Structure and Membrane Compartments

Keywords: lysosome-related organelles, exosomes, melanosomes, electron microscopy, immunocytochemistry, prion proteins, endosomes

Read the scientific activity report. (pdf 1.2Mo, last update 2th, february 2009)

The organelles of the endocytic pathway serve many ‘housekeeping' functions such as taking up nutrients, controlling signalling pathways and degrading unwanted macromolecules. But aside from these tasks, endocytic organelles can also serve specific functions in certain circumstances. The major goals of our research are to gain a better understanding of the biogenesis and functions of two such specialised endosomal organelles: exosomes, which are secreted from multivesicular bodies, and the lysosome-related organelles called melanosomes (Fig. 1).

We hope to unravel novel trafficking pathways and to understand how different molecular machineries evolve together to control trafficking events. Altogether these integrated studies provide for a conceptual working model to analyse the modifications of intracellular trafficking occurring during cell transformation and the cellular basis of lysosomal diseases and new insights will hopefully contribute to the development of novel therapeutic strategies.

Fig. 1 

In several cell types, the multivesicular bodies (MVBs) fuse with the cell surface and release intraluminal vesicles that are then called exosomes. Lysosome-related organelles (LROs) are generated in some cells of the hematopoietic and non-hematopoietic lineage. Melanosomes in melanocytes are an example of LROs.

Our group specializes in electron microscopy; the laboratory is equipped with high performance instruments for morphological and immunocytochemical analysis of cellular compartments, by using mainly immunogold labelling of ultrathin cryosections and whole cells. We study a variety of human and mouse cell types in culture by combining electron microscopy with light microscopy, biochemistry and siRNA methods.

Endosomes, exosomes and pathogens

In certain cell types, the multivesicular bodies of the endosomal pathway fuse with the cell surface to release into the extracellular environment small vesicles (50-90 nm) called exosomes. Many cells use exosomes to transfer proteins and lipids between them. We discovered that the glycophosphatidylinositol (GPI)-linked prion protein is released from cells in association with exosomes. Prions cause neurodegenerative disorders due to accumulation in the central nervous system of an abnormally folded form of the cellular prion protein called PrP^Sc, which is thought to be the infectious agent responsible for transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transmission of PrP^Sc are poorly understood.
Our findings suggest that exosomes might be vehicles for transmission of the infectious protein from one cell to another, so bypassing the need for direct cell-cell contact (Fig 2). Our recent studies indicate that retrovirus infections increase the release of prions associated with viral particles and exosomes.

Fig. 2 

Our studies throw light on the complexity of the endosomal system in some specialised cell types and demonstrate how endocytic organelles may be usurped not only by viruses but also by unconventional pathogens such as prions. A better knowledge of the mechanisms involved in exosome formation, secretion and targeting should help us to develop ways to interfere with transmission of these unconventional pathogens. We are currently studying prion protein trafficking in the endosomal system, with the same goal in mind.

Melanosomes, an example of lysosome-related organelles

Melanosomes are lysosome-related organelles (LROs) that synthesise and store the pigment melanin in melanocytes. In the skin, they are also secretory organelles, transferring melanin to keratinocytes and, thereby, generating the pigmentation of skin and hair. Melanosomes develop in stages defined by their structure and the enzymes they contain (Fig 3).

Fig. 3 

Distinct subdomains of endosomes are involved in sorting melanosomal proteins and in generating premelanosomes and mature melanosomes. Certain diseases associated with hypopigmentation are due to mutations in proteins involved in the regulation and specialisation of the endocytic pathway in melanocytes. Our recent studies provide evidence that the heterotetrameric adaptor proteins AP-3 and AP-1, the small GTPases Rab38/Rab32 and the BLOC (biogenesis of lysosome-related organelles complexes) are essential for the post-Golgi, endosomal sorting of melanosomal enzymes and other cargo such as the ATP7A copper transporter (Fig 4).

Fig. 4 

Our recent studies indicate that the enzymes are sorted from subdomains of early endosomes ; transport of TRP1 and tyrosinase results in maturation of premelanosomes to stage III and stage IV melanosomes. The adaptors AP-3 and AP-1, the BLOC complexes and the small GTPases Rab38/Rab32 are involved in the post-Golgi endosomal sorting of the melanosomal enzymes.

Studies of melanocytes by our team and others indicate that novel membrane trafficking regulators are involved in generating LROs. We aim to define better the different steps at which these proteins act, and how they co-ordinate with other traffic regulators such as Rabs and SNAREs. We also intend to image the three-dimensional organisation of the endosomal-melanosomal membrane system at high resolution using electron tomography.

Together, these studies provide a working model which helps further analysis of both the cellular basis of lysosomal diseases and the modifications of intracellular membrane trafficking that occur during cell transformation.

Last update: February 2009

Key publications

2007

• Raposo, G., and Marks, M.S.
  Melanosomes--dark organelles enlighten endosomal membrane transport
  Nat Rev Mol Cell Biol, 8, 786-797

2006

• Theos, A.C., Truschel, S.T., Tenza, D., Hurbain, I., Harper, D.C., Berson, J.F., Thomas, P.C., Raposo, G., and Marks, M.S.
  A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle morphogenesis
  Dev Cell, 10, 343-354

2004

• Fevrier, B., D. Vilette, F. Archer, D. Loew, W. Faigle, M. Vidal, H. Laude, and G. Raposo.
  Cells release prions in association with exosomes
  Proc Natl Acad Sci U S A., 101:9683-8