Imaging of PC12 cells and membrane sheets

To obtain a basic insight into the morphology of PC12 cells, intact cells were imaged by atomic force, bright field and phase contrast microscopy. A representative image of a fixed wild type cell (PC12-WT-1) is shown in Figure 4.1. The topographical AFM height image in Figure 4.1 a indicates a cell diameter of about 14 µm and a height of about 3 µm. Some folds on the cap of the cell are already visible in the height image but they become even clearer in the error image, i.e. the deviation of the PSD voltage from the setpoint, in Figure 4.1 b.

These structures, which might represent microvilli or sites of exocytosis, can also be

identified in the rendering of the height data in Figure 4.1 c. This representation indicates a rather flat shape of the cap of the cell. In Figure 4.1 d a phase contrast image is shown. Cells as shown in Figure 4.1 were subject to the sonication procedure described in section 3.2.2 to produce membrane sheets.

Figure 4.1: Morphological investigation of a fixed native PC12 cell. The cell was imaged by contact mode AFM. a shows the height image, b the corresponding error image. c shows a rendering of the height data presented in a. A phase contrast image of the cell is shown in d. Scale bars: 10 µm.

The epifluorescence micrographs of the membrane label (octadecyl rhodamine B chloride) of the obtained membrane sheets (Figure 4.2 a, b and c) are already distinct from fluorescence images of cells (not shown). When compared with PC12-WT-2 cells, PC12-WT-1 cells most times showed a more homogeneous surface, as indicated by Figure 4.2 b and Figure 4.2 a, respectively. However, in most PC12-WT-2 membrane sheets this heterogeneity was even more pronounced (data not shown). On the other hand, PC12-WT-1 membrane sheets more often possessed regions of brighter fluorescence at the rim of the sheets, as shown in Figure 4.2 a. These structures might correspond to overlapping membranes. Since the presence of such a structure indicates that the outer membrane leaflet might be facing upwards in these regions, care was taken that the MR-AFM measurement was performed in a central region of such a membrane sheet to avoid a measurement on the outer membrane leaflet, which does not contain accessible syntaxin-1 molecules. Figure 4.2 d-f show fluorescence micrographs of the actin label (Alexa-Fluor^®-488-phalloidin) and Figure 4.2 g-i depict an overlay of the membrane and the actin image. In general, the sheets of PC12-WT-2 cells contain less actin than PC12-WT-1 sheets. The membrane sheet shown in Figure 4.2 c, f and i, stemming from a PC12-WT-1 cell, bears a huge amount of actin. Such a sheet was not used for further

4.1 Heterogeneity and Clustering in PC12 Membrane Sheets

measurements or analyses. Furthermore, actin was enriched in regions of brighter fluorescence in the membrane channel, as evident from Figure 4.2 g and i. That might be explained by actin wrapped with membrane which, therefore, could not be removed.

Figure 4.2: Fluorescence micrographs of PC12 membrane sheets obtained by epifluorescence microscopy. The magenta channel shows the fluorescence of the membrane label (a, b and c), the green one actin (d, e and f) and the last row is an overlay of both channels (g, h and i). The sheet shown in a, d and g is derived from a PC12-WT-1 cell, the one in b, e and h from a PC12-WT-2 cell and the one in c, f and i again stems from a PC12-WT-1 cell, but contains a much larger amount of actin. Scale bars: 10 µm.

To get a more detailed insight into the morphology of PC12 cell membrane sheets, CLSM and AFM imaging was employed. Figure 4.3 a and b shows confocal micrographs of PC12-WT-1 membrane sheets. The membrane image in Figure 4.3 a indicates some heterogeneous structures. Some membrane sheets exhibit larger accumulations of fluorescent material or regions depleted in the same (Figure 4.3 b). Due to low quality, such sheets were as well discarded from further analysis.

A more precise view of the morphology can be gained by AFM height imaging (Figure 4.3 d) of the membrane sheet shown in Figure 4.3 c. The borderline of the membrane sheet and the presence of holes within is generally confirmed by the AFM height image. The membrane sheet has a height plateau at about 20 nm as compared with the height of the substrate.

Some small structures even reach heights above 100 nm. Nevertheless, a tremendous decrease of the height as compared with the native cell shown in Figure 4.1 is evident, confirming a successful production of a membrane sheet. Regions of brighter fluorescence signals in Figure 4.3 c do occasionally, but not entirely, colocalise with elevated regions in the AFM image. This shows that accumulations of membrane material play a role for the surface structure of the membrane sheets but also indicates that further structures might contribute as well, which is analysed in more detail in section 4.1.9 and discussed in section 5.1.1.

Figure 4.3: CLSM and AFM imaging of membrane sheets. In all cases sheets derived from PC12-WT-1 cells are shown. The confocal images depict a rather smooth homogeneous membrane sheet (a) and a more heterogeneous one (b), which would not have been used for further measurements. A further membrane sheet shown by an epifluorescence micrograph (c) was imaged in contact mode AFM to yield a height image (d). Scale bars: 10 µm.

4.1 Heterogeneity and Clustering in PC12 Membrane Sheets

To visualise the protein clusters to be investigated by MR-AFM, STED microscopy was performed employing immunofluorescence with an antibody directed against syntaxin-1.

Figure 4.4 shows a representative example of a membrane sheet derived from a PC12-WT-2 cell (N = 9 membrane sheets from two glass cover slips of a single preparation). The green channel, corresponding to the immunofluorescence of the syntaxin-1 label (Alexa-Fluor^® -488-goat-anti-mouse-IgG), shows a heterogeneous distribution with tiny regions of accumulated fluorescent probes. These structures are probably clusters of syntaxin-1 which are aimed to be investigated by MR-AFM in the present thesis.

Figure 4.4: STED image of syntaxin-1 clusters in a membrane sheet. The fluorescence signal of the membrane marker is shown in magenta, the STED immunofluorescence signal of the syntaxin-1 marker in green. Note the tiny segregated regions of intense immunofluorescence. Scale bar: 5 µm.