| The altered cancer metabolism is a hallmark of malignant phenotype. To develop a strategy targeting metabolism is a promising approach for effective cancer therapy.Numbers of findings have demonstrated the close relation between structural protein of caveolae: Caveonlin-1 (Cav-1) and cellular metabolism. Moreover, serving as a bridge linking cells and their external environment, the motility of caveolae is crucial. In the present study, we showed the role of Cav-1 played in cellular metabolism and the key value of its motility by revealing the signaling transduction involved in Cav-1/caveolae dependent endocytosis and trafficking, as well as the association of Cav-1 and mitochondria. The results are shown as follows:1. RalA regulates Cav-1/caveolae dependent endocytosis: (a) By quantifying RalA activatity during BSA (bovine serum albumin) induced endocytosis, we showed that the process of endocytosis activates RalA. (b) By comparing the level of BSA endocytosis and RalA/Cav-1 binding before and after expressing constitutively active and dominate negative RalA, we aim to prove that RalA activity determines both endocytosis and RalA/Cav-1 binding. (c) We treated cells with siRNA to inhibit RalA and rescued its expression by plasmid transfection afterwards. Through comparing the level of endocytosis in RalA knockdown and rescued cells, we demonstrated that RalA is a crucial regulator of Cav-1/caveolae dependent endocytosis.2. RalA activates its downstream effector PLD (Phospholipase D) to mediate Cav-1 dependent endocytosis: (a) To show that activation of PLD promotes endocytosis,non-specific PLD inhibitor was introduced. (b) The PLD1 and PLD2 specific inhibitor was employed to illustrate that only PLD2 regulates endocytosis. (c) The PLD2 inhibitor and mutant suppressing Cav-1-RFP motility confirms the regulating role of PLD2. (d) Using PA biosensor GFP-PASS (phosphatidic acid biosensor with superior sensitivity), we monitored the generation of PLD downstream factor PA (phosphatidic acid) in caveolae which therefore promotes Cav-1/caveolae dependent endocytosis.3. The mitochondrial morphology alteration induced by Cav-1 knockdown: (a)Mitochondria showed both fragmented and elongated morphology by siRNA mediated down regulation of Cav-1. (b) PA-GFP (photoactive-GFP) was introduced here to measure the degree of mitochondrial content mixing which represents mitochondrial dynamics. A higher dynamic level was detected for loss of Cav-1. (c) The mobility of mitochondrial matrix protein was tested by FRAP (fluorescence recovery after photobleaching) to reinforce the conclusion: low Cav-1 makes more dynamic mitochondria.4. Cav-1 regulates mitochondrial dynamics by repressing Mfn2 and Drpl translocation to mitochondria. (a) A higher expression level of Mfn2 and Drpl was detected in isolated mitochondrial fraction from low Cav-1 cells. (b) To test the direct association of Cav-1 with Mfn2 and Drpl, Co-immunoprecipitation and FRET(fluorescence resonance energy transfer) was introduced. (c) The result of immunostain showed increased mitochondrial translocation of Drpl caused by Cav-1 inhibition too.(d) The artificial linker of ER and mitochondria: OMM-ER-mRFP was employed here.By expressing the linker molecular, Mfn2 trafficking backing to ER in low Cav-1 cells suggests that the regulation role of Cav-1 in Mfn2 distribution relies on adjusting ER and mitochondrial coupling.5. The loss of Cav-1 leads to mitophagy: (a) A less mitochondrial mass was found in low Cav-1 cells. (b) A greater autophagy level was detected in Cav-1 knockdown cells by examining expression level of autophagy marker. (c) By observing ratio change of mt-Keima, we suggested that low Cav-1 might cause mitophagy.6. RalA mediates mitochondrial function and cell metabolism by Cav-1: (a)RalA and Cav-1 expression was separately and co-inhibited by siRNA. (b) A decrease of MMP and ATP production as well as an increase of H2O2 and L-lactate production was detected in RalA and Cav-1 down-regulated cells by MMP, ATP, H2O2and L-lactate assay. No difference was detected between knockdown of Cav-1 and co-inhibition of both RalA and Cav-1 suggesting RalA functioning through Cav-1.Altogether, through analyzing the related signaling pathway we established the essential regulation role of RalA in Cav-1/caveolae endocytosis which was performed by its downstream effector PLD catalyzing PA production in caveolae. While studying the interaction between Cav-1 and mitochondria, we elucidated that Cav-1 mediates mitochondrial dynamics and mitophagy by repression Mfn2 and Dip1 targeting. At last,we relate RalA to Cav-1 to demonstrate that both Cav-1 expression and motility can greatly impact mitochondrial function and cellular metabolism. |
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