Imaging dynamic molecular signaling by the Cdc42 GTPase within the developing CNS

Abstract

Protein interactions underlie the complexity of neuronal function. Potential interactions between specific proteins in the brain are predicted from assays based on genetic interaction and/or biochemistry. Genetic interaction reveals endogenous, but not necessarily direct, interactions between the proteins. Biochemistry-based assays, on the other hand, demonstrate direct interactions between proteins, but often outside their native environment or without a subcellular context. We aimed to achieve the best of both approaches by visualizing protein interaction directly within the brain of a live animal. Here, we show a proof-of-principle experiment in which the Cdc42 GTPase associates with its alleged partner WASp within neurons during the time and space that coincide with the newly developing CNS.

Figure 1. Cdc42 and WASp.

A. Cdc42 becomes active when it replaces GDP with GTP. B. WASp receives phosphorylation and can associate with active Cdc42 through its CRIB domain. C Localization of Cdc42 (elav-GAL4/UAS-mCherry::Cdc42). D Localization of WASp2 (elav-GAL4/UAS-mCherry::WASp). Dashed line indicates midline in the CNS segment.

Figure 2. Low-dosage expression.

A. Transgene stock carrying two GAL4-responsive transgenes (for example UAS-mEGFP::Cdc42 and UAS-mCherry:WASp). B. GAL driver stock (elav-GAL4). C. Experimental animal with single transgene dosages.

Figure 3. Minimum artefacts.

A. Embryo to larva survival rate. Suspected as an early-stage lethality (†). B. Larva to adult survival rate. C. Adult fertility and expected offspring genotype occurrence rate. Viability is the survival rate of the experimental animals as compared to both parental controls.

Figure 4. Direct molecular imaging in a live animal.

FRET detection within a live whole Drosophila embryo by 3D frequency-domain FLIM (fluorescence lifetime imaging microscope). Pockels cell electro-optic modulator and gated intensifier are synchronized at approximately 100 MHz. The relatively low light intensity facilitates efficient averaging of the lifetimes from individual fluorescently-labeled proteins in a large number of confocal pixels.

Figure 5. Lifetime quantification.

A. mEGFP emission is collected at four phase-shifted points. B. Mean fluorescence lifetime in a CNS segment examined (see A).

Figure 6. Sensitivity to detect protein-protein interaction.

A mEGFP lifetime by itself. B. As used as tag for Cdc42. C. With co-expression of cytoplasmic mCherry. D–E. FRET is detected as a drop in donor fluorescence lifetime with co-expression of WASp tagged with mCherry at amino (D) or carboxyl (E) terminus. F–G. Point mutations in Cdc42 prevent it from associating with WASp. H. A point mutation in Cdc42 promotes its associating with WASp. Each protein (pair) displays a mean lifetime (mean±s.d.) in n embryos at hour 15 (see Fig. 5 for scale bar).

Figure 7. Spatial resolution of interaction.

A. Lifetime is concentration-independent. B. Concentration varies from pixel to pixel and requires correction for FRET. C. Interaction is a product of lifetime and concentration.

Figure 8. Pixel-resolution analysis.

A. Neuropil emerges near the center of the CNS as a plasma membrane-enriched region by hour 21 (dotted line). B. Samples from a given experiment show both variability and consistency. C. Pixoplot displays individual pixels from multiple samples (n) along the axes of interaction and spatial position within the bilateral CNS. Black color indicates pixels with neuropil assignment.

Figure 9. Cdc42-to-WASp interaction.

A–C. Interaction between Cdc42 and WASp before (A), at the onset of (B), and after the formation of neuropil (C). Arrow points to the threshold based on background fluorescence.

Figure 10. WASp-to-Cdc42 interaction.

A–C. Interaction between Cdc42 and WASp before (A), at the onset of (B), and after the formation of neuropil (C).

Figure 11. Percentage of interaction.

A. All pixels from an image are plotted on a single Polarplot. Averages of fluorescence lifetime in individual pixels cluster around a ‘mean’ lifetime (red arrow). B. The known baseline lifetime, i.e., 2.56 ns, defines 0% FRET percentage on the semicircle (black open circle). A straight line projecting through the ‘mean’ lifetime (black circle) intersects with the semicircle at 100% FRET percentage (grey open circle). The position of the ‘mean’ in respect to the 0% and100% FRET is the estimate of percentage of interaction between the pair of proteins examined.