In situ quantitative imaging of cellular lipids using molecular sensors.
Membrane lipids are dynamic molecules that play important roles in cell signalling and regulation, but an in situ imaging method for quantitatively tracking lipids in living cells is lacking at present. Here, we report a new chemical method of quantitative lipid imaging using sensors engineered by labelling proteins with an environmentally sensitive fluorophore. A prototype sensor for phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2))--a key signalling lipid in diverse cellular processes--was generated by covalently attaching a single 2-dimethylamino-6-acyl-naphthalene group to the N-terminal α-helix of the engineered epsin1 ENTH domain, a protein that selectively binds PtdIns(4,5)P(2). The sensor allows robust and sensitive in situ quantitative imaging in mammalian cells, providing new insight into the spatiotemporal dynamics and fluctuation of this key signalling lipid. Application of the sensor to immune cells reveals the presence of a local threshold PtdIns(4,5)P(2) concentration required for triggering phagocytosis. This sensor strategy is generally applicable to in situ quantification of other cellular lipids.
Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Youngdae
Y
Yoon
Park J
PJ
Lee
Svetlana
S
Kurilova
Wonhwa
W
Cho
D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D010587:Phagocytosis; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020123:Sirolimus / Q000494:pharmacology; D020349:Surface Plasmon Resonance
D033942:Adaptor Proteins, Vesicular Transport; D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D008055:Lipids; D019269:Phosphatidylinositol 4,5-Diphosphate; C114154:epsin; D020123:Sirolimus