Abstracts (complete list) - Wissenschaft Online

Stefanie Margraf, Carsten Watzl
Investigation of membrane microdomains surrounding
Natural Killer cell receptors
Natural killer (NK) cells play a central role in linking the adaptive and the acquired
immune system. They represent the first lymphoid subpopulation in the defence against
tumors and viral infection. The activity of NK cells is regulated by the interplay of
activating and inhibitory surface receptors. The engagement of these receptors as well
as the localization within different membrane areas is crucial for signalling. We have
recently shown that the activating receptors 2B4 (CD244) and NKG2D are localized in
so called ‘lipid rafts’ upon ligand binding or stimulation by mAbs. The localization in
these specialized membrane domains is crucial for the function of these receptors. Lipid
rafts are typically analyzed via cold non-ionic detergent lysis and sucrose density
gradient centrifugation. However, this method has been criticized to result in artefacts.
A limitation of this method is the isolation and examination of all lipid raft membrane
fractions and their associated proteins within a cell. Here we use a new approach to
isolate membrane fragments surrounding a specific surface receptor without destroying
its natural composition. NK cell receptors are stimulated by antibodies coupled to
magnetic beads. NK cells are then physically disrupted in a nitrogen-cavitation bomb
and membrane fractions surrounding the stimulated receptor are immunoisolated by
separating the magnetic beads. The protein composition of the purified membrane
clusters is then analyzed via 2D-Gelelectrophoresis. Examination of the natural
environment of activating and inhibitory NK cell receptors will give us a new insight into
the structure and composition of lipid microdomains and will help us to understand their
role in the regulation of NK cell activity.