Session 1

Osteoclast recycling and the osteomorph:  mechanism behind the therapeutic response to denosumab withdrawal

Michelle M. McDonald

The anti-RANKL treatment Denosumab (Dmab) is an effective treatment for osteoporosis. However, rapid bone loss and increased fracture have been associated with treatment withdrawal. To examine this in real-time in vivo, we developed a novel intravital imaging methodology to visualize osteoclast dynamics in tibia in live mice. We showed that in addition to apoptosis, osteoclasts undergo fission and recycle their cellular constituents (osteomorphs) as they re-fuse, and used scRNAseq to define these novel osteomorphs. We also showed accumulation of osteomorphs and their re-fusion following withdrawal of RANKL inhibition, providing a mechanism for rebound bone loss and following Denosumab withdrawal.

Lighting up the pathways of caspase activation

Lisa Bouchier-Hayes

The caspase family of proteases are essential for the initiation and execution of apoptosis and innate immune signaling. Initiator caspases are the first activated in their pathway and are activated by proximity-induced dimerization upon recruitment to large molecular weight activation platforms. We have adapted bimolecular fluorescence complementation (BiFC) to measure caspase induced proximity, the first step in initiator caspase activation. By fusing non-fluorescent fragments of Venus to each caspase, we monitor the induced proximity of specific caspases through the refolding of Venus and resulting fluorescence. This allows for accurate spatial and temporal measurements of specific caspase activation in live cells. 

Deterministic early endosomal maturation driven by EEA1 noise suppression

Mr Harrison York, PhD Student, Monash University

Co Authors
Mr Kunaal Joshi, Purdue University
Dr Charles Wright, Purdue University
Mr Ullhas Moorthi, Monash University
Dr Hetvi Gandhi, Monash University
Mr Abhishek Patil, Monash University
Dr Srividya Iyer-Biswas, Purdue University
Dr Senthil Arumugam, Monash University

Endosomal trafficking in single cells is built of generation of membrane vesicles, their motor protein mediated transport, morphological alterations such as tubulation, fusion and fission, and dynamic maintenance of various identities, which is defined by the lipid composition and localization of specific proteins on them. Endosomal maturation is a major process in endosomal trafficking in which endosomes shed one specific protein and acquire another, resulting in an identity change. Individual processes that build up endosomal trafficking, including conversions, are interlinked and are inherently stochastic. While the general biochemical interactions have been well described, how all the events come together to overcome the inherent noise and stochasticity is much less explored. Here, capitalising on the rapid volumetric imaging capability of Lattice-light sheet, we capture whole-cell volumes, enabling post-acquisition analysis of all conversion events as well as other dynamic characteristics. We show that early endosome maturation is driven by endosomal collision-induced conversions. Furthermore, using live-cell Förster Resonance Energy Transfer, we demonstrate that this is underpinned by cyclical conformational changes in EEA1, which promotes the biochemical maturation of these vesicles through its asymmetric binding capacity and clustering on the endosomal membranes. Using simulations, we recapture the experimentally observed characteristics in the reaction scheme and the activity of EEA1. Based on these experiments, we describe an EEA1-dependent mechanism that enables deterministic outcomes in ensemble endosomal conversions in an otherwise stochastic system.

Shedding light onto the structural secrets inside pluripotent stem cells in real-time

Dr Jennifer Zenker, Group Leader, Australian Regenerative Medicine Institute, Monash University

Co Authors
Dr Asma Aberkane, Monash University
Ms Azelle Hawdon, Monash University
Ms Gemma Stathotos, Monash University
Dr Jessica Greaney, Monash University
Ms Yi Louise Li, Monash University

The organisation of a cell’s interior, the cytoskeleton and organelles, is fundamental for every cell’s functionality. However, unlike most differentiated cells, our knowledge about the contribution of the sub-cellular architecture to pluripotency remains scarce. Using advanced live imaging, we discovered polarised non-centrosomal microtubules as central player for the pluripotent state of the in vivo early mammalian embryo and induced pluripotent stem cells (iPSCs). Each cell contains an apical pole highly enriched with CAMSAP3-nucleated microtubules, growing in a longitudinal direction towards the base of PSCs. These non-centrosomal microtubules initiate an asymmetry of organelles and metabolites. We further establish their rearrangement upon differentiation in a germ layer-specific manner. Dissecting how intrinsic cellular regulation contributes to pluripotency will lead a revolutionary era of regenerative and reproductive medicine.