Donna Rose Whelan

La Trobe University · La Trobe Institute of Molecular Sciences (LIMS)

Single molecule localization microscopy (SMLM) and Synchrotron Fourier transform infrared (S-FTIR) spectroscopy are two techniques capable of elucidating unique and valuable biological detail. SMLM provides images of the structures and distributions of targeted biomolecules at spatial resolutions up to an order of magnitude better than the diffract...

DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function...

Single molecule localization microscopy (SMLM) techniques allow for sub-diffraction imaging with spatial resolutions better than 10 nm reported. Much has been discussed relating to different variations of SMLM and all-inclusive microscopes can now be purchased, removing the need for in-house software or hardware development. However, little discuss...

Homologous recombination (HR) is a crucial pathway for the repair of DNA double-strand breaks. BRCA1/2 breast cancer proteins are key players in HR via their mediation of RAD51 nucleofilament formation and function; however, their individual roles and crosstalk in vivo are unknown. Here we use super-resolution (SR) imaging to map the spatiotemporal...

DNA double-strand breaks (DSBs) are toxic DNA lesions, which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homol...

The 5-hydroxytryptamine 3 (5-HT3) receptor belongs to the pentameric ligand-gated cation channel superfamily. Humans have five different 5-HT3 receptor subunits: A to E. The 5-HT3 receptors are located on the cell membrane, but a previous study suggested that mitochondria could also contain A subunits. In this article, we explored the distribution...

Lipid droplets (LDs) are upregulated by host cells in the face of pathogen infection, however, the reason for this phenomenon remains largely unknown. Here, we demonstrate that virally induced LDs house a distinct and dynamic proteome containing key antiviral signalling pathway members, including the essential pattern recognition receptor; RIG-I, k...

Objective Centrally administered estrogen can increase sympathetic nerve activity to brown adipose tissue, resulting in thermogenesis. The central thermogenic effects of estrogen have not been investigated in males. Therefore, this study sought to investigate the effects of peripherally and centrally administered estrogen on thermogenesis, heart ra...

Single molecule (SM) super-resolution microscopies bypass the diffraction limit of conventional optical techniques and provide excellent spatial resolutions in the tens of nanometers without overly complex microscope hardware. SM imaging using optical astigmatism is an efficient strategy for visualizing subcellular features in 3D with a z-range of...

Background The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and...

Lipid droplets (LDs) are increasingly recognized as critical organelles in signalling events, transient protein sequestration and inter-organelle interactions. However, the role LDs play in antiviral innate immune pathways remains unknown. Here we demonstrate that induction of LDs occurs as early as 2 h post-viral infection, is transient and return...

Lipid droplets (LDs) have traditionally been thought of as solely lipid storage compartments for cells; however, in the last decade, they have emerged as critical organelles in health and disease. LDs are highly dynamic within cells, and their movement is critical in organelle-organelle interactions. Their dynamics are known to change during cellul...

Super-resolution microscopy (SRM) comprises a suite of techniques well-suited to probing the nanoscale landscape of genomic function and dysfunction. Offering the specificity and sensitivity that has made conventional fluorescence microscopy a cornerstone technique of biological research, SRM allows for spatial resolutions as good as 10 nanometers....

Significance DNA resection is an initial, decisive step in the homologous recombination repair pathway of DNA double-strand breaks (DSBs). However, the individual roles and cross-talk of key proteins in this process remain unclear. To resolve the spatiotemporal dynamics of this intricate process, we applied multicolor single-molecule localization m...

The role of central orexin in the sympathetic control of interscapular brown adipose tissue (iBAT) thermogenesis has been established in rodents. Stimulatory doses of caffeine activate orexin positive neurons in the lateral hypothalamus, a region of the brain implicated in stimulating BAT thermogenesis. This study tests the hypothesis that central...

Single-molecule super-resolution microscopy (SRM) combines single-molecule detection with spatial resolutions tenfold improved over conventional confocal microscopy. These two key advantages make it possible to visualize individual DNA replication and damage events within the cellular context of fixed cells. This in turn engenders the ability to de...

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to dir...

Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, their side effects require the development of safer drug regimens that still retain clinical efficacy. Currently, many questions remain regarding microtubule-interacting drugs at clinically relevant and ultra...

Functional brown adipose tissue (BAT) was identified in adult humans only in 2007 with the use of fluorodeoxyglucose positron emission tomography imaging. Previous studies have demonstrated a negative correlation between obesity and BAT presence in humans. It is proposed that BAT possesses the capacity to increase metabolism and aid weight loss. In...

Lipid droplets (LDs) are increasingly recognized as critical organelles in signalling events, transient protein sequestration and inter-organelle interactions. However, the role LDs play in antiviral innate immune pathways remains unknown. Here we demonstrate that induction of LDs occurs as early as 2 hours post viral infection, is transient, and r...

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork (RF) stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). To avoid mutagenesis, these breaks require repair...

The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imagi...

The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imagi...

Light microscopy has long been at the forefront of biological research, perhaps most significantly in the form of fluorescence microscopy. This technique, paired with the ongoing discovery and synthesis of increasingly brilliant fluorophores, allows for visualization of the internal machinations of cells with molecular specificity. However, until r...

The Beta-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bac- terial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ . Single-cell analysis showed that discrete membrane prec...

DNA double-strand breaks (DSBs) are toxic DNA lesions which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homolo...

We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF fo...

We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF fo...

Although microtubules (MTs) are known to have important roles in intracellular transport of many viruses, a number of reports suggest that specific viral MT-associated proteins (MAPs) target MTs to subvert distinct MT-dependent cellular processes. The precise functional importance of these interactions and their roles in pathogenesis, however, rema...

div class="title">Single Molecule Localization Microscopy of DNA Damage Response Pathways in Cancer. - Volume 22 Issue S3 - Donna R. Whelan, Yandong Yin, Keria Bermudez-Hernandez, Sarah Keegan, David Fenyö, Eli Rothenberg

Synchrotron radiation-Fourier transform infrared (SR-FTIR) microscopy coupled with multivariate data analysis was used as an independent modality to monitor the cellular bystander effect. Single, living prostate cancer PC-3 cells were irradiated with various numbers of protons, ranging from 50-2,000, with an energy of either 1 or 2 MeV using a prot...

Fourier Transform Infrared (FTIR) micro-spectroscopy is an emerging technique for the biochemical analysis of tissues and cellular materials. It provides objective information on the holistic biochemistry of a cell or tissue sample and has been applied in many areas of medical research. However, it has become apparent that how the tissue is handled...

Application of single-molecule fluorescence detection has led to the development of light microscopy techniques that make it possible to study fluorescent samples at spatial resolutions significantly improved upon the diffraction limit of light. The biological and materials science applications of these “super-resolution” microscopy methods are vas...

Synchrotron Radiation Fourier Transform Infrared (SR-FTIR) spectra of single human prostate adenocarcinoma PC-3 cells, irradiated with a defined number of 2 MeV protons generated by a proton microbeam along with non-irradiated control cells, were analysed using multivariate methods. A number of different Principal Component Analysis (PCA) models we...

The role that DNA conformation plays in the biochemistry of cells has been the subject of intensive research since DNA polymorphism was discovered. B-DNA has long been considered the native form of DNA in cells although alternative conformations of DNA are thought to occur transiently and along short tracts. Here, we report the first direct observa...

Interferon (IFN)-mediated immunity is a central mode of defense against viral infection and evasion of this immune response is critical to the pathogenicity of viruses. IFN-antagonist proteins have recently been shown to interact with host microtubules (MTs) demonstrating a novel mechanism for subverting the IFN response (1). Using super-resolution...

The ability to accurately detect DNA both quantitatively and qualitatively inside cells using Fourier transform infrared (FTIR) spectroscopy has been disputed. Recently, we have demonstrated that the variability of DNA absorptions is due to the dehydrated nature of biological samples prepared for FTIR spectroscopic measurement [1]. We have further...

The application of FTIR spectroscopy to disease diagnosis requires a thorough knowledge of the spectroscopy associated with the cell cycle to discern disease markers from normal cellular events. We have applied synchrotron FTIR spectroscopy to monitor cells at different phases of the cell cycle namely G1, S and G2 phases. By applying Principal comp...

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with res...

A technique capable of detecting and monitoring nucleic acid concentration offers potential in diagnosing cancer and further developing an understanding of the biochemistry of disease. The application of Fourier transform infrared (FTIR) spectroscopy has previously been hindered by the supposed non-Beer-Lambert absorption behavior of DNA in intact...

The ability to detect DNA conformation in eukaryotic cells is of paramount importance in understanding how some cells retain functionality in response to environmental stress. It is anticipated that the B to A transition might play a role in resistance to DNA damage such as heat, desiccation and toxic damage. To this end, conformational detail abou...