PALM imaging and cluster analysis of protein heterogeneity at the cell surface.
The authors employed photoactivatable localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM) imaging and image analysis based on Ripley's K-function to quantify the distribution and heterogeneity of proteins at the cell plasma membrane. The membrane targeting sequence of the N-terminal region of the T cell receptor-pathway kinase Lck fused to the photo-convertible fluorescent protein tdEos (Lck(N10)-tdEos), clusters into sub-100 nm regions which cover approximately 7% of the cell surface. 2-channel PALM imaging of Lck(N10)-tdEos and the N-terminus of the kinase Src (Src(N15)-PS-CFP2) are demonstrated. Finally, T cell microclusters at the immune synapse are imaged at super-resolution using dSTORM, showing that conventional TIRF images contain unresolved, small clusters. These methods are generally applicable to other cell and fluorophore systems to quantify 2-D molecular clustering at nanometer scales.
Centre for Vascular Research, University of New South Wales, Sydney, New South Wales, Australia. dylan.owen@unsw.edu.au
Dylan M
DM
Owen
Carles
C
Rentero
Jérémie
J
Rossy
Astrid
A
Magenau
David
D
Williamson
Macarena
M
Rodriguez
Katharina
K
Gaus
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis*; D000818:Animals; D037421:Anthozoa; D002462:Cell Membrane / Q000737:chemistry*; D016000:Cluster Analysis; D003198:Computer Simulation; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence / Q000379:methods*; D055095:Optics and Photonics / Q000379:methods; D013269:Stochastic Processes; D013601:T-Lymphocytes / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C110810:LAT protein, human; D008164:Luminescent Proteins; D008565:Membrane Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases