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Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling
Poster Abstracts
25-POS
Board 25
Phosphatidylinositol-4,5-bis(phosphate)-induced Moesin Adsorption on Supported Lipid
Bilayers : Role of Moesin Phosphorylation
Quentin Lubart
1,2
, Fabien DALONNEAU
1
, Laurent BLANCHOIN
2
, Catherine PICART
1
.
1
Laboratoire des matériaux et du génie physique, Grenoble, France,
2
CEA, Grenoble, Isère,
France.
Moesin protein from the ezrin/radixin/moesin family (ERM) link the cellular membrane to the
cytoskeletal actin filaments via the phosphatidylinositol-4,5-bis(phosphate) (PIP2). This link
induces many cellular events, such as the immunological synapse and microvilli formation. ERM
can be in a closed or in an open conformation. The open protein can interact with PIP2 and can
be phosphorylated at one or two threonine (T) residues. This phosphorylation helps to maintain
the open ERM conformation, which enables the proteins to be involved in other cellular events
and plays a role in the formation of protein dimers.
Our aim was to reconstitute in vitro the interactions between the plasma membrane and moesin
and to study the role of the specific phosphorylations.
To this end, wild-type moesin and double phosphomimetic mutant, where T235 and T558 were
replaced by aspartic acid (moesin-DD), were produced. We used PIP2-containing large
unilamellar vesicles (LUVs) and supported lipid bilayers (SLBs) to mimic the cell membrane.
Cosedimentation assays were used to quantify the affinity constant between the proteins and
PIP2-LUVs. Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to
investigate in real time the formation of PIP2-SLB and to quantify moesin adsorption. The
oligomerization of moesin was studied by analytical ultracentrifugation.
The affinity constant obtained by QCM-D and by cosedimentation experiments were in
agreement. The moesin-DD mutant formed more dimers in solution than wild-type moesin, had a
higher affinity for PIP2-containing membranes and interacted in a cooperative manner with the
PIP2-SLB.
These results suggested that phosphorylation may play a role in stabilizing moesin in an open
conformation at the plasma membrane. Our findings may be relevant for the formation in vivo of
the immunological synapse and microvilli.