The Morphological Research Of Microtubules Cytoskeleton Around Giant Vacuoles In Microglia

Objective:Cytoskeleton play a vital role in keeping cells morphology, intracellular transport and cell signaling deliver. Some reseach indicate that actin filaments in cell cortical, which are the main driving force, regulate endocytosis delicately, while microtubules emit from the cell central to edge in all directions as traits for intracellular transport. Preliminary work in our lab found that there were many macropinosomes when microglia migrated. Still now there is no record show the location of microtubules in microglia’s macropinocytosis. Our experiment focus on the morphology of microtubule cytoskeleton in primary cultured microglia which was induced giant vacuoles by ATP.Methods:A mixed glial culture was prepared from the cortices of neonatal Sprague-Dawley rats and maintained for7to10days in MEM containing10%FBS. The cells floating over the mixed glial culture were collected and transferred to appropriate glass coverslips. After4hours, culture cells were used for immunostainning or living cell assay. Cultures were preincubated with dextran and ATPyS, then were recorded living cell images by confocal microscope. Cultures were stained for several antibodies (Acetylated-tubulin, Tyrosinated-tubulin, phalloidin) overnight at4℃. Then the cultures were incubated with the fluorescence-conjugated secondary antibodies for1h at room temperature next day. After washing with PBS, cells were imaged with the confocal microscope and Super Resolution Structure Illumination microscope to check the location of microtubules and microfilaments. FV10-ASW4.0Viewer were used to analyze the position, size of giant vacuoles and distribution of actin and tubulin. Several GTPases Rab antibodies were used to study the relationship between giant vacuoles induced by ATPyS in microglia and the well-known endosomes. And we reconstructed the cytoskeleton by using IMARIS.Results:1. ATPyS induced a great deal of giant vacuoles in primary cultured microglia. These vacuoles could be loaded with dextran, so they were thought to be pinosomes.2. Part of the giant vacuoles were surrounded with microtubules.3. According to the statistics of vacuoles’size and position, giant vacuoles whose diameter was about4micrometers were preferred to be surrounded by microtubules. Compared to small vacuoles, which had no microtubules surrounded, these giant vacuoles were closer to cell edge..3. Microtubules around the giant vacuoles might relate to vesicules internalization.Conclusion:We found that ATPyS induced giant vacuoles whose diameter was about4micrometers in primary cultured microglia. There were microtubules around the vacuoles, indicating that microtubules might take part in vesicules internalization.