Session 8

Imaging of interactions and inhibition of hGIIA in prostate cancer cells

Mr Timothy Mann, PhD Student, Western Sydney University

Co-authors
A/Prof Kieran Scott, Western Sydney University
Dr William Church, Sydney University
Dr Anya Salih, Fluoresci Research
Ms Mila Sajinovic, Ingham Institute
A/Prof Albert Mellick, UNSW
Dr Ryung Rae Kim, SpeeDX

Inflammation, a hallmark of cancer, is driven in part by activation of a fatty acid oxidation pathway that generates a myriad of signalling lipids termed eicosanoids. The eicosanoid pathway, begins with the rate-limiting step of release of the 20-carbon fatty acid arachidonic acid from phospholipid membranes, predominantly by phospholipase A2 (PLA2). The secreted PLA2,? hGIIA, is aberrantly over-expressed in prostate cancer (PCa), increases eicosanoid production and PCa cell proliferation, making it an attractive target for inhibition.

This research uses live imaging of tagged hGIIA in prostate cancer cell lines to identify that hGIIA enters the cell through autocrine and paracrine pathways, and is sequestered into the caveolae. Vesicle tracking found that hGIIA docks to vimentin and is trafficked around the cell. Direct binding of hGIIA to vimentin is confirmed with FLIM-FRET and Co-IP. Furthermore, the cyclic peptide c2, currently in clinical trial for the treatment of PCa, inhibits the hGIIA/vimentin interaction in vitro. c2 is autofluorescent with a unique fluorescent lifetime, allowing live cell imaging of c2 entry into the cell which colocalises with hGIIA in caveolae. Treatment of cells with c2 also results in altered vimentin filament organisation and hGIIA movement around the cell. These studies show that vimentin is likely involved in hGIIA’s proliferative effect, and that c2’s mechanism of action is likely through perturbation of the novel hGIIA vimentin interaction.

Building and Using a Multifunctional Single Molecule/Super-Resolution Microscope

Dr Donna Whelan, DECRA Fellow, La Trobe University

This seminar will detail the design, build, and use of a bespoke, multimodal/super-resolution fluorescence microscope which we have established at La Trobe, Bendigo. In particular, I will describe how this setup was built with increased flexibility and a more user-friendly nature, compared to previous custom and commercial options. I will also detail the pipeline for our current single-molecule/super-resolution experiments from sample preparation and design, through acquisition and image rendering, and finally analysis. This includes recent results in the field of DNA damage and repair which identified previously unknown mechanisms in damage mitigation and disease avoidance(1, 2). Other applications that will be highlighted – all using the same microscope – have made use of fluorescence single particle tracking and darkfield microscopy in studies addressing motor neuron disease(3), anti-viral response(4), and metabolism(5, 6).

1.Whelan DR, et al. Plos Genetics. 2020;16(12): e1009256.
2.Whelan DR, Rothenberg E. Proceedings of the National Academy of Sciences. 2021;118(11):e2021963118.
3.Konopka A, et al. Molecular Neurodegeneration. 2020;15(1):51.
4.Monson EA, Whelan DR, Helbig KJ. Int J Mol Sci. 2021;22(9).
5.Sievers W, et al. Plos One. 2020;15(7):e0236286.
6.Van Schaik L, et al. Scientific Reports. 2021;11(1):113.

A super-resolution microscopy study of drug-induced microtubule filament dysfunction

Dr Ashley Rozario, Research Assistant, Monash University

Co-authors
Dr Sam Duwé, Hasselt University, Diepenbeek, Belgium
Mr Cade Elliott, Monash University
Mr Riley Hargreaves, Monash University
Dr Gregory Moseley, Monash University
A/Prof Peter Dedecker, KU Leuven, Leuven, Belgium
Dr Donna Whelan, La Trobe University
Dr Toby Bell, Monash University

The microtubule (MT) network comprises tubular filaments ~25 nm wide and serves vital roles in cellular transport, structural integrity and cell division. The importance of MTs for cell health has motivated the characterization of MT dysfunction caused by anticancer drugs to better understand drug mechanisms. Direct observation of individual MT filaments in situ can be achieved using super-resolution microscopies that surpass the 200 - 300 nm diffraction limit of conventional imaging methods. Single molecule localization microscopy (SMLM) reaches as good as 20-nm spatial resolution, ideal for interrogating individual MT filaments in fixed cells. To image MTs in live cells, fluctuation correlation-based microscopy (SOFI) provides milder imaging conditions (i.e. low laser power) for subdiffraction time-lapse movies of live-cell MT filament dynamics.

Here we apply complementary super-resolution imaging strategies for a holistic perspective of MT dysfunction induced by low doses of antimitotic drug colcemid. SMLM shows MT filament curvature to be more pronounced, increasing with colcemid concentrations from 7 - 80 nM. Aberrant filament curvature induced by 50 - 80 nM of colcemid, quantified using SIFNE, was found to reach up to 2 µm/rad, a value associated with MT filament breakage. Higher colcemid doses at 100 and 200 nM revealed short and few MT filaments, suggesting MT fragmentation as a possible drug mechanism. Live cell SOFI reveals MT filament dynamics to be suppressed with as low as 18 nM of colcemid without aberrant filament curvature present.

Cellular remodelling during necroptosis characterized by live cell imaging

Dr Ying Zhang, Research Officer, Walter and Eliza Hall Institute

Co-authors
Dr Niall Geoghegan, Walter and Eliza Hall Institute
Dr Andre Samson, Walter and Eliza Hall Institute
Dr Kelly Rogers, Walter and Eliza Hall Institute
A/Prof James Murphy, Walter and Eliza Hall Institute
Prof Guillaume Lessene, Walter and Eliza Hall Institute

Necroptosis is a form of caspase-independent programmed cell death that has been suggested to play a role in the pathogenesis of various diseases. Necroptosis is executed by the activation of RIPK3 and MLKL that leads to the assembly of necrosome and subsequent rupture of plasma membrane. Due to the release of cell content, such as damage-associated molecular patterns (DAMPs), from ruptured membrane, necroptosis triggers inflammatory responses. Currently, the key biological markers that can distinguish necroptosis from other types of cell death is MLKL phosphorylation and membrane translocation, while the morphological changes, the release of DAMPs and the process of MLKL activation have not been well characterized.

Here, we employed Lattice Light Sheet Microscopy (LLSM) that can quickly generate three-dimensional (3D) super-resolution images with very limited phototoxicity to monitor the process of necroptosis in live cells. We observed a series of necroptosis-specific morphological changes and reorganization of actin cytoskeleton and further explored their relationships with DAMPs release. Our study also focuses on MLKL and investigated the involvement and the role of this pseudo-kinase in the process of cellular remodelling. This work provides new insight into the cellular events associated with necroptotic cell death with potential implications for fundamental and translational research.

Latest developments of Luxendo lightsheet in advancing Life Science applications

Zheng Chao, Scitech

In the past few years, light sheet microscopy has greatly expanded its use in biological research. Its unique advantage of capturing images of whole organisms and tissues in real time has brought researchers into an exciting new era. This discussion focusses on recent developments of lightsheet microscopy from Luxendo. Some of the exciting new developments including photo-manipulation module, TAG lens and MEMS module, Lattice lightsheet module as well as optimized big data processing algorithms. Brief introduction of the technologies, functionalities and applications will be presented in the workshop.

Adaptive Optics and Adaptive Illumination - Key Ingredients for 3D and Live cell STED superresolution

Carola Thoni, Lastek

Imaging Biological Structures of cells below 100nm using SIM

Gavin Symonds, Carl Zeiss

There are a number of imaging techniques that provide resolution below the diffraction limit of the optical microscope. However imaging of biological structures below 100nm, not only for fixed samples but especially for live cells, presents a number of challenges. These challenges include the choice of special fluorescent markers and sample chemistry, potentially high laser powers at the sample plane, and extended image acquisition times depending on the technique. Double the traditional SIM resolution is now possible with a new dual iterative processing method implemented as SIM^2. With this presentation we will briefly introduce the concept of SIM and SIM^2 processing, and show several examples.