Microtubules Growth Rate Alteration in Human Endothelial Cells
Figure 2
Selective visualization of growing microtubule plus-ends in human EC. EB3-GFP was used as a marker of growing distal tips (plus-ends) of microtubules. HPAEC were transfected with the plasmid expressing EB3-GFP and the cells growing in monolayer were selected for analysis. Not all cells in the monolayer expressed the construct, and, therefore, could be detected in the fluorescence micrographs. Persistent microtubule growth was confirmed by long EB3-GFP tracks. EB3-GFP movement was analyzed by time-lapse microscopy. Images were acquired every 1 second. ((a), (b)) Two neighboring EC expressing EB3-GFP, low magnification. (a) EB3-GFP tracks oriented radially from the centrosome can be seen to elongate persistently. EB3-GFP is presented at microtubule plus-ends during growth phases but disappears after transition from growth to pause or shortening phase. (b) EB3 tracks obtained by EB3-GFP patches displacement on time-lapse series during 60 second. Scale bar, 10 m. ((c)–(e)) High magnification of left cell from two EB3-GFP expressing EC shown in ((a), (b)): (c) first frame; (d) the same frame with EB3-GFP patches marked with red circles for analysis; (e) EB3 tracks obtained by EB3-GFP patches displacement on time-lapse series during 60 seconds (are colored individually). Scale bar, 10 m. ((f), (g)) High magnification of the frames 1–10. (f) Ten consecutive frames (1–10 sec.) showing the movement of EB3-GFP comet on microtubule tip growing radially from the centrosome region. EB3 tracks (purple) obtained by EB3-GFP patches displacement during 10 seconds. (g) EB3 tracks (pink) obtained by EB3-GFP patches displacement during 10 second near the cell margin. (h) Quantification of plus-ends displacement of microtubules shown in (f). (i) Quantification of plus-ends displacement of microtubules shown in (g).
