Reasons for large ratio between unspecific and specific events

As stated in section 5.1.2 it is unlikely that specific events make up a large amount of the events observed in MR-AFM experiments. Thus, in this section possible reasons for the frequent occurrence of unspecific and, if present at all, low amount of specific events are considered.

Apart from the observation that the frequency of events is found to be smaller on the same membrane sheet after the addition of free IgG-antibodies in solution there is hardly any evidence for the detection of specific events when performing MR-AFM experiments with anti-Syx-ABs. By the non-regioselective binding of amine groups on IgG-antibodies the molecules might be bound in a way which does not promote interactions with the antigen, for example by tethering the antibody by its variable domains or by the antibody lying flat on the solid surface.^[155] By changing to nanobodies which are coupled regioselectively to the cantilever by their C-terminal cysteine residue this problem should be excluded. Furthermore, immunostaining experiments (section 4.1.4) indicate that the antibody used for MR-AFM experiments might be less effective in recognising syntaxin-1 than another clone available, which might explain an insufficient detection of syntaxin-1 in MR-AFM force maps.

However, even the utilisation of nanobodies did not result in a discrimination between specific and unspecific interactions.

To exclude some expected reasons for a possible lack of specific interactions experimental precautions were taken. First, for almost all preparations a sufficient concentration of nanobodies was verified after size exclusion chromatography (section 4.1.5.1). Second, a hindrance of specific interactions by the fixation was tested and shown to be quite unlikely as for silicon nitride cantilevers with coupled anti-Syx-ABs the frequency of events was even

5.1 Heterogeneity and Clustering in PC12 Membrane Sheets

reduced in measurements on unfixed membrane sheets (section 4.1.3.2). Furthermore, this is also indicated by successful immunostaining experiments.

As already discussed in section 5.1.2 many elevations were discovered on membrane sheets (Figure 4.31), which were identified before to be, at least in parts, secretory vesicles.^[153]

Obviously these vesicles which dock to syntaxin-1 might hinder the interaction between the nanobodies on the cantilever and the target protein sterically. Even if parts of the structures stem from protein aggregates it is reasonable that larger proteins also block specific interactions and the detection of clusters in a similar way. However, the combination of AFM and STED imaging shown in Figure 4.34 indicates that syntaxin-1 is also abundant in regions of the membrane sheet which are not occupied by large elevations. Therefore, one might argue that in some regions of the membrane sheet steric blocking of specific interactions should be a less dominant phenomenon.

Not only plasma membrane proteins but also larger proteins on the secretory vesicles might be capable of inducing unspecific interactions on their own. Unspecific interactions of proteins and other biomolecules with cantilevers are well described in literature. For example, Oesterhelt et al. employed physisorption of bacteriorhodopsin on a silicon nitride cantilever to study the unfolding of the protein. In their experiments a dwell time of 1 s at a force of 1 nN resulted in a binding frequency of 15 % between the cantilever and the protein.^[156] Dufrêne and coworkers observed a tremendously larger frequency of interactions of the cantilever with an extracellular matrix made up of polysaccharide intercellular adhesins compared with the cell surface of Staphylococcus aureus.^[157] Moy and coworkers identified that upon interaction of a BSA-functionalised cantilever with a load force of 200 pN a dwell time of less than about 1-4 s is needed to keep the frequency of events below 17 % when interacting with a BSA-coated glass surface. When all other parameters were kept constant the dwell time could be increased to more than 10 s and to almost 50 s when the substrate was replaced by a PEG-coated glass and by an agarose bead, respectively.^[158] These findings show that unspecific interactions are a quite common phenomenon in AFM measurements. When regarding the low probability of unspecific interactions on the agarose bead one can hypothesise a reason why so many unspecific interactions are observed in the present study: When taking the above mentioned results of Moy and coworkers into account,^[158] the presence of glycolipids and glycoproteins on the outer leaflet of a cell membrane^[33] may point to a larger risk of MR-AFM experiments being biased by unspecific interactions when measuring on the inner leaflet of a cell membrane as done in the present study. On the other hand, glycoproteins are also described to hinder specific interactions^[154]

or to induce adhesive interactions on their own.^[157,159] Contamination of the cantilever by adsorption of molecules which might alter the interactions with the sample are described as

well.^[160] By Creasey et al. it was noted that especially when employing standard force mapping one has to take care that only specific interactions are analysed.^[101] This was implemented in the present thesis beginning with the manual check of every single force curve and exclusion of force curves that are very unlikely to represent specific interactions (see section 3.4.5). The most sophisticated approach was the fit of the force curves with a WLC model and the subsequently performed variation procedure of threshold values of the involved quantities in section 4.1.8.3. Interestingly, even structures remaining after protein digestion by proteases can induce strong unspecific interactions (Figure 4.33). This shows that unspecific interactions are a huge bias when performing MR-AFM experiments on membrane sheets. Notably, unspecific interactions were probably not caused by preceding interactions like breakthrough events or sliding away of vesicles visible in the approach force curve (section 4.1.8.1).

The protein dependent structures discussed in section 5.1.1 probably influence interaction events. It was observed that the frequency of events on membrane sheets treated with proteases differs significantly from that on untreated membrane sheets (sections 4.1.6.4 and 4.1.9.2). However, the distribution of maximum interaction forces is almost unchanged. It has already been noted in section 4.1.9.2 that this similarity points to the existence of the same kind of interactions in both cases. Since it is not expected that syntaxin-1 withstands this procedure, the finding mentioned before further supports the hypothesis that no or only very few specific interactions are detected in untreated membrane sheets. Notably, the rupture distance is significantly larger after protein digestion. It is unlikely that proteins are unfolded by proteases but the polypeptide chains withstand the treatment and thereby looser and unfolded filaments still interact with the cantilever in a similar way as before and thus causing the same maximum interaction force, but that due to the unfolded structure the rupture takes place at a larger distance. Instead, facilitation of lifting of the membrane in the altered membrane sheets might be a plausible explanation for the larger rupture distances. This is reasonable since the poly-L-lysine which tethers the membrane sheet to the glass cover slip should be affected by the protein digestion by the proteases as well and therefore the contact between the membrane sheet and the support should be weakened.

Evidence was found that structures residing on the membrane and not the lipid membrane itself causes the observed events. Interactions with pure lipid membranes take place at smaller maximum interaction forces and smaller rupture distances (Figure 4.33). On GUV-MPs the latter are much more similar to the length of the PEG chain than on membrane patches, supporting the notion that proteins on membrane sheets cause interactions with the cantilever. However, interactions with lipids present in membrane sheets but not in the GUV-MPs might also cause the observed differences. On the other hand, especially the long

5.1 Heterogeneity and Clustering in PC12 Membrane Sheets

rupture distances in the case of membrane sheets and complex shapes of some force curves (arrowhead in Figure 4.13 a) are in agreement with unfolding of proteins by AFM described in literature.^[156,161] The quite symmetric distribution of maximum interaction forces found for measurements on GUV-MPs points to a simple kind of interactions like van-der-Waals forces between macroscopic bodies but not to multiple parallel bonds. Measurements on GUV-MPs with anti-Syx-NB functionalised gold coated cantilevers show a larger frequency of events as compared with anti-Syx-AB functionalised silicon nitride cantilevers. This might be explained by the five times larger Hamaker constant of an interaction between two gold surfaces in water compared with two silicon nitride surfaces.^[162,163]

Unspecific interactions might also be induced by actin or other filaments present on the membrane sheets. The presence of actin on membrane sheets was revealed in several experiments (Figure 4.2 and Figure 4.31). Furthermore, the detection of quite heterogeneous structures in MR-AFM measurements on membrane sheets from knockdown cells might indicate adhesive interactions with filamentous structures (see elongated structures in Figure 4.25 d). On the other hand, it has to be noted that the amount of erroneous height values in the FD-AFM image in Figure 4.31 is quite low in regions of abundant actin. This points to only few very strong interactions with actin since extraordinary strong adhesive interactions might lead to errors in FD-AFM images. Furthermore, the small persistence length determined (vide infra) does not point to an interaction with actin when taking its large persistence length of about 17 µm into account.^[164]

In Figure 4.22 cumulative probabilities from different experiments performed with varying cantilever functionalisation methods and experimental parameters are plotted. A varying mixture of aminoalkanethioles used for the functionalisation and different contact times can lead to varying numbers of parallel bonds. This has to be considered when comparing measurements performed with different conditions. The similarity of the distribution of the forces and the fact that most force curves only show a single peak do not point to the formation of different numbers of bonds or to multiple bonds at all. However, the observed tail towards large forces in the histograms points to the existence of multiple bonds for a subset of force curves. Next, in most experiments a large frequency of events was observed which renders the formation of multiple bonds very likely. Furthermore, the persistence lengths listed in Table 4.5 are significantly smaller than that of a PEG chain (Lp(PEG) = 0.38 nm).^[150] Considering a parallel alignment of springs in equation (3.11), about 20 parallel bonds would be necessary to reduce the apparent persistence length to the values determined from the fits. However, such a situation does not fit to the rather small interaction forces determined. Thus, the WLC model does probably not accurately describe the interactions observed in the MR-AFM experiments. This is further indicated by the large

number of force curves with a small coefficient of determination. The obtained values of the persistence length and of the contour length are probably further biased by the finite and constant slope of the force curves at larger D (see Figure 4.13), which might stem from an additional elastic element like the lifted membrane.

Very likely experimental biases causing the lack of detection of syntaxin-1 in MR-AFM measurements on PC12 membrane sheets are illustrated schematically in Figure 5.1.

Figure 5.1: Schematic illustration of possible reasons for the large ratio between unspecific and specific interactions. The nanobody (magenta) on the cantilever is sterically hindered from the interaction with its binding partner syntaxin-1 (green) in the membrane, by synaptic vesicles (blue), other proteins (red) and filaments (cyan).

Furthermore, proteins (shown for the cyan filaments) can bind to the cantilever and induce unspecific interactions. Adhesive interactions between the vesicles and the cantilever are likely as well. The different elements are not to scale.

A remaining concern is that the functionalisation of the cantilever might not have been intact or that the nanobodies do not bind their target any more when coupled to a surface, i.e. the tip. Varying frequencies of events among different cantilevers (Figure 4.23) do not necessarily point to more or less successful functionalisations of the cantilever but can also be caused by differences in the tip radius as a larger tip radius should lead to a larger probability of any kind of interaction. As already mentioned in section 4.1.7 a chemical characterisation on small surfaces such as AFM cantilevers and especially their curved tips is hard and often replaced by an indirect verification by observed interaction events and their successful competition.[27,28,101,110] For the sake of completeness it should be noted that for the present thesis in single experiments it was attempted to characterise functionalisations performed by the methods described in section 3.2.5 on oxidised silicon or gold coated

5.1 Heterogeneity and Clustering in PC12 Membrane Sheets

wafers (not described in previous chapters). By AFM imaging few tiny structures of a few nanometer in height and lateral size were found on an unfunctionalised gold coated wafer and on a wafer presumably functionalised with nanobodies. By a qualitative investigation the amount of these structures was not increased on the presumably functionalised wafer which might point to a non-successful functionalisation. However, since a discrimination between nanobodies and the structures on the unfunctionalised wafers might be difficult, this kind of experiment was not performed any longer. On the other hand, in an attempt to couple avidin to oxidised silicon wafers, measured contact angles increased or decreased with each step of functionalisation as it was to be expected. This might point to a successful functionalisation, but this measurement was not repeated and further experiments with avidin were not carried out. The amination of gold coated cantilevers by aminoalkanethioles was, in contrast to most reports, not performed in ethanol but in water. Due to less concerns about evaporation of the solvent, this method was adapted from Wirde et al. who revealed the coupling of cysteamine to gold surfaces in water to be as efficient as in ethanol.^[165]

Furthermore, in situ coupling of cysteine residues of a protein to gold coated cantilevers in aqueous buffer is also described.^[166] Thus, in principle this reaction can be expected to be successful. Even if the first steps of cantilever functionalisation were not successful, they would be circumvented by the utilisation of cantilevers which had already been functionalised with a maleimide group by the manufacturer. However, since the experiments performed with those cantilevers did not lead to a detection of specific interactions either, the first steps of functionalisation were either not the cause of a lack of detection of specific events or there has to be another problem. Hinterdorfer and coworkers reported an average interaction force between a bare or a PEG-functionalised cantilever and a mica surface which was coated with chromatin of (118 ± 134) pN and (116 ± 63) pN,^[28] respectively. For the corresponding antibody-antigen interaction they obtained an average force of (57 ± 20) pN.^[28] In the 15 maps recorded with anti-Syx-NB functionalised cantilevers and a retraction speed of 1 µm∙s^–1 in the present study the determined maximum interaction force is (179 ± 375) pN (mean ± SD). This points to a non-successful functionalisation of the cantilever, however, for an accurate evaluation a cumulative probability plot of the data described by Hinterdorfer and coworkers would be beneficial, since the median force observed in the present study matches the average reported by Hinterdorfer and coworkers for the antibody-antigen interaction quite well.

Provided that the reaction between the maleimide and the nanobody is successful, a lack of specific interactions might still originate from non-functional nanobodies for example caused by denaturation of the nanobodies on the solid surface of the cantilever tip.

It has to be noted that some experiments suffer from few repetitions. Therefore, results of some kind of experiments can only be regarded as preliminary and more repetitions would be necessary to certainly state on the indications presented before. However, the vast amount of different indications obtained from varying kinds of experiments and approaches all pointing to the lack of specific and to abundant unspecific interactions together draw a clear picture.