Research Network for Metals in Medicine

 

 

Dr Sharon La Fontaine

Position: Senior Research Officer, (NH&MRC R. Douglas Wright Fellowship)

Affiliation: Deakin University, School of Biological and Chemical Sciences

Postal Address:
School of Biological and Chemical Sciences
221 Burwood Highway
Burwood Victoria, 3125
AUSTRALIA

Phone: +61 (03) 9251 7348
Fax: +61 (03) 9251 7328
Email: sharonl@deakin.edu.au
Webpage: http://agrippina.bcs.deakin.edu.au/ASP_pages/staffsystem/showprofile/default.asp?Member=335


Research Profile

My research career began with a prestigious postgraduate scholarship and led to five publications, one patent and several awards. This level of productivity, recognized by further awards, continued into postdoctoral research in Prof Julian Mercer's laboratory (The Murdoch Institute [Dec. 1994-1998] and Deakin University [1999-present]). During this time I also gave several invited presentations (University of Utrecht, The Netherlands, 1997; The 1st International Meeting on Copper Transport and its Disorders: Molecular and Cellular Aspects, Italy, 1997; ASBMB, Adelaide, 1998; FASEB Summer Conference, USA, 1998, and UCLA, USA, 1999), and authored 3 book chapters. During July 1999 to July 2000 I studied copper metabolism in the unicellular green algae, Chlamydomonas reinhardtii, in Prof. Sabeeha Merchant's laboratory, UCLA, USA, which led to a key publication (SL10) and a review of copper and iron metabolism in plants for a book chapter (SL2). In 2000 I was awarded an NH&MRC R. Douglas Wright Fellowship that has enabled me to identify novel proteins which interact with the Menkes and Wilson ATPases. These are currently being characterized to further elucidate the details of the copper pathway in cells and will form a substantial research program that I will lead.

I have spent 9 years in the field of copper metabolism, mostly working with Prof Julian Mercer on mammalian copper metabolism, with one year spent working with Prof Sabeeha Merchant, UCLA, USA on copper and iron metabolism in the model photosynthetic organism, the unicellular green algae, Chlamydomonas reinhardtii. In Prof. Merchant's laboratory, I studied several genes involved in copper and iron metabolism in this organism. This work demonstrated for the first time a role for copper in iron metabolism in a photosynthetic species, but also led us to propose the existence of an additional copper-independent iron assimilation pathway in this organism.

In Prof Mercer's laboratory I developed mammalian expression vectors (La Fontaine et al, 1998, Plasmid 39:245-251) that were pivotal in establishing expression systems for structure/function studies of the Menkes (MNK) and Wilson (WND) proteins, and have formed the basis for the research activities and productivity of our group (as evident from publication output). These plasmids solved the plasmid stability problems in Escherichia coli, which had plagued experiments both here and overseas, and continue to be employed by our local and international collaborators.

I successfully expressed the Menkes protein in Chinese Hamster Ovary cells (La Fontaine et al, 1998, Hum. Mol. Genet. 7:1293-1300) which enabled us to demonstrate its intracellular localization and its association with vesicles within the cell, an important step towards elucidating the pathway of copper transport within the cell. An important contribution was the demonstration that MNK and WND could restore copper transport to Menkes patient fibroblasts, confirming for the first time that mutations within the MNK gene are solely responsible for the defective copper transport and the diverse phenotypes of Menkes patients, and that the closely related WND could functionally substitute for MNK (La Fontaine et al, 1998, J. Biol. Chem. 273:31375-31380). This expression system continues to be central to all of the ongoing projects in our laboratory aimed at investigating the cell biology of MNK and WND and the functional effects of mutations in these molecules (eg. Petris et al., 1998 Hum. Mol. Genet. 7:2063-2071; SL3-6). More recently I have become involved in investigations of the molecular and cellular basis for copper toxicity in sheep, as well as in a comparative study of the cell biology of the WND orthologues from species (mice, sheep, human) with distinct copper metabolism. As my major project (funded by an NHMRC R.D. Wright Award) I am currently characterizing several novel proteins that I have identified to interact with MNK and WND which will make a substantial and important contribution to this field, as new molecules implicated in the pathway of cellular copper transport.

Selected Publications

  1. S. La Fontaine, S.D. Firth, P.J. Lockhart, H. Brooks, J. Camakaris and J.F.B. Mercer Functional analysis of the Menkes disease protein. Adv. Exp. Med. Biol. 448, 67-82 (1999)
  2. D. Strausak, S. La Fontaine, J. Hill, S.D. Firth, P.J. Lockhart and J.F.B. Mercer The Role of GMxCxxC Metal Binding Sites in the Copper-induced Redistribution of the Menkes Protein (MNK or ATP7A). J. Biol. Chem. 274, 11170-11177 (1999).
  3. S. La Fontaine, S. D. Firth, P. Lockhart, H. Brooks, J. Camakaris, J.F.B. Mercer Intracellular localization and loss of copper responsiveness of Mnk, the murine homologue of the Menkes protein, in cells from blotchy (Moblo) and brindled (Mobr) mouse mutants. Hum. Mol. Genet. 8, 1069-1075 (1999)
  4. S. La Fontaine, M.B. Theophilos, S.D. Firth, R. Gould, R.G. Parton, and J.F.B. Mercer* Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copper ATPase. Hum. Mol Genet. 10, 361-370 (2001)
  5. I. Voskoboinik, M. Greenough, S. La Fontaine, J.F.B. Mercer and J. Camakaris* Functional studies on the Wilson copper P-type ATPase and Toxic Milk Mouse mutant. Biochem. Biophys. Res. Commun. 281, 966-970 (2001)
  6. K.D. Bissig, S. La Fontaine, J.F. Mercer, and M. Solioz. Expression of the human Menkes ATPase in Xenopus laevis oocytes. Biol Chem. 382, 711-714, (2001)
  7. P.J. Lockhart, S. La Fontaine, S.D. Firth, M. Greenough, J. Camakaris, and J.F. Mercer* Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase. Biochim Biophys Acta. 1588,189-94, (2002).
  8. S. La Fontaine, J.M. Quinn, S.S. Nakamoto, M.D. Page, V. Göhre, J.L. Moseley, J. Kropat, and S. Merchant. Copper-dependent iron assimilation pathway in the model photosynthetic eukaryote Chlamydomonas reinhardtii. Eukaryot Cell. 1, 736-757, (2002).
  9. M.C. Pascale, S. Franceschelli, O. Moltedo, F. Belleudi, M.R. Torrisi, C. Bucci, S. La Fontaine, J.F.B. Mercer and A. Leone. The endosomal trafficking of the Menkes copper ATPase ATP7A is mediated by vesicles containing the Rab7 and Rab5 GTPase proteins Exp. Cell Res. Exp. Cell Research 291, 377-385 (2003).


Facilities

The Centre for Cellular and Molecular Biology (CCMB) at Deakin University, in which our laboratory is based, is fully furnished with general equipment necessary to undertake biochemical, molecular and cellular biology projects, and includes an atomic absorption spectrophotometer for metal analysis, and a recently purchased state-of-the-art Leica TCS SP2 confocal laser microscope with acousto-optic beam splitter providing significantly increased sensitivity over standard models to obtain excellent quality images of protein localization and trafficking.