Identification And Characterization Of Tunneling Nanotubes In Osteocytes

Objectives:This study aimed to identify tunneling nanotubes(TNTs)between mouse osteocyte-like cell line MLO-Y4 cells,analyze their general properties such as morphological characteristics,composition and formation mechanism,investigate the factors affecting the formation of TNTs,such as stress conditions like nutrition,p H and oxidative stress,and further analyze their material transport properties,to provide a more comprehensive theoretical basis for understanding the structure and function of osteocytes and their communication network in bone tissue.Methods:1.Identification and general characterization of intercellular TNTs in osteocytesThe MLO-Y4 mouse osteocyte-like cell line was cultured,and were identified under laser confocal microscope by labeling fibrillar actin(F-actin)with phalloidin and nuclei with DAPI.The morphological characteristics such as length,diameter,presence or absence of branches and the form of connections were analyzed.The composition of intercellular TNTs in MLO-Y4 cells was further analyzed by labeling cytoskeletal α-microtubulin using anti-α-microtubulin antibody.The mechanism of formation of intercellular TNTs in MLO-Y4 cells was investigated using a live cell imaging system.MLO-Y4 cells were cultured in culture medium containing 0,0.25,0.5 and 1 μmol/L cytochalasin B for 24 h,respectively,and the percentage of cells containing TNTs and the average number of TNTs contained per cell were observed.2.Factors influencing the formation of intercellular TNTs in osteocytesMLO-Y4 cells were cultured in low-serum culture medium,acidic culture medium and acidic culture medium with low serum for 24 h,respectively,to observe the percentage of cells containing TNTs and the average number of TNTs contained per cell.MLO-Y4 cells were cultured in oxidative stress medium containing 100 and200 μmol/L H2O2 for 24 h and 2 h,respectively,and the percentage of cells containing TNTs and the average number of TNTs per cell were observed.3.Study of the material transport role of TNTs between osteocytesDiD-labeled MLO-Y4 cells were used as donor cells and EGFP-transfected MLO-Y4 cells or unlabeled MLO-Y4 cells were used as recipient cells,which were co-cultured at a 1:1 ratio for 24 h.DiD-positive recipient cells were observed;To further quantify direct or indirect communication-mediated material transport,the percentage of DiD-positive cells was detected by flow cytometry after setting up the conditioned media set,and the percentage of DiD-positive cells in the recipient cells was counted.DiD-labeled donor cells were co-cultured with EGFP-transfected recipient cells or unlabeled recipient cells at a 1:1 ratio for 16-24 h.The formation of TNTs between donor and recipient cells and the vesicle transport mediated by them were observed using a live cell imaging system with DIC.Results:1.Identification and general characterization of intercellular TNTs in osteocytesTNTs between MLO-Y4 cells were identified by fluorescence staining using laser confocal microscopy combined with three-dimensional reconstruction with the following criteria: at least two cells connected,containing F-actin,and not in contact with the bottom of the culture dish.The average diameter of the TNTs we identified in MLO-Y4 cells was 366 nm(210～1430 nm)and the average length was 5.7 μm(1.1～129 μm).Most of the TNTs(97.5%)had no branching structures,and a few(2.5%)had branches.Formation of TNTs could be between dendrites(52.1%),between dendrites and cell bodies(42.4%),and between cell bodies(5.5%),and TNTs connected through between cell bodies were significantly larger in diameter and length than the other two types of TNTs.Except for those containing F actin,the thicker TNTs between MLO-Y4 cells also contained α-microtubulin.The mechanism of formation of intercellular TNTs in MLO-Y4 cells included cell movement mechanism and protrusion elongation mechanism.The percentage of cells containing TNTs and the average number of TNTs per cell were significantly reduced after 24 hours of cytochalasin B action at 0.5 and 1 μmol/L compared with the control group.2.Factors influencing the formation of intercellular TNTs in osteocytesCompared with the control group,the percentage of cells containing TNTs and the average number of TNTs per cell were significantly higher in MLO-Y4 cells cultured in low-serum culture medium,acidic culture medium and acidic culture medium with low serum after 24 h.Moreover,compared with the culture in low-serum culture medium or acidic culture medium alone,MLO-Y4 cells cultured in acidic culture medium with low serum contained a percentage was greater.The percentage of cells containing TNTs was significantly higher in MLO-Y4 cells cultured in oxidative stress medium containing 200 μmol/L H2O2 for 24 h compared with the control group.3.Study of the material transport role of TNTs between osteocytesAfter 24 hours of co-culture of DiD-labeled donor cells with EGFP-transfected recipient cells,most of the recipient cells contained DiD-labeled vesicles.To further quantify the two modes of communication,indirect and direct communication,DiD-labeled donor cells were used to co-culture with unlabeled recipient cells,and flow cytometry assays revealed a significant increase in the percentage of DiD-positive cells in the recipient cells after 24 h of co-culture compared to 0 h of co-culture between the two cells;to exclude the effect of indirect communication between cells,conditioned medium from the donor cells was added to the recipient cells for 24 h.It was found that the percentage of DiD-positive cells in the recipient cells was significantly reduced compared to 24 h of direct co-culture between the cells.After co-culture of DiD-labeled donor cells with EGFP-transfected recipient cells for16-24 h,TNTs were present between donor cells and recipient cells containing donor cell-derived DiD.After co-culture of DiD-labeled donor cells with unlabeled recipient cells for 16-24 h,continuous movement of DiD-labeled vesicles within the TNTs was observed,and vesicles from one cell directly transported through the TNTs to the other cell.Conclusions1.There is a structure of TNTs between MLO-Y4 osteocytes,and its morphological properties are osteocyte-specific.2.Stress conditions such as low nutrition,acidic p H or oxidative stress can promote the formation of TNTs between MLO-Y4 osteocytes.3.MLO-Y4 osteocytes can undergo direct material transport via TNTs.