A Two‐Photon Fluorescent Probe for Lipid Raft Imaging: C‐Laurdan

Kim, Hwan Myung; Choo, Hyo Jung; Jung, Sung-No; Ko, Young Gyu; Park, Won Hwa; Jeon, Seung Joon; Kim, Chul Hoon; Joo, Taiha; Cho, Bong Rae

doi:10.1002/cbic.200700003

Cited by 226 publications

(249 citation statements)

References 28 publications

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“…This behavior is similar to that observed for laurdan, with the exception that the fl uorescence spectrum of the latter probe in water is more shifted to the blue than expected if only considering the solvent polarity ( 22 ). Also, C-laurdan presents enhanced photostability under two-photon excitation and higher water solubility than laurdan due to the fact that one of the methyl groups of the dimethylamine moiety of laurdan was replaced by a carboxylic group in C-laurdan ( 22 ). These properties make C-laurdan an extremely useful probe for studying lipid membrane properties, and thus it is starting to be used in studies of membrane organization in living cells ( 22,23 ).…”

Section: Confocal Microscopy Measurements and Gp Determinationssupporting

confidence: 83%

“…Recently Kim et al ( 22 ) have synthesized a new fl uorescent probe, 6-dodecanoyl-2-[ N -methyl-N -(carboxymethyl) amino]naphthalene (C-laurdan), designed for imaging and sensing lipid organization in membranes.…”

Section: Confocal Microscopy Measurements and Gp Determinationsmentioning

confidence: 99%

“…Also, C-laurdan presents enhanced photostability under two-photon excitation and higher water solubility than laurdan due to the fact that one of the methyl groups of the dimethylamine moiety of laurdan was replaced by a carboxylic group in C-laurdan ( 22 ). These properties make C-laurdan an extremely useful probe for studying lipid membrane properties, and thus it is starting to be used in studies of membrane organization in living cells ( 22,23 ).…”

Section: Confocal Microscopy Measurements and Gp Determinationsmentioning

confidence: 99%

See 2 more Smart Citations

Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Traian

Flecha

Levi

2012

Journal of Lipid Research

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Section: Confocal Microscopy Measurements and Gp Determinationssupporting

confidence: 83%

Section: Confocal Microscopy Measurements and Gp Determinationsmentioning

confidence: 99%

Section: Confocal Microscopy Measurements and Gp Determinationsmentioning

confidence: 99%

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Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Traian

Flecha

Levi

2012

Journal of Lipid Research

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“…These highly ordered structures are proposed to function in dynamic cellular processes such as membrane trafficking, signal transduction, and regulation of membrane protein activity. 40 Although the existence of membrane microdomains in live cells has been controversial, some studies [41][42][43] provided experimental evidence for these microdomains in living cell membranes.…”

Section: Discussionmentioning

confidence: 99%

Cystin Localizes to Primary Cilia via Membrane Microdomains and a Targeting Motif

Tao

Yang³

et al. 2009

Journal of the American Society of Nephrology

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Primary cilia are dynamic, complex structures that contain Ͼ500 proteins, including several related to polycystic kidney disease. How these proteins target to cilia and assemble is unknown. We previously identified Cys1 as the gene responsible for disease in Cys1 cpk mice, a mouse model of autosomal recessive polycystic kidney disease; this gene encodes cystin, a 145-amino acid cilium-associated protein. Here, we characterized the localization of cystin in the embryonic kidney and liver, in isolated renal collecting ducts, and in an inner medullary collecting duct mouse cell line. Because endogenous levels of cystin expression are low, we generated inner medullary collecting duct cell lines that stably express enhanced green fluorescence protein-tagged constructs of wild-type cystin or various truncation mutants. We determined that cystin is myristoylated at its G2 residue and that N-myristoylated cystin fractionates with membrane microdomains. Furthermore, the N-myristoylation signal is necessary but not sufficient to target cystin to the primary cilium. Analysis of deletion and chimeric constructs identified an AxEGG motif that is necessary to target and retain cystin in the cilium. Derangement of these localization motifs may lead to cystic kidney disease.

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“…Despite its many applications in model (30,32) and cellular membranes (29,33,34), Laurdan has some disadvantages on a methodological level, including low solubility in aqueous media, low fluorescence intensity, and photo-bleaching, which were overcome by the addition of a carboxyl-headgroup (6-dodecanoyl-2-methylcarboxymethylaminonaphthalene; C-laurdan) (35).…”

mentioning

confidence: 99%

Order of lipid phases in model and plasma membranes

Kaiser

Lingwood

Levental

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

425

396

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Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165-3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective.generalized polarization value ͉ giant unilamellar vesicle ͉ membrane organization ͉ lipid sorting ͉ lipid raft T he lipid raft hypothesis postulates that selective interactions among sphingolipids, cholesterol, and membrane proteins contribute to lateral membrane heterogeneity (1). A tenet of the model is that small, dynamic cholesterol-sphingolipid-enriched assemblies can be induced to coalesce into larger, more stable structures through clustering of domain components (2). Although experimental data support cholesterol-dependent nano-scale membrane heterogeneity (3-8) and selective domain formation upon raft cross-linking (9-12), the mechanisms that govern such associations in cell membranes remain unclear.On the molecular level, a key feature that is thought to contribute to raft assembly is the propensity of cholesterol to pack tightly with saturated acyl chains of lipids causing them to adopt an extended conformation (13,14). In multi-component model membranes (n Ͼ 2), this interaction can lead to microscopically separate fluid membrane phases: the liquid-ordered (Lo) phase, enriched in saturated (sphingo-)lipids and cholesterol in a highly condensed state, and the liquid-disordered (Ld) phase, enriched in unsaturated glycerophospholipid in a disordered state (15)(16)(17).Several features of the Lo phase in model membranes correspond to the predicted properties of lipid rafts in cel...

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A Two‐Photon Fluorescent Probe for Lipid Raft Imaging: C‐Laurdan

Cited by 226 publications

References 28 publications

Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Cystin Localizes to Primary Cilia via Membrane Microdomains and a Targeting Motif

Order of lipid phases in model and plasma membranes

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