Cell surface membrane homeostasis and intracellular membrane traffic balance in mouse L929 cells

We have developed a simple method for synchronizing L929 mouse fibroblasts. Cultured as monolayers, these cells stop growing at confluency and arrest at the end of the G(1) phase. Upon seeding at low density, they enter the S phase simultaneously. Using these cells we then looked at the evolution of the surface membrane area during the cell cycle using the fluorescence mem brane probe TMA-DPH. In contact with cells, this probe partitions between t he membrane (probe fluorescent) and the external medium (non-fluorescent), delivering a signal proportional to the membrane area. This area was consta nt until just before mitosis, when it increased at once. With the same prob e as an endocytic marker, we examined how this membrane homeostasis could b e consistent with intracellular membrane trafficking. The study was limited to one selected period of the cell cycle (6-9 hours). We observed that 14% of the membrane endocytosed was not recycled, but was replaced at the cell surface by newly formed membrane from biosynthetic pathways. Brefeldin A m odified the membrane traffic, but not the overall membrane homeostasis. The results are discussed in the framework of a maturation model.