Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles

Abstract

Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured cells, were observed to reversibly phase segregate at temperatures significantly below 37 °C. In this contribution, we compare the temperature dependence of fluid phase segregation in HeLa and Rat basophilic leukemia (RBL) cells. We find an essentially monotonic temperature dependence of the number of phase separated vesicles in both cell types. We also observe a strikingly broad distribution of phase transition temperatures in both cell types. The binding of peripheral proteins, such as cholera toxin subunit B (CTB), as well as annexin V, is observed to modulate phase transition temperatures, indicating that peripheral protein binding may be a regulator for lateral heterogeneity in vivo. The partitioning of numerous signal protein anchors and full length proteins is investigated. We find Lo phase partitioning for several proteins assumed in the literature to be membrane raft associated, but observe deviations from this expectation for other proteins, including caveolin-1.

A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains

“…Posttranslational modifi cation with multiple saturated fatty acid motifs confers to some proteins an affi nity for lipid raft domains, as demonstrated by a number of biochemical, biophysical, and imaging approaches (43). In the case of Lck, a variety of studies indicate that its N-terminal targeting motif is suffi cient to effect its localization in lipid rafts ( 6,17,24,25,42 ). Indeed, biophysical studies using fl uorescent ( Fig.…”

“…Like the previously characterized fl uorescent reporter Lck-GFP ( 16 ), Lck-BAP-GFP incorporates the fi rst 26 N-terminal amino acids of the human Lck protein, which includes its post-translationally modifi ed membrane-targeting motif. This short N-terminal sequence has been shown in the context of chimeric proteins to be myristoylated, palmitoylated, and lipid-raft targeted ( 6,24,25 ). The Lck-BAP-GFP raft reporter developed here incorporates BAP and GFP modules ( Fig.…”

Glycolipid Crosslinking is Required for Cholera Toxin to Partition into and Stabilize Ordered Domains

“…This preference of CTxB for the l o phase is thought to arise in part from the intrinsic phase preference of GM1 itself (9). Moreover, binding of CTxB can drive the formation of both nanoscale and large-scale domains in cell-derived vesicles and model membranes that are close to a demixing point (6)(7)(8)10). Thus, CTxB not only functions as a raft marker but also as a domain inducer and raft stabilizer.…”

“…T misc is defined as the temperature at which 50% of the GPMVs contain coexisting l o and l d phases and provides a measure of the energetics of mixing behavior of the two phases (12,13). Treatment of GPMVs with WT CTxB has been previously reported to increase T misc , indicating that rafts are stabilized in the presence of CTxB (7). Consistent with this, we found that GPMVs isolated from COS-7 cells and subsequently treated with WT CTxB exhibited an increased T misc .…”

“…Its homopentameric B subunit (CTxB) recognizes and binds up to five molecules of its glycolipid receptor ganglioside GM1 (5). Previous studies have established that CTxB partitions into the l o phase, defining it as a raft marker (6)(7)(8). (Working definitions of terminologies used are in the Supporting Material.)…”