| Objective:To study the effect of DRAM1 on the structure of Golgi apparatus its associated vesicle transport,then to preliminary explore the mechanism by which DRAM1 affects the Golgi structure.Methods:Immunofluorescence was used to detect the co-localization of endogenous or exogenous DRAM1 with Golgi apparatus through confocal microscopy.DRAM1 was knocked down by siRNA interference or lentivirus infection,and then subjected to immunofluorescence treatment for morphological observation of Golgi apparatus.The vesicular trafficking related to Golgi apparatus was studied by the transport of ts045-VSVG-GFP and CI-M6PR in cells.The interaction between DRAM1 and structural proteins on Golgi apparatus was investigated with immunoprecipitation.Results:In this study,the co-localization of endogenous and exogenous DRAM1 with different parts of Golgi apparatus was confirmed.DRAM1 was partially localized to the Golgi apparatus.Immunofluorescent or electron microscopic results showed that Golgi fragmentation occurred after DRAM 1 knockdown.Subsequently,studies revealed that with Golgi apparatus was disrupted,the vesicular trafficking closely related to Golgi apparatus was influenced,as reflected in the delayed transport of ts045-VSVG-GFP from Golgi apparatus to plasma membrane,moreover,the endocytosis of CI-M6PR from cell surface to trans-Golgi was also disrupted.Finally,our studies demonstrated that DRAM1 did not regulate the structure of Golgi apparatus through microtubules or microfilament,further results indicated the Golgi fragmentation caused by DRAM1 knockdown was not dominated by part of DRAM1 directly located in the Golgi apparatus.Conclusions:DRAM1 participated in the maintenance of structure integrity of Golgi structure,deficiency of DRAM 1 caused Golgi fragmentation,affected vesicular transport associated with Golgi apparatus.This effect of DRAM1 was independent with cytoskeleton,and the regulation of Golgi architecture by DRAM1 was mediated by DRAM1 outside of Golgi. |
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