Single-Molecule Diffusion Reveals Similar Mobility for the Lck, H-Ras, and K-Ras Membrane Anchors

Abstract

Recent evidence on the occurrence of small (5-700 nm diameter) lipid microdomains in the exoplasmic leaflet of the plasma membrane has evoked interest in the possibility that similar domains may also be present in the cytoplasmic leaflet of the plasma membrane. However, current knowledge about these "lipid rafts", in live cells is limited. One way to obtain insight into the occurrence and the size of lipid rafts is the use of single-molecule microscopy, which allows one to study the diffusive motion of individual molecules with high positional and temporal accuracy. Using this technique, we compared the diffusion behavior of the Lck membrane anchor, which has a high affinity for lipid rafts, to the diffusion behavior of the K-Ras membrane anchor, which has negligible affinity for rafts and compared the results with those of the H-Ras membrane anchor. Surprisingly, we found only minor differences in the diffusion behavior of the various lipid anchors, indicating that putative cytoplasmic leaflet lipid rafts would have to be small (<137 nm diameter) and do not affect the mobility of membrane-anchored molecules much on timescales up to 60 ms.

Alternate raft pathways cooperate to mediate slow diffusion and efficient uptake of a sphingolipid tracer to degradative and recycling compartments

“…A number of approaches could be used to test this model, including colocalization by super-resolution fluorescence imaging methods, or fluorescence cross-correlation spectroscopy, in parallel with studies of uptake kinetics. Similar bimodal diffusion behaviors to that displayed by SBD have been seen before, notably in a recent study by Pinaud et al (Pinaud et al, 2009), and earlier by Lommerse and colleagues (Lommerse et al, 2006). Other authors have recently derived diffusion constants from anisotropy and fluorescence recovery after photobleaching (ARAP and FRAP) (Goswami et al, 2008) and high-speed single-particle tracking (Umemura et al, 2008) on raftand non-raft molecules; however, these either showed complete immobility associated with nanoclusters, or uniformly distributed hop-diffusion behaviors, irrespective of raft or non-raft localization.…”

Signalling complexes and clusters: functional advantages and methodological hurdles

“…Segregation of Ras and RasGEFs into small plasma-membrane domains would also increase the efficiency and fidelity of the system, because new activated Ras molecules are rapidly generated at the boundary between the cluster and the inactive substrates that are homogenously distributed throughout the membrane. Clustering and increased fidelity of the system only occurs in the presence of slow reactant diffusion, emphasising the importance of membrane anchorage of Ras molecules and their effectors (Das et al, 2009b;Lommerse et al, 2006).…”

Plasma membrane regulates Ras signaling networks

“…An example of this comes from the comparison of Lck, which localizes to lipid rafts, and K-Ras using high accuracy position and temporal resolution single-molecule microscopy. 49 This study revealed only minor differences in the diffusion behavior, suggesting that lipid rafts must be very small (<137 nm in diameter) and do not affect the mobility of Lck and K-Ras. Modern super high resolution imaging techniques, such as photo-activated localization microscopy or stochastic optical reconstruction microscopy that can resolve structures as small as 10 nm in diameter, 50 will be very useful in addressing the questions in regards to Ras membrane localization.…”