Download Role of Cystinosin in Vesicular Trafficking and Membrane Fusion

Cystinosis Research Foundation
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Principal Investigator (s): Corinne Antignac, MD, PhD
Project Title: “Role of cystinosin in vesicular trafficking and membrane fusion”
Objective/Rationale: The cystinosis gene encodes a lysosomal cystin transporter, cystinosin.
Cells overexpressing cystinosin fused to a green-fluorescent protein (cystinosin-GFP) to allow
its easy identification under fluorescent microscopy, displayed aggregation of lysosomes, which
suggests the role of cystinosin in membrane fusion events. Moreover, little is known about the
way cystinosin is targeted to the lysosomes and the role of its two sorting signals in this
process. Our proposal is a one-year follow up of the initial project, to complete the data on
lysosomal targeting of cystinosin. Moreover, we will analyze the possible function of cystinosin
in mechanisms of vesicle fusion.
Project Description: The studies on targeting of lysosomal membrane proteins indicate the
existence of direct (intracellular) and indirect (via plasma membrane) pathways by which
proteins can be sorted to these organelles, mediated by distinct adaptor protein complexes. To
verify the way cystinosin is targeted to lysosomes, we will analyze the impact of depletion of
different adaptor proteins on the possible mislocalization of cystinosin-GFP to cellular
compartments other than lysosomes by confocal microscopy. Our previous study indicates that
cystinosin is mainly targeted via the direct pathway omitting plasma membrane. This will be
further analyzed using TIRF (total internal reflection fluorescence) microscopy and cell surface
biotinylation, both methods permitting to determine the level of cystinosin at the cell surface.
Moreover, when overexpressing cystinosin-GFP, we could observe the expansion of the Vti1b
positive compartment. Vti1b had recently been described as the protein involved in the fusion
of autophagosomes with lysosomes, suggesting that enlarged structures observed could be of
autolysosomal origin. We will further analyze if these vesicles are functional autolysosomes and
the role of cystinosin in their formation.
Relevance to the Understanding and/or Treatment of Cystinosis: The study of cystinosin
trafficking will help us to better understand process targeting cystinosin to the lysosome and
especially the role of the unconventional lysosomal targeting motif cystinosin bears. Moreover,
the finding of additional functions for cystinosin could explain why some symptoms of
cystinosis (i.e. the Fanconi syndrome) are not improved by lysosomal cystine depletion using
cysteamine, and provide insight into the complex processes governing vesicular fusion.
Anticipated Outcome: We expect to unravel the way cystinosin is targeted to the lysosomes
and the role of its both sorting motifs in this process. Moreover, by analyzing the impact of
cystinosin in vesicular fusion, we hope to better understand its function additional to the cystin
transport and thus the pathophysiological mechanisms underlying cystinosis.