Abstract
Lipid rafts are suggested to be sphingolipid and cholesterol rich domains that
segregate out from the bulk plasma membrane by forming a more ordered lipid
phase. Lipid raft-like domains have been described as cell signalling platforms and
have been implicated in the regulation of an array of signal transduction events. This
study investigates the association of two classes of protein, 14-3-3 proteins and
plasma membrane SNAREs, with lipid raft-like domains. The 14-3-3 family of
proteins are important regulators of numerous cell signalling pathways and are
essential for cell survival; recently there has been some interest in the roles of these
soluble proteins at the membrane, though this area remains poorly characterised.
14-3-3 has also been linked to CJD progression, which is directed by the lipid raft
associated prion protein. SNAREs are essential mediators of exocytosis, a process
that is also reported to depend on cholesterol, implying lipid raft involvement.
SNAREs have also been isolated in detergent resistant membranes (DRMs) that are
believed to represent clustered lipid rafts.
To examine the association of 14-3-3 and SNAREs with lipid raft-like domains in
N2a and PC 12 cells two approaches were taken. Initially, detergent resistant
membranes were isolated and analysed for protein association. The second approach
involved quantitative analysis of the colocalisation of 14-3-3 and SNAREs with
membrane domains in intact cells by confocal microscopy, using the lipid raft marker
cholera toxin B subunit (CTXB). Discrepancies between results from these two
methods add to evidence implying that DRMs do not necessarily represent pre¬
existing membrane domains. Cholesterol depletion, which affects the integrity of
lipid raft-like domains, caused a rearrangement CTXB labelled clusters in N2a and
PC 12 cells. The colocalisation of 14-3-3 with CTXB was unaffected by cholesterol
depletion, a result which does not support the localisation of 14-3-3 to lipid raft-like
domains. Interestingly however, the membrane distribution of the lipid raft marker
Thy-1, a GPI-anchored protein, was also unaltered when cholesterol was depleted. In
contrast to previous reports, disruption of SNAP-25 or syntaxinla (SNARE) clusters was not observed following cholesterol depletion. However, in N2a cells, the
colocalisation of SNAP-25 with CTXB was reduced, though this was not the case in
PC 12 cells. Taken together these results suggest that cholesterol depletion may affect
various raft-associated proteins and cell types in different ways. The findings from
N2a cells indicate a role for lipid raft-like domains in controlling the spatial
distribution of SNAP-25 on the plasma membrane. The membrane distribution of
syntaxinla appears to be differently regulated from that of SNAP-25, which may
have implications for the regulation of exocytosis.
