FM dyes, their analogs and fluid phase markers fail to label endocytosis in IHCs . 55

As mentioned in the Introduction, the initial motivation to develop a customized membrane/endocytosis marker was the interest to study synaptic vesicle recycling and membrane trafficking in the auditory IHCs. Up to know this issue has been elusive due to the permeation of commonly used dyes through mechanoelectric transduction (MET) channels located at the hair bundle of these cells (Nishikawa and Sasaki, 1996; Gale et al., 2001;

Meyers et al., 2003). Hence, incubation with these dyes results in a very strong cytoplasmic labeling (due to dye molecules permeating the MET channels) that masks the fluorescent signal contributed by truly endocytosed dye molecules. The first step in this project was to revisit the application of such dyes in our own working conditions and setups. Preliminary work was performed in a previous PhD project carried out in our laboratory (Kamin, 2011).

Dr. Dirk Kamin applied a series of commercial dyes on the mouse organ of Corti (OC), the sensory epithelium that hosts IHCs, to evaluate their diffusion properties into such structure and their permeation into the IHCs. A list of the dyes he used can be found in Table 3.1.

The first group included dyes from the FM family. These are styryl molecules that reversibly bind to membranes and have been widely used in the last two decades to study synaptic vesicle recycling in synaptic boutons (Betz et al., 1992; Hoopmann et al., 2012). Dr. Kamin found that except for FM 3-25, all other dyes from the FM family that were evaluated produced a strong and fast staining of the IHC cytoplasm, being much stronger than the intensity found in the surrounding cells (e.g. pillar and supporting cells). By its characteristics, this labeling is difficult to reconcile with an endocytosis-dependent pathway and suggests that the dye molecules rather reach the interior of the IHCs by permeating their MET channels. The same results were obtained using other small membrane-binding molecules like DCF and Di-2-ANEPEQ. The first conclusion of these results is that dyes in the size range of the 450-550 Daltons are small enough to pass through the MET channels. The

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second conclusion is that a long and highly hydrophobic molecule like FM 3-25, with two octadecyl chains, is retained in the outermost regions of the tissue and therefore does not diffuse freely into the OC. In his study, Dr. Kamin also found that soluble compounds like calcein or fluorescein-coupled dextrans could successfully reach the fluid space around the IHCs without permeating them, but would not be taken up into endocytic organelles. Being calcein a relatively small molecule (622 Da), it was concluded that membrane binding is important for MET channels permeation, and efficient endocytic uptake (Kamin et al., 2014).

Table 3.1 List of commercial dyes tested on IHCs

Dye Molecular Weight

FM 4-64FX (fixable version of FM 4-64) 448

FM 1-84 478 Di-2-ANEPQ (also known as JPW1114) 549 Fluid phase

markers Calcein 622

3000 Da Dextran - Fluorescein ~1500-3000 (range of sizes obtained through a

viscosity-based purification)

To strengthen these results and prove that this fluorescence signal is mainly generated by artifactual labeling of the cytoplasm and in a less extent by dye endocytosis, I tested the probes that showed IHCs permeation (FM 1-43, AM 1-43, FM 4-64, FM 4-64FX, FM 1-84, DCF and Di-2-ANEPEQ) at low temperature, at which endocytic processes should be inhibited. I found that at such conditions all FM dyes permeated IHCs (Figure 3.1A), in a similar fashion that at RT and comparable to a previous study reporting FM 1-43 permeation at 4°C in bullfrog saccular hair cells (Meyers et al., 2003). The same strong labeling was also seen for DCF and Di-2-ANEPEQ (Figure 3.1B). Quantification of the fluorescence intensity levels for RT and low temperature incubations showed no significant difference in the amount of labeling, confirming that IHC staining cannot be exclusively explained by endocytic processes (Figure 3.1C).

57 Figure 3.1 Commercial membrane markers label IHCs in an endocytosis-independent process.

A. Dyes from the FM family different in size and structure (450-480 Da) were incubated on IHCs at low temperature to inhibit endocytosis. Strong IHC labeling suggests permeation of the dyes through MET channels. Scale bar, 10 µm. B. Two membrane-binding probes, DCF and the voltage sensor Di-2-ANEPEQ, labeled the cytoplasm of IHCs in a similar fashion to FM dyes. Scale bar, 10 µm. C. Analysis of fluorescence intensity for dyes that gave a strong labeling inside IHCs. ROIs were selected from the cell cytoplasm, avoiding the nuclear area. Fluorescence values are expressed as fold over background. The black bars represent experiments performed at RT. The gray bars represent experiments performed at low temperature (2-4°C). Data analysis was performed using the following numbers of IHCs per condition. FM 1-43: 26 at RT, 19 on ice; AM 1-43: 20, 16; FM 4-64: 13, 23; FM 4-64FX: 8, 20; FM 1-84: 7, 27; DCF: 8, 15; Di-2-ANEPEQ: 30, 35.

From these results I concluded that none of the commonly used commercial dyes tested here is suitable for assessing endocytosis in IHCs, and that a better approach requires the design of customized membrane-binding fluorescent tools.

I then set out to target the different parameters that would define a suitable endocytosis

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marker for IHCs. In section 3.2 I will describe the steps that led me to successfully develop such marker, mainly based on the implementation of membrane labeling and uptake assays in cultured mammalian cells. In section 3.3 I will show how the obtained marker was applied to the study of membrane trafficking in IHCs.

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3.2 Design, synthesis and evaluation of novel membrane-binding probes

3.2.1 Requirements for the generation of suitable membrane probes