Swinburne University of Technology Bionanoengineering Group Group Leader: Dan Nicolau Postdoctoral Researchers: Kristi Hanson Prashant Sawant Postgraduate Researchers: Luisa Filipponi Peter Livingston Gerardin Solana Codrin Mocanasu

Primary ‘Fluorescence-Relevant’ Research Application of lithographic techniques for micro/nano- structuring of polymer surfaces to: Control of in vitro motility of protein molecular motors Confinement of live cells, esp. fungi Spatial control of protein attachment with retention of bioactivity Actin filaments (green) moving on a patterned myosin surface. BSA (red) was patterned by  CP and used as a myosin blocking agent. Actin filaments moving in tracks created by laser ablation of AAPO. Fungal filament growth in confined PDMS microstructures. Selective adsorption of FITC-avidin on hydrophobic micro-scale patterns created by bi- layer lithography (PtBMA – DNQ).

Progress, Challenges and Approaches Fungi Progress Growth of fungi in microconfined conditions has revealed a number of unusual phenomena:  Collision-induced branching  Directional memory  Intra-hyphal coordination Challenges Live imaging of cytoskeletal dynamics desirable Requires development and optimization of suitable fluorescent probes Approaches Phalloidin conjugated probes  Issues with toxicity and growth disruption GFP tagged proteins  Would require extensive experimental work to develop GFP constructs specific to application – relatively little work in this area with fungi ??? Molecular Motors Aim Development of nano-devices able to detect single biomolecules based on actin-bound antibody-antigen interactions. Challenges Requires ability to image multiple fluorophores with sufficient temporal and spatial resolution High temporal resolution required to detect effects of interactions on motility Large field of view at high spatial resolution = low temporal resolution Confocal imaging of only one label at sufficient temporal resolution is already problematic –made worse by addition of multiple flourophores Also complicated by interference from polymer substrate, even with confocality, due to nm-scale separation between substrate and protein. Approaches ???? Growing hyphal tips stained with FM4-64, showing apical vesicle cluster, vacuolar membranes, and mitochondria (from Fischer-Parton et al., 2000).

Challenges Specific Challenges Common theme is certainly development of better labels for live cell imaging Chrombodies? Nanoparticle probes? Overall Network Benefit primarily from having access to expertise and resources in other areas, not necessarily requiring extensive development E.g., FCS for monitoring actin-bound antibody/antigen interactions