Investigation Of The Endocytic Pathway Of Different Sized Fluid Phase Solutes And The Function Of SEC10 In The Endocytosis Of Mammalian Cell

Endocytosis plays a key role in cellular activities. When the process of endocytosis is blocked or the cargo is transported to the wrong destination, it will lead to various diseases. Much work has been focused on clathrin-mediated endocytosis(CME), which is utilized by the internalization of specific membrane receptors and extracellular nutritional molecules. The classic definition for macropinocytosis is the bulk and non-selective uptake of extracellular fluid, macropinosomes formed during this process vary in size from 0.2 to 5 ?m in diameter due to the absence of the induction by the particles ingested or the nucleation of coats formed on the plasma membranes.The capacity to dissect the contribution of macropinocytosis to cellular processes has been hampered by a lack of unique molecular markers and defining features. In this study, we investigated the endocytosis of two differently sized fluorescence-tagged dexrans(10 kDa and 70 kDa, respectively), two widely used fluid phase molecules. Our studies suggested that dynamin-dependent micropinocytosis and–independent macropinocytosis are required for small-sized fluid phase molecules. When either one is blocked, the other would be up regulated. Yet for large-sized fluid-phase molecules, macropinocytosis is the predominant way. So large-sized fluid-phase molecules will be the better tracer for macropinocytosis.Exocytosis is another important physiological process. The exocyst complex is considered to mediate the tethering of vesicles to the membrane prior to fusion. Exocyst is a multimeric protein complex highly conserved from yeast to mammals. However, several recent studies suggested that exocyst may have a role in the endocytic pathway. Using RNAi technique, we studied the role of SEC10 in mammalian cells, which is one component of exocyst. We found that SEC10 participated in clathrin-mediated endocytosis pathway, probably by an effect on the earlier stage of post-endocytic transport of molecules, which indirectly led to the impairment of endocytosis.