localization, and that the Pcnt- sfGFP+/+ cell line can be used as model for study of the PCM in fixed or live cell imaging. Figure 1 GFP-tagged pericentrin forms ring like structure in DT40 centrosomes. DT40 Pcnt-GFP+/+ cells were stained with anti-GFP (green) and anti-Cep135 (red) antibodies and imagined using SIM. In the next part of the project we investigated if DNA damage induces changes in the PCM structure. The cells were exposed to 5 Gy of X-ray radiation, and stained for centrosomal marker, Cep135, 24h post treatment. The PCM was then visualized using SIM. As expected, γ-irradiation induced centrosomal amplification. We observed distortion of the pericentrin ring structure in cells exposed to DNA damage but not in the control cells, suggesting involvement of PCM in DNA damage response. Further experiments are necessary to establish the connection between changes in PCM structure and DNA damage induced cell cycle arrest. We plan to test whether PCM responses to genotoxic stress depend on integrity of the DNA damage response. We will examine the PCM structure in cells that lack components of the DNA response network, microcephalin and Chk1. .
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