Lessons from protein-based probes

For a first exploration of membrane binding and fixability properties two chimeric membrane-binding molecules were synthesized:

- Insulin-palmitoyl-Atto 647N (IPA): coupling of human insulin, palmitic acid and the dye Atto 647N-NHS ester. Estimated size 6.9 kDa.

- Transferrin-palmitoyl-Alexa 594 (TfPA): coupling of human transferrin-Alexa 594 with palmitic acid. Estimated size 80.6 kDa.

Additionally, the B subunit of cholera toxin (CTB) conjugated to Alexa 594 (CTBA) was included in the experimental setup. While the cholera toxin A subunit is responsible for the pathogenic effects such as efflux of chloride ions and H2O release to the intestinal lumen via its catalytic activity on ADP-ribosylation, the B subunit facilitates the internalization of the toxin into cells of the host (Sanchez and Holmgren, 2011). CTB binds to the plasma membrane by recognition of the pentasaccharide chain of GM1 gangliosides (Eidels et al., 1983). Although initial studies proposed an exclusive caveolar endocytosis pathway for CTB (Tran et al., 1987), more recent studies propose also uptake by clathrin-dependent, and caveolin- and clathrin- independent mechanisms (Torgersen et al., 2001). Thus, CTB has been used as endocytosis tracer to study early endosome sorting and budding (Barysch et al., 2009). At neutral pH the CTB subunit (11.4 kDa) can also exist as pentamers (57 kDa), ranging in size between IPA and TfPA.

Cultured hippocampal neurons were incubated for 5 minutes with IPA, TfPA or CTBA and fixed for 30 minutes with a 4% PFA solution. After quenching with 100 mM NH4Cl, coverslips were placed on glass slides using Mowiol for embedding. Considering that the reaction performed to produce IPA and TfPA results in bulk labeling of the proteins with a variable number of fluorophore copies per molecule, it was difficult to determine the final concentration of the products in the output solution. IPA, TfPA and CTBA were diluted in Tyrode’s buffer at different concentrations and evaluated under epifluorescence microscopy to establish a concentration giving appropriate fluorescence intensity.

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Figure 3.2 Differences in fixability and labeling distribution among protein-based membrane-binding probes.

Insulin and transferrin molecules conjugated to fluorescent and lipid molecules (IPA and TfPA, respectively), were compared to the fluorescently labeled cholera toxin B subunit (CTBA) in a membrane labeling assay. Probe incubations were performed on neuronal rat hippocampal cultures. Homogeneity of membrane labeling was found to be size-dependent and preserved upon fixation and embedding in Mowiol. Permeabilization, as used in immunostaining procedures, detached unfixed probe molecules and mobilized them into the cell interior. This effect was worsened by embedding with melamine resin after permeabilization. Improving fixation with glutaraldehyde helped to keep the probes bound to membranes. Scale bar, 10 µm.

While IPA and CTBA gave a homogenous labeling of neuronal membranes, TfPA gave a dotted discontinuous labeling pattern (Figure 3.2, first column). This could be explained by the larger size of TfPA, which seems to precipitate on the cellular membranes forming protein clusters. These results already suggest the inconvenience of TfPA for homogeneously reporting all endocytic events happening at the cell surface.

63 It is also important to highlight that IPA and TfPA were normally found on the plasma membrane of the intact cells, and only rarely in endocytosed structures. This would suggest that their relatively large sizes, when compared to dyes from the FM family, might hamper their uptake, being this lower than the non-conjugated native molecules.

When fixation was followed by a permeabilization procedure (0.1% Triton X-100 plus 2.5%

BSA, 1.5 hours) and Mowiol embedding, IPA was found inside the cell soma and processes, indicating very poor fixability. TfPA and CTBA remained on the membranes (Figure 3.2, second column).

As explained in the Methods section, the OC is a complex and thick tissue that would be difficult to study in detail under STED microscopy. For obtaining detailed images of recycling organelles, resin embedding followed by thin sectioning is a good option.

Melamine is a non-fluorescent water-baser resin that offers good sample preservation.

When labeled, fixed and permeabilized neurons were embedded in melamine instead of Mowiol, the probe molecules were mobilized into the cell somas and processes, with stronger effects again on IPA (Figure 3.2, third column). This effect was largely reduced by including a post-fixation step with 2.5% glutaraldehyde after the permeabilization, and before the Melamine embedding (Figure 3.2, fourth column).

Important aspects of probe-membrane interaction can be deduced from these results:

- Large probes like TfPA (80.6 kDa) have difficulties to homogeneously distribute along plasma membranes, reducing their ability to report endocytic events.

- Although IPA has a smaller size (6.9 kDa) and offers a continuous membrane labeling, it was not properly fixed. CTBA was better preserved on membranes, but after melamine embedding its staining appeared less continuous and punctuated.

Deficient fixation of these molecules is probably due to concealing of amine groups by the protein’s tertiary and quaternary structures (in the case of CTBA), leaving only a few accessible groups for aldehyde crosslinking.

- Permeabilization washes off probe molecules that are not properly fixed, and relocalizes them in structures or compartments that were not labeled initially (e.g.

cytoplasm).

- Resin diffusion into cells and its polymerization impose additional factors for probe mobilization from membranes, which can be partly compensated for by using more efficient fixation protocols (e.g. using glutaraldehyde).

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The main conclusion of these experiments is that fixability and probe size are pivotal aspects for the successful labeling of cell membranes and its preservation for further sample processing (e.g. immunostaining).