Role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas.
We previously found that a directional movement of the raft component GD3 towards mitochondria, by its association with microtubules, was mandatory to late apoptogenic events triggered by CD95/Fas. Since CLIPR-59, CLIP-170-related protein, has recently been identified as a microtubule binding protein associated with lipid rafts, we analyzed the role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas. To test whether CLIPR-59 could play a role at the raft-microtubule junction, we performed a series of experiments by using immunoelectron microscopy, static or flow cytometry and biochemical analyses. We first assessed the presence of CLIPR-59 molecule in lymphoblastoid T cells (CEM). Then, we demonstrated that GD3-microtubule interaction occurs via CLIPR-59 and takes place at early time points after CD95/Fas ligation, preceding the association GD3-tubulin. GD3-CLIPR-59 association was demonstrated by fluorescence resonance energy transfer (FRET) analysis. The key role of CLIPR-59 in this dynamic process was clarified by the observation that silencing CLIPR-59 by siRNA affected the kinetics of GD3-tubulin association, spreading of GD3 towards mitochondria and apoptosis execution. We find that CLIPR-59 may act as a typical chaperone, allowing a prompt interaction between tubulin and the raft component GD3 during cell apoptosis triggered by CD95/Fas. On the basis of the suggested role of lipid rafts in conveying pro-apoptotic signals these results disclose new perspectives in the understanding of the mechanisms by which raft-mediated pro-apoptotic signals can directionally reach their target, i.e. the mitochondria, and trigger apoptosis execution.
Sorice
Maurizio
M
Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy. maurizio.sorice@uniroma1.it
Matarrese
Paola
P
Manganelli
Valeria
V
Tinari
Antonella
A
Giammarioli
Anna Maria
AM
Mattei
Vincenzo
V
Misasi
Roberta
R
Garofalo
Tina
T
Malorni
Walter
W
D017209:Apoptosis / Q000276:immunology* / Q000502:physiology; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D005732:Gangliosides / Q000502:physiology*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000502:physiology*; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D019014:fas Receptor / Q000276:immunology*
C449670:CLIP3 protein, human; D005732:Gangliosides; D008869:Microtubule-Associated Proteins; D019014:fas Receptor; C026226:ganglioside, GD3