Molecular recognition AFM with conventional IgG antibodies

As a first approach to map the distribution of syntaxin-1 molecules in membrane sheets derived from PC12-WT-1 cells (see section 3.2.2), mouse-anti-rat-IgG antibodies raised against syntaxin-1 (anti-Syx-AB) were coupled to AFM-cantilevers as described in section 3.2.5, and used to carry out MR-AFM experiments on membrane sheets as detailed in section 3.3.4.3. In initial experiments force maps of an array of 16×16 force curves (briefly called 16×16 force maps) were recorded to get a general overview over the interactions.

These experiments yielded a relative frequency of the occurrence of an event of fe = (33 ± 7) % (mean ± standard deviation (SD)) (N = 6 maps from three sheets and three independent preparations). A selection of force curves chosen arbitrarily from these maps is shown in Figure 4.7.

Figure 4.7: Arbitrarily chosen force curves obtained from MR-AFM experiments with conventional IgG antibodies against syntaxin-1 coupled to the cantilever on PC12-WT-1 membrane sheets. The force curves were recorded with a cantilever retraction speed of 1 µm∙s⁻¹. In a five randomly picked force curves per force map, 30 in total, are shown. A magnification close to the zero-force point is plotted in b. The colours of the force curves are chosen arbitrarily to provide better distinguishability.

To obtain a basic insight into the spatial distribution of these events, an initial MR-AFM imaging experiment with exactly the functionalisation strategy described in section 3.2.5 was performed by measuring a single 64×64 force map. In Figure 4.8 the Ripley analysis of this experiment is presented.

8 As the experiments described in this section are introductory ones, the reported results will, after the presentation of a selection of force curves, immediately be continued with observed binding efficiencies and clustering behaviour. For a more general introduction including a deeper explanation of the force curves and the presentation of a force map and force-distribution histograms, see the description of the main experiments in section 4.1.5.2.

4.1 Heterogeneity and Clustering in PC12 Membrane Sheets

Figure 4.8: Ripley analysis of a MR-AFM experiment with a conventional IgG antibody. a plots the L(r)−r values of the measured data (× and cyan line) and for homogeneous random data of the same number of events (○ and magenta line) as a function of r. In b the overlay of the Ripley density map, the corresponding clustered events (red) and all other events (black) is shown. For details see caption of Figure 3.18.

The Ripley analysis shows a non-homogeneous distribution of events at around 0.1 µm. By the subsequently performed spatial cluster analysis the presence of two segregated clusters in a central region of the investigated area and a tiny one close to the border was identified.

The relative frequency of events is as low as fe = 8.0 % in this map. In contrast to initial test experiments with smaller force maps which have not shown clear clusters (data not shown), this experiment implies the detection of clusters in PC12 membrane sheets. However, the specific nature of these events and the corresponding clusters has to be checked, additionally. To this end, some initial control experiments were performed which are described in the following sub-section.

4.1.3.1 Control experiments for MR-AFM by conventional IgG antibodies

First, to test whether the detected events are caused by the specific interaction of antibodies with syntaxin-1 molecules, a competition experiment was performed by the addition of 0.02 mg of the antibody to 2 ml measuring buffer and incubating for 1.5 h. Before and after the addition of the antibodies 16×16 force maps were recorded. This experiment showed, based on the comparison of the mean values and the corresponding standard deviations, no significant difference in the relative frequency of events (fe = (28 ± 4) % (mean ± SD) (N = 3 maps from the same sheet as used for three maps before the addition of the antibody)), as compared with the previously described experiment without antibody competition (see

section 4.1.3). However, when compared with the data stemming from exactly the same membrane sheet recorded before the addition of the antibodies (fe = (37 ± 4) % (mean ± SD) (N = 3 maps)), a significant difference is obtained (see Figure 4.9). Thus, comparing results from the same sheet the competition leads to a smaller amount of events. However, there are still many events left. These events might be caused by an incomplete competition or by unspecific interactions.

Figure 4.9: Relative frequency of events in MR-AFM with anti-Syx-ABs coupled to the cantilever on membrane sheets and related control experiments. Data of measurements with antibodies on the cantilever which are supposed to provide specific interactions are shown in blue, control experiments are shown in red. The caption of the bars denote: all: all six measurements supposed to show specific interactions with IgG-antibodies coupled to the cantilever, same: only those measurements supposed to show specific interactions which stem from the same membrane sheet used for the competition experiment with free antibodies, AB: free antibodies in solution, SDS: antibody denatured by sodium dodecyl sulfate, bare: non-functionalised bare silicon nitride cantilever. The bars show the mean values, the error bars correspond to the standard deviation and the grey circles show the values of individual force maps.

To shed more light on the latter hypothesis, measurements with non-functionalised cantilevers and denatured antibodies on the cantilever were performed. Due to the changed experimental approach of MR-AFM with nanobodies linked to the cantilever (see section 4.1.5) all experiments described in this section were only carried out a single time.

First, a completely functionalised cantilever was subject to denaturation of the bound anti-Syx-ABs by incubation in 2 % sodium dodecyl sulfate in ultrapure water over night.

Subsequent MR-AFM measurements of 16×16 force maps yielded a relative frequency of events of fe = (29 ± 42) % (mean ± SD) (N = 3 maps on three membrane sheets from a single preparation). Note the large deviation of the relative frequency among individual maps (see also Figure 4.9). Furthermore, a bare silicon nitride cantilever was used to probe unspecific interactions. The corresponding measurements of 16×16 force maps show a mean relative

4.1 Heterogeneity and Clustering in PC12 Membrane Sheets

frequency of occurrence of an event of f^e = (13 ± 10) % (mean ± SD) (N = 3 maps on three membrane sheets from a single preparation). Despite the large variations among individual maps, the mean relative frequency of events in the case of the denatured antibody is close to that of the non-denatured. However, this statement is not quite robust since the mean value is largely increased by a single value at about 80 %. Furthermore, the measurements with the bare cantilever show that a subset of the events observed in MR-AFM experiments with a native antibody might stem from non-functionalised parts of the silicon nitride cantilever. A deeper discussion of possible non-specific interactions will be given in section 5.1.3.

4.1.3.2 MR-AFM experiments with unfixed membrane sheets

Since control experiments and measurements with antibodies supposed to be coupled to the cantilever yielded similar relative frequencies of events, the question arose whether fixation might render the epitopes inaccessible for the antibodies or alter in a way that specific interactions are inhibited. If that was the case, a larger binding probability would be expected in unfixed membrane sheets. Therefore, during preparation (see section 3.2.2) the fixation was left out but instead the sheets were washed directly with buffer and stained afterwards.

In a single experiment 32×32 force maps were recorded whose analysis yielded a relative frequency of events of fe = (7 ± 7) % (mean ± SD) (N = 3 maps on three membrane sheets from a single preparation) and, therefore, about four times smaller than in the case of fixed membrane sheets. Thus, this finding contradicts the above hypothesis. It might be that the non-fixed structures described in section 4.1.9 slipped upon approach of the cantilever which could lead to the significantly reduced value of fe.