Whelan - Biophysics, DNA damage, fluorescence, single molecule imaging

Each day, the DNA of healthy cells suffers from endogenous replicative stress caused by competition between replication forks, transcription sites, and other DNA-binding proteins. This results in 5-50 DNA double strand breaks (DSBs) occurring in each human replicating cell, every 24 hours. Exogenous stressors such as cigarette smoke or alcohol only serve to increase the total amount of DNA damage. DNA DSBs need to be repaired without any loss or gain of genetic code because this could potentially engender a mutation leading to cell death of malfunction. Mutations and cell death caused by DNA misrepair, often exacerbated by deficiencies in repair pathways, underpin many cancers, autoimmune and neurodegenerative diseases.

Because of this, a plethora of interacting and redundant repair proteins work within cells to maintain genomic integrity, and much research has been undertaken to try to understand and manipulate these pathways – particularly because inducing genomic stress remains our primary method for treating cancer. However, studying these pathways in vivo has been challenging because of the high signal-to-noise within the dense, homogenous nucleus, and the low level of endogenous DSB induction.

Using innovative labelling and super-resolution assays that I developed during my postdoctoral studies I am now focussed on elucidating damage and repair events at the single molecule scale. In particular I aim to map the high-fidelity homologous recombination repair of collapsed replication forks, and to investigate the role of transcriptional silencing within the nucleolus.

External collaborator: Eli Rothenberg, NYU Langone Medical Center, USA

The ideal way to conduct research is to tailor experiments and techniques to best suit whatever question you are trying to answer. However this requires access to, and know-how of, an ever-expanding repertoire of scientific methods which cannot be contained within a single lab. Because of this, collaboration and the exchange of ideas that occurs at conferences are cornerstones of successful research programs. Within the fast-moving sphere of biophysical methods – specifically spectroscopies and microscopies – we are working to design a pipeline that will allow users to access several complementary techniques with single-cell, and even sub-cellular, correlation. This pipeline will incorporate several sequential techniques beginning with live-cell infrared and Raman spectroscopies. Infrared and Raman offer a wealth of information regarding the composition of a single live cell which can be interpreted to determine cell cycle, disease/stress state, and cell type.

Next we can visualize targets of interest within the same cell using live cell super-resolution fluorescence microscopy (50-100 nm resolutions with SOFI and PALM) and then the same or complementary targets using even higher-resolution fluorescence microscopy following cell fixation (20 nm resolution with dSTORM). This allows comparison between the native live cell state and after fixation. Next we aim to be able to assess the overall topography of intra-cellular environments using atomic force and electron microscopies (AFM and EM). Finally, we are also developing correlative Expansion microscopy to visualize substructures using fluorescence microscopies at enhanced resolutions. Developing protocols to conduct any and all of these methods on the same single cell will lead to a much better understanding and appreciation of preparative artifacts and ensure conclusions based on multiple experiments are more robust that those relying on any single technique.

External collaborators: Markus Sauer (Wurzburg University, Germany), Peter Dedecker (KU Leuven, Belgium), Toby Bell, Rico Tabor and Alison Funston (Monash University).

DNA double strand breaks occur regularly in healthy cells and need to be repaired without changes to the underlying genetic code. To achieve this, several redundant and complementary pathways exist, with the most complicated – homologous recombination (HR) – believed to be the highest fidelity and thus the preferred repair pathway. HR uses the broken end of the DNA to search the rest of the genome and find a homologous sequence (e.g. a sister chromatid or homologous gene). A single-stranded portion of the broken end then displaces the homologous sequence strand and uses its complementary strand as a repair template. The protein Rad51 and its mediator Brca2 are known to facilitate this complicated search and DNA strand invasion, however the dynamics and mechanisms of this process are not well understood. Deficiencies and mutations in Brca2 and Rad51 are well known as oncogenic, and so characterizing their roles in HR could lead to a better understanding, and new therapeutic approaches, for many cancers. FRET enables sub-nanometer dynamic distance measurements to be taken on a single molecule scale using pairs of fluorescent dyes. By labelling double stranded DNA molecules tethered to glass slides and determining the changing conformation of the DNA in the presence of an invading DNA strand and proteins we are investigating the precise dynamics of homology search and strand invasion.

Fluorescence microscopy is a cornerstone technique of various biomedical and biochemical research, allowing for the structures, distributions, and interactions of specific proteins and macromolecules to be visualized within cells, tissues, and intact organisms. However, the diffraction of light dictates that no matter the magnification and quality of microscope used, a fluorescence image of a cell will be limited to spatial resolutions worse than ~250 nanometers. What this effectively means is that fluorescence images suffer a blurriness that makes discerning details smaller than 250 nanometers extremely difficult. Super-resolution techniques circumvent this limitation in a number of ways: Expansion Microscopy (ExM) does so by physically making the cells, tissue or organism bigger, and can theoretically achieve sub-5 nanometer resolutions. SOFI (super-resolution optical fluctuation imaging), although not imparting exceptional resolution gains (typically 80-100 nanometers), offers the enormous advantage over other super-resolution techniques that it can be used to image live cells, at dynamic framerates. With only a handful of labs in the world using these techniques we are working to develop new labelling strategies based on innovative orthogonal chemistry and specialized fluorescent proteins, and to validate these approaches by visualizing key drug- and disease-targeted structures within cells.

Senior DECRA Fellow

Dr Donna Whelan

PhD student

Riley Hargreaves

Honours student

Esther Miriklis