Manos Mavrakis1, Richa Rikhy1,
Bob Phair2, Jennifer Lippincott-Schwartz1.
1) Cell Biology and Metabolism Branch, National Institute of Child
Health and Human Development, National Institutes of Health,
Bethesda, MD 20892; 2) Integrative Bioinformatics Inc, Los Altos, CA
94024.
In this study we focus on the organization of the plasma membrane
(PM) in the precellularizing Drosophila embryo. We have previously
shown that each nuclei in the early embryo possess functionally
compartmentalized secretory units in the absence of physical
barriers. It is unknown whether proteins delivered to the PM are
able to freely diffuse to all regions of the embryo, or whether the
PM is compartmentalized over individual nuclei. To follow plasma
membrane dynamics, we generated flies expressing
fluorescently-tagged integral
membrane proteins or fluorescently-tagged
proteins associated with the inner leaflet of the plasma membrane,
and performed photobleaching experiments. In FRAP experiments, both
classes of membrane-associated
proteins exhibited free lateral diffusion in the PM. FLIP assays
showed, however, that the lateral diffusion of these proteins was
restricted to regions of PM over individual nuclei. Finally, when a
PM pool was optically pulse-chased in
photoactivation experiments, proteins could freely diffuse in the
embryo PM, but only within the PM region over individual nuclei and
not across the PM over adjacent nuclei. Altogether our findings
suggest the presence of a plasma membrane diffusion barrier that
restricts lateral diffusion of proteins in the PM over multiple
nuclei. We are currently investigating the nature of such diffusion
barriers using a genetic approach, as well as by performing
ultrastructural studies. Implications of this plasma membrane
compartmentalization in the development of the early embryo are
discussed. |