Raposo

Structure and Membrane Compartments

Graça Raposo
Scientific keywords: amyloid fibrils, exosomes, intracellular trafficking, Lysosome-related organelles and associated diseases, melanosomes, protein processing
Technics Used in the Lab: Electron Microscopy: Conventional ultrastructural analysis, immunocytochemistry in resin embedded cells and tissues and ultrathin frozen sections (Tokuyasu). High Pressure Freezing, Freeze substitution, Electron Tomography. Photonic microscopy: immunofluorescence (3D deconvolution), live cell imaging (spinning disc, TIRF) Correlative Light Electron Microscopy (CLEM) Biochemistry: western blotting, immunoprecipitations, sucrose gradient organelles purification, Melanin quantification, production of recombinant proteins Cell culture: Transfection (plasmids, siRNAs), infection (shRNA), Melanocytic cell line, primary human melanocytes and keratinocytes, 2D co-culture.

The major goals of our research are to gain a better understanding of the biogenesis and functions of specialized endosomal organelles: the lysosome-related organelles of pigment cells called melanosomes and exosomes, which are secreted from multivesicular bodies. One focus is on elucidating mechanisms involved in early melanogenesis in particular those underlying the generation of physiological amyloids (sorting and processing mechanisms). Another aspect addresses how genes mutated in lysosomal diseases (Hermansky Pudlak Syndrom) regulate late stages of melanogenesis and how these novel trafficking regulators operate in concert with other ubiquitous components of the trafficking machinery (adaptors, Kinesins, Myosins, Rab GTPases and effectors). Our projects aim at over passing the current knowledge on the cellular and molecular basis of melanosome dysfunction in pigmentation related disorders and melanoma and the role of exosomes in pigmentation and amyloidogenesis. These studies will also contribute to enlighten generalized structural and functional modifications of the endosomal system in specialized cells. They aim at shedding light onto the molecular machinery that controls endosomal sorting and motility and the mechanisms underlying cellular communications within the epidermal-melanin unit by direct cell-cell contacts or via secretion of exosomes.

Fig.1 KIF13 is an endosomal recycling kinesin. It is required for endosome positionning close to melanosomes (live cell, electron tomography). It’s inactivation leads to decreased pigmentation

Fig.1 KIF13 is an endosomal recycling kinesin. It is required for endosome positionning close to melanosomes (live cell, electron tomography).
It’s inactivation leads to decreased pigmentation

Fig. 2. BACE2 in formation of amyloids by PMEL. BACE 2 KO mice show pigmentation defects. Pigment cells depleted for BACE2 display abnormal amyloid fibrils (electron tomography).

Fig. 2. BACE2 in formation of amyloids by PMEL. BACE 2 KO mice show pigmentation defects. Pigment cells depleted for BACE2 display abnormal amyloid
fibrils (electron tomography).