Monitoring lipid anchor organization in cell membranes by PIE-FCCS.
This study examines the dynamic co-localization of lipid-anchored fluorescent proteins in living cells using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and fluorescence lifetime analysis. Specifically, we look at the pairwise co-localization of anchors from lymphocyte cell kinase (LCK: myristoyl, palmitoyl, palmitoyl), RhoA (geranylgeranyl), and K-Ras (farnesyl) proteins in different cell types. In Jurkat cells, a density-dependent increase in cross-correlation among RhoA anchors is observed, while LCK anchors exhibit a more moderate increase and broader distribution. No correlation was detected among K-Ras anchors or between any of the different anchor types studied. Fluorescence lifetime data reveal no significant Förster resonance energy transfer in any of the data. In COS 7 cells, minimal correlation was detected among LCK or RhoA anchors. Taken together, these observations suggest that some lipid anchors take part in anchor-specific co-clustering with other existing clusters of native proteins and lipids in the membrane. Importantly, these observations do not support a simple interpretation of lipid anchor-mediated organization driven by partitioning based on binary lipid phase separation.
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California 94720, USA.
Sara B
SB
Triffo
Hector H
HH
Huang
Adam W
AW
Smith
Eldon T
ET
Chou
Jay T
JT
Groves
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D019169:Jurkat Cells; D008051:Lipid Bilayers / Q000378:metabolism; D058635:Lipid-Linked Proteins / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
D008051:Lipid Bilayers; D058635:Lipid-Linked Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)