Detergent-resistant membranes and the protein composition of lipid rafts

The plasma membrane of eukaryotic cells contains lipid rafts with protein and lipid compositions differing from the bulk plasma membrane. Several recent proteomic studies have addressed the composition of lipid rafts, but the different definitions used for lipid rafts need scrutinizing before results can be evaluated

Phospholipase D signaling: orchestration by PIP2 and small GTPases

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions

Cold-induced coalescence of T-cell plasma membrane microdomains activates signalling pathways

The plasma membranes of eukaryotic cells are hypothesised to contain microdomains with distinct lipid and protein composition known as lipid rafts. In T cells, cross-linking of lipid raft components triggers signalling cascades. We show that the T-cell antigen receptor (TCR) and a protein tyrosine kinase, Lck, have a patchy plasma membrane distribution in Jurkat T cells at reduced temperatures, although they have a continuous distribution at physiological temperature (37 degrees C). GM1 displays a patchy distribution at reduced temperature after Triton X-100 extraction. The archetypal non-lipid raft marker, the transferrin receptor, displays a more continuous plasma membrane distribution uncorrelated with that of Lck at 0 degrees C. Cold-induced aggregation of the lipid raft-partitioning proteins is accompanied by increased tyrosine phosphorylation and ERK activation, peaking at 10-20 degrees C. Tyrosine phosphorylation is further greatly increased by ligating the TCR with anti-CD3 at 10-20 degrees C. The tyrosine phosphorylation mainly occurred at the plasma membrane, was dependent on Lck and on the surface expression of the TCR. The activation of tyrosine phosphorylation and ERK by TCR ligation at reduced temperature also occurred in human primary T cells. These results support the concept that lipid rafts can form in membranes of live cells and that their coalescence stimulates signalling