Funded projects

Development of a minimal in vitro system of lipid membrane based amyloidogenesis

2012 - G. Raposo (UMR144), G. Van Niel (team Raposo UMR144), D. Levy (UMR168)

Study the molecular mechanisms involved in the formation of amyloid fibers in exosomes through the establishment of a minimal in vitro model of amyloidogenesis that can be further exploited to understand pathological situations.

1. Isolation of PMEL recombinant fragments

2. Reconstitution of a minimal in vitro system able to reproduce the multilayer structure associated with PMEL

3. Analysis of liposomes by cryoEM

4. SILAC to characterize the composition of the exosomes containing multilayer structures to isoolate putative partners involved in amyloidogenesis and functional testing (kd and overexpression)

Methods/Technologies:

  • Biomimetic in vitro systems
  • CryoEM
  • SILAC

Keywords: biomimetics, exosomes, amyloidogenesis, cryoEM

Formation of amyloid fibrils results from the aggregation of proteins and protein fragments and it is mainly associated with pathological situations such as Alzheimerʼs disease (AD). There are increasing evidences for the involvement of endosomes and the intraluminal vesicles present in their lumen in the initiation of amyloidogenesis. A specialized cell, the melanocyte exploits endosomes to produce amyloid fibers in a physiological manner1. These physiological fibers result from the sorting and processing of the melanosomal transmembrane protein PMEL (or Pmel17) in association with the intraluminal vesicles of the endosomes2,3. Our previous studies and preliminary data indicate several homologies between pathological and physiological amyloidogenesis4 and reveal that intraluminal vesicles are potential nucleator platforms for amyloid 5. Our project proposes to delineate the molecular components and events involved in the formation of amyloid fibers on intraluminal vesicles.

This project will benefit from the expertise of the Team of G. Raposo and G. van Niel (UMR 144) and of D. Levy (UMR 168) in intracellular trafficking and biophysics of membrane, respectively. It gathers concepts of cell biology involving multi-components and biophysics of controllable biomimetic systems through state of the art electron microscopy of the platform of cell imaging. Our strategy relies on the reconstitution of a minimal in vitro system able to reproduce the intraluminal vesicles and their environment. Compared to previous approaches, we focus on the formation of fibers in the presence of lipidic model membranes rather than in solution. This will first allow to strengthen the role of intraluminal vesicles and of each subdomain of PMEL luminal domain in amyloidogenesis and to test the role of the density and orientation of PMEL in this process. Next, this model will be then used as an efficient tool to study the effect of proteases and potential partners identified in former studies as well as the role of lipid composition, pH or ionic strenght on membrane based amyloidogenesis. The establishment of a minimal in vitro model of amyloidogenesis could be also further exploited to understand pathological conditions.

Representative scheme of the working model and strategy of the project. 1. Our past and present work establishes that the luminal amyloidogenic domain of PMEL is shed within endosomes and sorted on intraluminal vesicles (ILV). This process is thought to induce amyloid fibril formation from the surface of ILV. Amyloid fibrils are stored in a specialized compartment (Melanosomes). 2. The goals of our project are to produce recombinant luminal domain of PMEL (rPMEL) (A) in order to develop an minimal in vitro system able to reproduce membrane-based amyloid formation (B) that we will investigate by several technics among which cryo-electron microscopy (C).

Representative scheme of the working model and strategy of the project. 1. Our past and present work establishes that the luminal amyloidogenic domain of PMEL is shed within endosomes and sorted on intraluminal vesicles (ILV). This process is thought to induce amyloid fibril formation from the surface of ILV. Amyloid fibrils are stored in a specialized compartment (Melanosomes). 2. The goals of our project are to produce recombinant luminal domain of PMEL (rPMEL) (A) in order to develop an minimal in vitro system able to reproduce membrane-based amyloid formation (B) that we will investigate by several technics among which cryo-electron microscopy (C).

1 Watt, B., van Niel, G., Raposo, G. & Marks, M. S. Pmel: A Pigment Cell-Specific Model For Functional Amyloid Formation. Pigment cell & melanoma Research, doi:10.1111/pcmr.12067 (2013).

2 Berson, J. F. et al. Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis. J Cell Biol 161, 521-533 (2003).

3 Van Niel, G. et al. The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during melanogenesis. Developmental Cell 21, 708-721, doi:10.1016/j.devcel.2011.08.019 (2011)

4 Rochin, L. et al. BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells. Proceedings of the National Academy of Sciences of the United States of America 110, 10658-10663, doi:10.1073/pnas.1220748110 (2013).

5 Hurbain, I. et al. Electron tomography of early melanosomes: implications for melanogenesis and the generation of fibrillar amyloid sheets. Proc Natl Acad Sci U S A 105, 19726-19731, doi:0803488105 [pii] 10.1073/pnas.0803488105 (2008).