Science, Technology and Engineering

Ryan Laboratory

Department of Biochemistry

Research - Mitochondrial Biogenesis and Disease

Mitochondria are the primary site of cellular ATP synthesis, and as such are essential for cell viability. They are also important in calcium homeostasis and apoptosis and are involved in many diseases. Mitochondria are not created de novo, but require the constant synthesis of mitochondrial and nuclear-encoded proteins for their biogenesis. Our research falls around 4 main areas: Mitochondrial morphology and distribution Respiratory complexes and mitochondrial disease Protein import into mitochondria Mitochondrial outer membrane permeabilization in apoptosis Techniques: We use a variety of techniques including in vitro import and assembly assays, blue-native PAGE, mammalian cell culture, RNAi and lentiviral protein expression, analysis of patient cell lines, metabolic labelling, protein expression and purification, protein structure determination, mass spectrometry, cross-linking and affinity techniques, mouse genetics, yeast genetics, fluorescence microscopy and live cell imaging. Support: Our work is primarily supported by the National Health and Medical Research Council and Australian Research Council.

Ryan lab 2011

Mike leads the Biology Program in the Centre of Excellence for Coherent X-ray Science (www.coecxs.org). The ARC funded Centre brings physicists, chemists and biologists together to develop fundamentally new approaches to probing biological structures and processes. It combines world-class expertise in imaging, structural biology, laser science and molecular theory. The project aims to develop novel high-resolution imaging of biological structures using the Australian Synchrotron, and other sources.

Mitochondrial morphology and distribution

In mammalian cells mitochondria are generally found as a reticulated network radiating from the nucleus with individual mitochondria displaying morphological and functional heterogeneity. Their diversity in form reflects a multiplicity of roles in cell development and differentiation. Sperm cells, for example, contain elongate mitochondria wrapped in a helical sheath around the axoneme complex to provide ATP for flagellum movement, whilst pancreatic acinar cells exist in three functionally unconnected mitochondrial populations that sense and discriminate between Ca 2+ signals in the immediate environment.

Although the mechanisms by which mitochondria undergo structural changes within a cell are not known, they are primarily affected by (i) fusion and fission events, and (ii) interaction with cytoskeletal elements, kinesin and attachment proteins. Changes in mitochondrial morphology are also required for mitochondrial turnover and appear important during programmed cell death. We are characterising a number of molecules involved in mitochondrial morphology and distribution.

Significance: A number of proteins identified in mitochondrial shaping have been directly linked to diseases including Parkinson's disease and neuromuscular disorders. The exact function and regulation of mitochondria fission, fusion and distribution remains obscure and significant biochemical and cell biological analysis is required. Characterisation of the machineries will enable us to better understand an area of biology that is fundamental to life.

Respiratory chain complexes and mitochondrial disease (in collaboration with Dr Mat McKenzie)

Complex I (NADH-Ubiquinone oxidoreductase) of the mitochondrial respiratory chain is a ~900 kDa complex containing 45 different subunits in humans, 7 of which are encoded by mtDNA. The remaining 38 nuclear encoded proteins are translated in the cytosol and must be imported into the organelle. Thus, mechanisms must be in place that co-ordinate and regulate the assembly of nuclear and mtDNA encoded Complex I subunits into the functional complex. Little information is so far known about the assembly and turnover of human Complex I.

Mitochondria diseases occur ~1/5,000 live births with defects in Complex I being the most prevalent. Complex I deficiency often results in multi-system disorders with a fatal outcome. Defects in Complex I have also been implicated in Parkinson’s disease and in eliciting cell death. We believe that in some cases, Complex I deficiency can be attributed to defects in the cellular machinery involved in the assembly of Complex I.

We are investigating the biogenesis of Complex I (and other respiratory complexes) and characterising the proteins involved in its assembly. Through a collaboration with Dr David Thorburn from the Murdoch Children's Research Institute (Royal Children's Hospital), we are examining skin cells from patients with mitochondrial disease to determine how mutations in genes encoding Complex I assembly factors contribute to Complex I dysfunction.

Protein import into mitochondria (in collaboration with Dr Diana Stojanovski)

The movement of proteins in and across membranes is an essential process in even the simplest of living cells. The extensive compartmentalisation of eukaryotic cells with the presence of multiple membranes places a high demand on the fidelity of this process. A large percentage of the eukaryotic cell’s protein complement are inserted into, or translocated across membranes. Most proteins of the endoplasmic reticulum are inserted into this organelle during their synthesis off ribosomes. However, proteins destined for mitochondria are typically imported into the organelle after translation. Protein precursors that are targeted to the mitochondrial matrix passage through TOM and TIM channels with the assistance of chaperones that pull or ratchet the precursor across. However, a large number of mitochondrial precursor proteins do not enter the matrix but still must translocate the mitochondrial outer membrane. The driving forces and mechanisms that govern this remain largely enigmatic.

Using mammalian and yeast models along with protein structure and function studies, we aim to investigate how the cooperation of cytosolic factors, mitochondrial translocation machineries and chaperones drive the import of these select mitochondrial precursors.

Significance: The primary benefit of this work will be the expansion of our knowledge regarding protein trafficking with eukaryotic cells and specifically those regulating mitochondrial biogenesis. This project has the capacity to outline new themes and descriptions on the nature of events governing precursor transport through the cytosol and translocation into mitochondria, and can transform the classical textbook descriptions of these events. This work will also provide a means for research into targeting of apoptotic factors to mitochondria and the analysis of diseases that arise from defects in protein biogenesis

Mitochondrial outer membrane permeabilization in apoptosis

Bax and Bak are pro-apoptotic factors which upon activation induce mitochondrial outer membrane permeabilisation (MOMP) to mediate cytochrome c release. This release is the point of no return in the process of programmed cell death. While intensely studied, the molecular mechanism of MOMP is not understood, hindered by the lack of biochemical approaches to analyse Bax and Bak activation. Using blue-native PAGE, we have found that inactive Bak associates with VDAC2 in ~400 kDa complexes at the mitochondrial outer membrane and is released during apoptosis. We aim to employ our techniques to explore the molecular mechanisms for Bak and Bax targeting to mitochondria, activation by pro-apoptotic BH3 only proteins and their regulation by anti-apoptotic factors. Significance: Apoptosis is one of the major mechanisms of cell death in response to cancer therapy. However, changes in expression of Bcl-2 family members can lead to resistance to such therapies. A number of agents that block the functions of anti-apoptotic family members from functioning are undergoing clinical trials. Our approaches will aid in the refinement of the Bak/Bax activation models and will provide important new insights into the roles of mitochondrial proteins other than Bcl-2 family members in the regulation of MOMP which have not been adequately studied.