Roles Of ACTIN In Biogenesis Of Transport Vesicles On Mammalian Cells Early Endosomes

As to eukaryotic cells, the compartmentalized morphology structure on endocytic pathway is the understructural for the intracellular transport and sorting of endocytic molecular. Material transport and information exchange between compartmentalization morphology depends on the formation and transport of vesicles. EE as highly dynamic compartmentalized morphology structure on endocytic pathways, is among the most versatile organellesis, where is endocytic cargo sorting platform. They sort the cargo molecules for recycling pathway through highly dynamic tubular protrusions, or by intraluminal vesicle transport endocytic cargo to the lysosome for degradation. The sorting and transport on EE is a complex and refined process that requires a variety of proteins and lipids, which jointly regulate the sorting and vesicles formation. Some studies have indicated that in mammalian cells, ACTIN is closely related to the sorting and transport vesicles formation on the EE. However, ACTIN in the process of membrane remodeling for the transport vesicles formation, including tubulation and fission activities, remain to be elucidated.Hence, COS-7 and He La cells were utilized as research carrier, and the location of ACTIN on endosomal/lysosomal system in COS-7 and He La cells was examined through fluorescent labeling and laser confocal fluorescence microscopy techniques. Furthermore, in order to observe the dynamic behavior of ACTIN and its regulator WASH in the transport vesicles formation on EE,activated fluorescence labeled RAB5 protein(RAB5 Q79L), Life Act-m Kate 2 or GFP-WASH were transiently expressed, the expression of RAB5 Q79 L resulted in EE homologous integration and formation vacuoles,which conducive to observe the dynamic events on EE in the case of the limited resolution of fluorescent microscope. The mechanism of ACTIN in transport vesicles formation on EE was explored through ACTIN drug treatment experiments; simultaneously, we exploited drug treatment experiments to further explore the effect of ACTIN functional defects on Tfn and 10 KDa Dextran intracellular transport. Finally, we preliminary explored the dynamic behavior correlation between ACTIN and WASH in the events of transport vesicles formation on EE through the triple-labled and confocal time-lapse imaging techniques.ACTIN intracellular location assays suggested that the localization of ACTIN in COS-7 and He La cells presented consistently, mainly on EE, the distribution on RE was less, and the distribution on EE membrane was in the form of discrete patch. Fluorescent-labeled live cell time-lapse imaging data indicated that in COS-7 cells, ACTIN and WASH are closely related to the formation of transport vesicles on EE. Latrunculin B treatment experiments on COS-7 cells data showed that inhibited cell ACTIN dynamics, resulted in the long tubular structure formation on EE, may be due to the dysfunction of fission. Simultaneously, drug treatment on He La cells assays indicated that ACTIN dysfunction impacted the intracellular transport of Tfn and 10 KDa Dextran, and were trapped in EE. We first explored the ACTIN location in different species mammalian cells and the role of ACTIN in the formation of vesicles on EE. We initially explored the dynamic behavior of correlation between ACTIN and its NPF WASH in transport vesicles formation on EE by fluorescent labeling live cell imaging technology, which may provide a theoretical basis for later related research.