Navigating the nuclear highways
Liz has developed a method—microscopy combined with fluorescence fluctuation analysis—to track the movement of molecules around the complex DNA networks within the nuclei of live cells.
Inside the nucleus, DNA repair machines find sites of damage to prevent genetic mutations, and transcription factors search for target DNA sequences to maintain normal gene expression.
At any given time, thousands of molecules move around the nucleus. Building a complete road map for navigating the DNA network will help to understand how and why deregulated nuclear traffic leads to diseases like cancer.
By analysing changes in fluorescence intensities, Liz discovered that DNA networks rearrange to create highways for repair and transcription factors to arrive at target destinations.
The aim of our research is to delineate the role of a dynamic nuclear architecture in directing nuclear traffic. Given the multi-layered organisation of DNA into a three dimensional genome,a consensus has emerged that nuclear architecture encodes spatiotemporal information that informs DNA target search. The naturally occurring changes in genome organisation range from nanometre local chromatin fibre movements on a millisecond time scale, to global higher order chromatin rearrangements on a minute-to-hour timescale. While live cell imaging can capture snapshots of the changes in chromatin organisation associated with different cell cycle stages, the temporal resolution of a frame acquisition renders the micro- to millisecond chromatin dynamics relevant to gene expression ‘invisible’. In recent work we developed microscopy methods—based on fluorescence correlation spectroscopy—to monitor the real time rearrangements in chromatin structure with sub-micron resolution. With these new methods we have demonstrated that the chromatin network is not merely an obstacle to diffusion, but instead a dynamic entity, which through millisecond structural changes, mediates transcription factor–DNA binding activity and directs DNA repair factor recruitment to damage sites. Our goal is now to identify the aspects of nuclear architecture, which facilitate nuclear navigation, and how this ‘spatiotemporal map’ is compromised during oncogenic transformation.
About Elizabeth Hinde
In 2010 Elizabeth completed her PhD in fluorescence spectroscopy at the University of Melbourne and was then recruited to the University of California, Irvine, USA to pursue a post-doctoral fellowship under the mentorship of Professor Enrico Gratton. In the Gratton lab (2010-2013) Elizabeth developed methods based on fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) to quantify chromatin dynamics in live cells. With the aim of applying this technology to cell biology, Elizabeth returned to Australia in 2013 as a UNSW Vice Chancellor Fellow under the mentorship of Professor Katharina Gaus. At UNSW (2013-2015) Elizabeth’s research revealed insights into genome organisation that led to the award of a Cancer Institute of NSW Early Career Research Fellowship in 2015 and this enabled her to establish a research group within the EMBL Australia node for Single Molecule Science. Elizabeth’s group aims to investigate the role of nuclear architecture in facilitating genome function.
Hinde, E.*, Pandzic, E., Yang, Z., Ng, I.H.W., Jans, D.A., Bogoyevitch, M.A., Gratton, E. and Gaus, K.* (2016). Quantifying the dynamics of the oligomeric transcription factor STAT3 by pair correlation of molecular brightness. Nature Communications, 7 (11047):DOI:doi:10.1038/ncomms11047.
Hinde, E.*, Kong, X., Yokomori, K., & Gratton, E. (2014). Chromatin Dynamics during DNA Repair Revealed by Pair Correlation Analysis of Molecular Flow in the Nucleus. Biophysical Journal, 101(1), 55-65.
Hinde, E., Digman, M. A., Hahn, K. M. & Gratton, E*. (2013). Millisecond spatiotemporal dynamics of FRET biosensors by the pair correlation function and the phasor approach to FLIM. PNAS, 110(1), 135-140.
Hinde, E., Digman, M. A., Hahn, K. M. & Gratton, E*. (2012). Biosensor Förster resonance energy transfer detection by the phasor approach to fluorescence lifetime imaging microscopy. Microscopy Research and Technique, 75(3), 271-281.
Hinde, E., Cardarelli, F., Digman, M. A., & Gratton, E*. (2010). In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow. PNAS, 107(1),16560-16565.
Click here for full publication list
- UNSW Medicine, Dean's Rising Star Award, 2015
- Cancer Institute of NSW Early Career Fellowship, 2015
- Young Fluorescence Investigator Award, US Biophysical Society, 2014
- UNSW Vice Chancellor Fellowship, 2013
1.Impact of nuclear architecture on transcription factor target search.Transcription factors have evolved ‘target search’ strategies that allow them to efficiently navigate the nuclear space and arrive at their target sequence. This target search strategy is underpinned by molecular diffusion, which, in turn, is controlled by the nuclear architecture and oligomeric state of the transcription factor. While it is known from in vitro studies that transcription factors employ oligomerisation to modulate DNA binding activity, no imaging approach proposed so far can track the molecular mobility of protein oligomers within the nuclei of live cells. To address this research gap we recently established a new microscopy method termed pCOMB (pair correlation of molecular brightness) that can quantify the transport and binding dynamics of different oligomeric species in live cells. The overall aim of this project is to use to the pCOMB method to quantify the role of global chromatin organization in facilitating transcription factor target search. This work is in collaboration with A. Prof. Marie Bogoyevitch (Uni. Melb., Australia), Prof. David Jans (Monash, Australia) and Prof. Enrico Gratton (UCI, USA).
2. Role of chromatin dynamics during the DNA damage response.Tumour suppression relies on the preservation of genome integrity and this is maintained by a cellular surveillance system called the DNA damage response (DDR). While it is known that the DDR can instantaneously detect a genomic lesion, how it spatiotemporally coordinates DNA repair factor recruitment to that damage site is not currently understood. Given the high degree of genome organization in three dimensional space, a consensus is emerging that nuclear architecture encodes spatiotemporal information that informs DNA damage signalling and repair. In recent work we demonstrated a spatial heterogeneity in chromatin condensation in the region flanking a genomic lesion. The overall aim of this project is to investigate how these changes in chromatin compaction modulate genome topology to facilitate the DDR. By use of a fluorescence lifetime imaging microscopy (FLIM)-based assay we will test the hypothesis that spatial heterogeneity in chromatin compaction during the DDR serves to facilitate DNA repair factor access whilst blocking transcription of neighbouring genes. This work is in collaboration with Dr. Anthony Cesare (CMRI, Australia).
3. Aspects of genome topology compromised during oncogenesis.DNA targeting drugs need to enter the cellular nucleus. However, how to design carriers such as nanoparticles for nuclear access is poorly understood. Given the infectivity of pathogens depends on nuclear entry, pathogen-shaped drug carriers may offer a unique solution to directly deliver and release chemotherapeutic agents into the nucleus. In recent work we established pair correlation analysis to map the molecular mobility of nanoparticles with millisecond and sub-micron resolution. Using this assay we demonstrated that nanoparticle shape influences nanoparticle mobility across the plasma membrane, escape from the endosomal pathway and access to the nucleus. The aim of this project is to explore how nanoparticle geometry can be used, as a tool, to selectively target DNA targeting drugs such as doxorubicin, to cancerous nuclei. This work is in collaboration with Dr. Cyrille Boyer (UNSW, Australia), Prof. Justin Gooding (UNSW, Australia) and Prof. Katharina Gaus (UNSW, Australia).
- Kitiphume Thammasiraphop (Honours student)
- Erol Dalkic (Honours student)