Checkpoint signalling

DNA damage checkpoint and non-genetic heterogeneity

Cell signalling and cell fate choices exhibit significant variability within identical clones. Indeed, non-genetic heterogeneity is a very common phenomenon in biological systems and an emerging concept underlying early oncogenesis, tumour progression and treatment of cancers. Unfortunately, such non-genetic heterogeneity remains refractory to current analytic methods. Therefore, we have developed a unique biochemical imaging platform, which includes genetically encoded probes, innovative hardware and analytical tools, that permits us to manipulate and probe biochemical networks in living single cells over time. We have observed how cells respond with intrinsic (non-genetic) heterogeneity resulting in a complex phenotypic landscape in response to DNA damage, ranging from DNA damage repair and survival, induction of alternative tumour suppressive mechanisms (apoptosis, necrosis or senescence), and delayed cell death after continued proliferation in the presence of unrepaired DNA damage. We are now revealing phenotypes previously concealed by population measurements and linking diverse phenotypic-response to an identical stimulus to specific patterns of biochemical (signalling and metabolic) networks. Revealing mechanisms that support the fitness of cancer cell lineages, mechanisms otherwise hidden by population averages, has profound implications on our understanding of therapy and the process of oncogenesis.

Graphical abstract

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Publications

  • Maximilian W Fries, Kalina T Haas, Suzan Ber, John Saganty, Emma K Richardson, Ashok R Venkitaraman, and Alessandro Esposito,*, “Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate”, online at bioaRxiv [BIORXIV/2018/427237] while under review
  • Siddharth De, Callum J Campbell, Ashok R Venkitaraman, and Alessandro Esposito,*, “Pulsatile MAPK signalling modulates p53 activity to control cell fate decisions at the G2 checkpoint for DNA damage”, in review
  • Callum J. Campbell, Ashok R. Venkitaraman and Alessandro Esposito*, “Checkpoint non-fidelity induces a complex landscape of lineage fitness after DNA damage”, online at bioaRxiv [BIORXIV/2018/431486 while under review
  • Alessandro Esposito. “Cooperation of partially-transformed clones: an invisible force behind the early stages of carcinogenesis”,  online at bioaRxiv [BIORXIV/2018/431478 while under review
  • Liang H, Esposito A, De S, Ber S, Collin P, Surana U, Venkitaraman AR, “Homeostatic control of the G2 checkpoint via polo-like kinase 1 engenders non-genetic heterogeneity in its fidelity and timing”, Nat Comms. 5, 4048

DNA Damage Response and Repair

While setting-up new imaging technologies at the MRC Cancer Unit, we have worked to maintain and expand state-of-the-art imaging facilities. We have thus deployed a variety of techniques to understand various aspects of the DNA damage response, including mobilization of DNA repair factors using fluorescence correlation spectroscopy, checkpoint fidelity using sensitized emission FRET and the nucleation and extension of RAD51 and RPA on DNA with single-molecule localization microscopy. 

Graphical abstract

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On the left, an early collaboration with Anand (PhD student in the Venkitaraman lab) soon after I joined the Director’s lab. In the middle, a work published 8 years later with Kalina with a more established team of people funded within the Director’s group. On the right, a collaborative work with Hongqing (PhD student in the Venkitaraman lab) while I was still working as an individual post-doctoral scientist on establishing novel imaging technologies.

Publications

  • Kalina T. Haas, MiYoung Lee, Alessandro Esposito† and Ashok R. Venkitaraman† (2018), “Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites”, Nucleic Acids Research 46(5):2398–2416
  • Liang H, Esposito A, De S, Ber S, Collin P, Surana U, Venkitaraman AR, “Homeostatic control of the G2 checkpoint via polo-like kinase 1 engenders non-genetic heterogeneity in its fidelity and timing”, Nat Comms. 5, 4048
  • Jeyasekharan AD, Ayoub N, Mahen R, Ries J, Esposito A, Rajendra E, Hattori H, Kulkarni RP, Venkitaraman AR, “DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells”, Proc. Natl. Acad. Sci. USA, 107(50): 21937–21942.