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20
Novel fluorescent probes to study sphingomyelin
dynamics in living cells
Claudio Giraudo 1 , Lavan Khandan 1 , Jeff Coleman 1 , James Rothman 1 .
Yale University, New Haven, CT 06520 1
Abstract:
Sphingomyelin is a highly abundant lipid in eukaryotic membranes and has been implicated in
modulating signal transduction, enzymatic reactions and viral budding from infected cells.
Sphingomyelin, cholesterol and glycosphingolipids share the ability to decrease membrane
fluidity by segregating laterally in the plane of the membrane and give rise to domains ranging
from 20-100nm that are enriched in these lipids and specific proteins. The synthesis of most of
these lipids is carried out in the Golgi Complex and from there, they are transported and
concentrated at the plasma membrane. To investigate the dynamics of sphingomyelin in living
cells and its ability to form nanodomains within the Golgi membranes we generated a fusion
construct coding for a signal sequence, a fluorescent protein and the non-toxic region of lysenin
toxin, which specifically binds to sphingomyelin. Expression of this construct in cultured cells
showed a spotted distribution mainly concentrated in the perinuclear area with a scarce colocalization
with Golgi markers. Temperature blockage at 20°C for 3 h considerably increased
the level of co-localization with Golgi markers. Preliminary results showed that knocking down
the expression of the sphingomyelin synthase-1 and -2 genes by using siRNA, and thus
inhibiting sphingomyelin synthesis, resulted in a dispersal of the fluorescent marker throughout
the cells. Further studies are required to determine whether Golgi components and cargo
molecules are differentially included/excluded from sphingomyelin rich domains within the
secretory pathway. Altogether, these results set the stage for future experiments to study the
dynamics of sphingomyelin in living cells and can be used as springboard to study the dynamics
of other lipids by using appropriate toxins that bind to them.