Dataset Information

Monitoring lipid anchor organization in cell membranes by PIE-FCCS.

ABSTRACT: 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.

SUBMITTER: Triffo SB

PROVIDER: S-EPMC3626236 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Monitoring lipid anchor organization in cell membranes by PIE-FCCS.

Triffo Sara B SB Huang Hector H HH Smith Adam W AW Chou Eldon T ET Groves Jay T JT

Journal of the American Chemical Society 20120614 26

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-corr ...[more]

PMID: 22631607

Similar Datasets

Project description:Ras GTPases play a crucial role in signal transduction cascades involved in cell differentiation and proliferation, and membrane binding is essential for their proper function. To determine the influence of the nature of the lipid anchor motif and the difference between the active (GTP) and inactive (GDP) forms of N-Ras on partitioning and localization in the lipid membrane, five different N-Ras constructs with different lipid anchors and nucleotide loading (Far/Far (GDP), HD/Far (GDP), HD/HD (GDP), Far (GDP), and HD/Far (GppNHp)) were synthesized. Using the surface plasmon resonance technique, we were able to follow the insertion and dissociation process of the lipidated proteins into and out of model membranes consisting of pure liquid-ordered (l(o)) or liquid-disordered (l(d)) phase and a heterogeneous two-phase mixture, i.e., a raft mixture with l(o) + l(d) phase coexistence. In addition, we examined the influence of negatively charged headgroups and stored curvature elastic stress on the binding properties of the lipidated N-Ras proteins. In most cases, significant differences were found for the various anchor motifs. In general, N-Ras proteins insert preferentially into a fluidlike, rather than a rigid, ordered lipid bilayer environment. Electrostatic interactions with lipid headgroups or stored curvature elastic stress of the membrane seem to have no drastic effect on the binding and dissociation processes of the lipidated proteins. The monofarnesylated N-Ras exhibits generally the highest association rate and fastest dissociation process in fluidlike membranes. Double lipidation, especially including farnesylation, of the protein leads to drastically reduced initial binding rates but strong final association. The change in the nucleotide loading of the natural N-Ras HD/Far induces a slightly different binding and dissociation kinetics, as well as stability of association, and seems to influence the tendency to segregate laterally in the membrane plane. The GDP-bound inactive form of N-Ras with an HD/Far anchor shows stronger membrane association, which might be due to a more pronounced tendency to self-assemble in the membrane matrix than is seen with the active GTP-bound form.

Dataset Information

Monitoring lipid anchor organization in cell membranes by PIE-FCCS.

Publications

Monitoring lipid anchor organization in cell membranes by PIE-FCCS.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets