Discussion and outlook

An assay for coupling streptavidin coated microspheres to GFP-coupled membrane proteins was established in order to make 2D-TNIM experiments on membrane molecules possible.

Control experiments indicated that 80–90% of the spheres bind specifically to the protein of interest. Thus, when evaluating 2D-TNIM experiments it must be kept in mind that every 6^th–7^th might report on the mobility of plasma membrane components distinct from the re-spective molecule of interest. Such nonspecific binding is a typical challenge when working with relatively large marker particles. Suzuki et al. (2000) reported for antibody mediated coupling of 730 nm latex spheres to cell surface proteins a specificity of 6:1 (in presence of antibody, binding of spheres was enhanced 6 fold). Murase et al. (2004) reported for antibody mediated coupling of 40 nm gold spheres to cell surface proteins a specificity of 5:1. This shows that a specificity between 6:1 and 7:1, which was obtained here, meets current requirements.

The binding of the streptavidin microspheres to the GFP coupled membrane proteins is me-diated by biotinylated monoclonal GFP antibodies. Specific binding of the spheres to the cell surface proteins was only achieved if (i) the cell surface proteins were incubated with the antibody (see also section D) and (ii) the streptavidin spheres were bound to the antibodies that already recognised their GFP substrates on cell surface (preincubation of spheres with antibodies was not successful, probably due to mechanical constraints that are experienced by the antibodies that are bound to the sphere). Using this assay, it can not be excluded that one antibody (which is intrinsically bivalent) is bound to two GFP-coupled cell surface proteins simultaneously. As a consequence, the experiments in section 4.4 may underestimate (maximally by a factor of 2) the diffusion coefficient of a monomeric EGFR, which is expected to diffuse faster than two EGFR that are coupled by an antibody. This uncertainty could be avoided in future experiments by developing a method that allows one to determine the exact number of molecules that are bound to the microsphere at a time, e.g. using the fluorescence of the GFP that is coupled to the proteins. This is however challenging as the GFP-proteins

Figure 4.3: TNIM at a cell membrane. A) Schematics. The diffusion of a streptavidin-microsphere is confined by the optical trap (red gradient). The trap is positioned in bulk solution and then moved to the surface of a cell expressing GFP-tagged membrane molecules bound to biotinylated GFP-antibody. B) Measured position time traces of the Brownian motion of the sphere in the optical trap (y(t)not shown). Positions are shown every 1 ms as lines between data points. At the cell surface, thez-position fluctuations are confined to the space above the cell surface. Transient binding (*) to a membrane molecule confines the motion to the cell surface. C) Enlarged view of the binding event in (B). Positions are shown every 2.5µs as lines between data points. D) Histograms of the spheres center position calculated from 4 s (left and middle panel) or 200 ms (right panel) position fluctuations, respectively. The sphere is indicated as a black circle. The center of the optical trap is marked by a red cross.

The cell surface is indicated in grey. Membrane heterogeneities hindering the lateral motion of the sphere-molecule complex are indicated in dark grey. Experimental details: sphere positions were monitored at 400 kHz; histogram binning is 5 nm; the sphere was binding to a GFP-EGFR transfected Cos7 cell; sphere radius was 125 nm.

that are bound to the microsphere would have to be distinguished from unbound GFP-proteins that diffuse in the membrane area below or next to the microsphere. It is a general challenge in SPT experiments to determine the exact number of molecules that are bound to the marker particle. In conventional SPT, marker particles are incubated with cells for minutes. Murase et al. (2004) reported that particles could be only identified as bound if they stayed at least 3 seconds on the membrane. Otherwise it was not possible to distinguish them from unbound marker particles diffusing next to the membrane. This is problematic because after the forma-tion of the first bond, the close vicinity of the marker particle to the membrane facilitates the formation of further bonds to additional membrane molecules. Thus, to investigate diffusion of single/few membrane molecules it is advantageous to observe the motion of the marker as fast as possible after the formation of the first bond. The TNIM permits one to detect the initial binding of the marker particle and membrane molecule with millisecond temporal resolution (see Fig. 4.3). Apart from the long term observations that are discussed in section 4.3.3, only 2D-TNIM data directly (less than 1 second) after binding were evaluated ensuring that only a minimal number of membrane proteins were bound to the sphere. In fact, it was observed in most experiments that the mobility of the bound sphere gradually decreased (on a seconds timescale) after binding, indicating the formation of further bonds. In some cases a step-like reduction in the mobility was observed, indicating that the formation of individual bonds can be monitored (not shown). It would be an interesting future project to analyse this data with regards to the fundamental physics that govern the motion of a complex of proteins in a fluid lipid bilayer and also with regards to cellular signalling (see the ”oligomerisation induced trapping model” in Fig. 1.1D).

Fig. 4.3B shows a transient binding event of ca. 200 ms. The large subsequent z-position fluctuations indicate that the bond is released, probably due to the dissociation of the an-tibody from the GFP. In fact, an analysis of the distribution of binding times can reveal detailed information on antibody antigen interactions on a single molecule level as has been demonstrated by Kulin et al. (2002). Here, bonds that were stable for more than 500 ms were selected to investigate the lateral mobility of the attached membrane proteins.

The TNIM makes it possible to image the local flatness of the cell surface by the sphere’s thermal position fluctuations before binding (see Fig. 4.3A). This is valuable, because a ma-jor goal of 2D-TNIM experiments is to identify structures within the plasma membrane that restrict the lateral mobility of the embedded molecule. It is therefore useful to control local membrane flatness, because large membrane protrusions could restrict the lateral mobility of the marker sphere. Such restrictions that act on the marker sphere would hamper the inter-pretation of the joint motion of marker and molecule. This is a major technological advance as video-microscopy based SPT typically lacks the axial z resolution that would be necessary

to control the local plasma membrane flatness (Saxton and Jacobson, 1997).

In conclusion, a robust and general assay for binding marker particles to GFP coupled mem-brane proteins was established. The specificity of the assay is comparable or better compared with what is reported in recent SPT literature. Moreover, the TNIM makes it possible to observe the actual binding event with millisecond temporal resolution and to control the lo-cal flatness of the plasma membrane on a slo-cale of tens of nanometers, thereby significantly facilitating the interpretation of the motion of the marker-protein complex.