Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study

Abstract

We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with approximately 200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis.

Fig. 1

Topographical imaging of endocytic pits in living cells by SICM. a Schematic diagram of the scanning ion conductance microscope. b SICM topographical image of live Cos-7 cell. c High resolution topographical SICM image of live Cos-7 cell membrane revealing numerous pits. d High resolution topographical SICM image of a fixed Cos-7 cell membrane revealing numerous pits. e Zoomed image showing a single pit (top). Topographical profile of a pit in a live cell (bottom). f Zoomed image showing two pits (top). Topographical profile of pits in fixed cell (bottom)

Fig. 2

Topographical and fluorescent imaging of clathrin coated pits in fixed clathrin-GFP transfected Cos-7 cells by scanning surface confocal microscope. a Schematic diagram of SSCM. b Topographical image of cell. c Fluorescent image of the clathrin-GFP transfected cell shown in b. d 3D representation of overlaid topographical and fluorescent images shown in b and c, respectively. e High resolution topographical image of cell surface revealing numerous clathrin-coated pits. f Same topographical image as in e but inverted and presented in a red palette. g Overlaid inverted topographical and fluorescent images shown in f and g, respectively. The image reveals that the pit topography matches the clathrin-GFP fluorescence. h The distribution of clathrin-coated pit width calculated from the SICM topographical images

Fig. 3

Topographical and fluorescent imaging of caveolin pits in fixed caveolin-GFP transfected Cos-7 cells by SSCM. a Topographical image of cell. b. Fluorescent image of caveolin-GFP transfected cell shown in a. c 3D representation of overlaid topographical and fluorescent images shown in a and b, respectively. d High resolution topographical image of cell surface. e High resolution fluorescent image of caveolin-GFP transfected cell shown in d. f Caveolin pit width distribution histogram calculated from SICM topographical images. g Digital zoom of the topographical image shown in d (dotted square) revealing numerous caveolin pits. h Same zoomed topographical image as in g but inverted and presented in a red palette. i Overlaid inverted topographical image shown in h and digitally zoomed fluorescent image from the area shown in e (dotted square). The image reveals that pits match the caveolin-GFP fluorescence

Fig. 4

Topographical and fluorescent imaging of fixed flotillin-GFP transfected Cos-7 cells by SSCM. a Topographical image of cell. b. Fluorescent image of flotillin-GFP transfected cell shown in a. c 3D representation of overlaid topographical and fluorescent images shown in a and b, respectively. d High resolution topographical image of the cell surface revealing numerous indentations. e Same topographical image as in d but inverted and presented in red palette. f Overlaid inverted topographical image shown in e and high resolution fluorescent image of flotillin-GFP acquired from the same area. The image reveals that some indentations on the cell surface match flotillin-GFP fluorescence (white arrows). g High resolution topographical image of cell surface revealing numerous indentations (solid arrows) as well as two protrusions (hollow arrows). h Same topographical image as in g but high-pass filtered. i High resolution fluorescent image of caveolin-GFP transfected cell shown in g. The arrows point to indentations that match flotillin-GFP fluorescence. Hollow arrows point to protrusions that match flotillin-GFP fluorescence

Fig. 5

Live topographical and fluorescent imaging of clathrin coated pits in clathrin-GFP transfected Cos-7 cells by SSCM. a High resolution topographical image of live cell membrane revealing numerous clathrin-coated pits. b. Same topographical image as in a but inverted and presented in red palette. c Overlaid inverted topographical image shown in a and fluorescent image of the same area. The image reveals that, on live cells, we can detect that the pits’ topography match clathrin-GFP fluorescence. d Sequence of topographical images of live cell membrane revealing dynamics of the clathrin-coated pits. The images are separated by 10 min