Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation

Abstract

Studying gene function in the post-genome era requires methods to localize and inactivate proteins in a standardized fashion in model organisms. While genome-wide gene disruption and over-expression efforts are well on their way to vastly expand the repertoire of Drosophila tools, a complementary method to efficiently and quickly tag proteins expressed under endogenous control does not exist for fruit flies. Here, we describe the development of an efficient procedure to generate protein fusions at either terminus in an endogenous genomic context using recombineering. We demonstrate that the fluorescent protein tagged constructs, expressed under the proper control of regulatory elements, can rescue the respective mutations and enable the detection of proteins in vivo. Furthermore, we also adapted our method for use of the tetracysteine tag that tightly binds the fluorescent membrane-permeable FlAsH ligand. This technology allows us to acutely inactivate any tagged protein expressed under native control using fluorescein-assisted light inactivation and we provide proof of concept by demonstrating that acute loss of clathrin heavy chain function in the fly eye leads to synaptic transmission defects in photoreceptors. Our tagging technology is efficient and versatile, adaptable to any tag desired and paves the way to genome-wide gene tagging in Drosophila.

Figure 1.

Tag-selection cassettes and strategy to insert N- (Top) or C- (Bottom) terminal protein tags in P(acman) rescue fragments for Drosophila transgenesis. (A) N-tag- and C-tag-template vectors, containing tag and a kanamycin (Kan) selection marker, were generated and can be used to create N- or C-terminal protein fusions of a gene of interest. (B) Using the N- or C-tag-template vectors (A) and the primers indicated in (B) tag-selection cassettes are PCR amplified. The 70-bp primers harbour 20-bp homology to the tag-template vectors (blue arrowhead) required for PCR amplification and 50-bp homology to the target region (black box with left homology arm, L; and right homology arm, R). These 50-bp homology arms are required for recombineering. (C) The PCR fragment is subsequently transformed in recombination competent bacteria that harbour P(acman) containing the gene of interest, previously obtained by gap repair. (D) Recombination between the homology arms in the PCR fragment and the GOI occurs and recombination events are selected using kanamycin; correct recombination events are identified by PCR using GOI-reco-F/PL452-5′Seq-R in one PCR reaction and PL452-3′Seq-F/GOI-reco-R in a second PCR reaction. (E) Finally, the Kan cassette is removed using Cre recombinase expressing bacteria and verified by PCR using primers GOI-reco-F and -R. In each case, the final product is a gene fusion between the tag and gene of interest, with an in-frame LoxP site as linker. Fusion protein expression is controlled by endogenous elements. (F) N- and C-terminal protein fusions harbor a 20 and 30 amino acid peptide linker, respectively. Remaining LoxP site is indicated in red and the corresponding amino acid residues are indicated. Amino acids identical in both peptide linkers are indicated in green.

Figure 2.

Fusion proteins generated using the tagging strategy are expressed. (A–C) Overview of transgenic strategies resulting in P(acman) containing Clathrin heavy chain (chc) N-terminally tagged with a FLAG-4C tag (4C-chc⁺) (A), Endophilin (endo) C-terminally tagged with a CyPet fluorescent protein (endo-CyPet) (B) or Synaptojanin (synj) C-terminally tagged with a YPet fluorescent protein (synj-YPet) (C). Corresponding fragments obtained by gap repair from respective BAC's are indicated; tag is in yellow, LoxP site is red. (D) Synaptojanin, a synaptic protein involved in endocytosis is enriched in the neuropile of dissected live third instar larval ventral nerve cords, in line with the endogenous localization of synaptojanin. (E–F) Synaptojanin (E, green in F) is also detected at the presynaptic side of neuromuscular junction boutons and is surrounded by post-synaptic DLG labeling (magenta). (G–H) Ectopically provided chc, expressed from the 4C-chc⁺ transgene, is detected using FlAsH reagent at live neuromuscular junction preparations incubated with FlAsH. Non-bound FlAsH was washed away and fluorescence was detected using YFP optics. While wild types (w) (G) treated with FlAsH do not show significant fluorescence, transgenic chc (H) is ubiquitously expressed but significantly enriched in synaptic boutons, similar to endogenously expressed chc.

Figure 3.

Fusion proteins generated using the tagging strategy are functional. (A–B) Transgenic Drosophila that harbor P(acman) containing Clathrin heavy chain N-terminally tagged with a Flag-4C tag (4C-chc⁺) or Synaptojanin C-terminally tagged with a YPet fluorescent protein (synj-YPet) are fully viable and show no obvious behavioral defects, including climbing behavior after being tapped down (A) and flight ability (B). (C) Schematic diagram of ERG recordings. ERG are field recordings of the retina layer in response to a light flash. (D) A normal ERG response shows a depolarization in response to turning light on and a repolarization in response to turning light off. In addition, activation of the post-synaptic cells located under the photoreceptors also generates an ‘on’ transient and an ‘off ’ transient that coincides with turning light on or off, respectively (arrowheads). While ERGs of flies with defective endocytosis in their photoreceptors show no on and off transients (asterisk), the ERGs of flies with 4C-chc⁺ or synj-YPet constructs are indistinguishable from controls.

Figure 4.

FlAsH-FALI of 4C-chc in photoreceptors leads to defects in synaptic transmission. (A) Strategy to inactivate 4C-chc using FlAsH-FALI. (i) Flies are immobilized on a microscope slide with Pritt glue and microinjected with FlAsH (diluted in HL-3) or with HL-3 (controls) underneath the photoreceptor layer. Injected flies are left to equilibrate and (ii) chc is subsequently photoinactivated by illuminating the entire eye for 5 min with 500 ± 12 nm epifluorescent light using a 10× lens. ERGs in response to a 1 s green light pulse are recorded by placing a recording electrode on the eye and a reference electrode in the thorax. (B) ERGs of FlAsH or HL-3 injected w (top) or w chc¹; 4C-chc⁺ (bottom) flies with or without illumination with 500 ± 12 nm light. Note that ‘on’ and ‘off ’ transients are apparent in all conditions, except when chc in w chc¹; 4C-chc⁺ animals is photoinactivated using FlAsH-FALI (asterisks). For all conditions, at least 10 flies were injected and their ERG recorded (flies that did not survive injection were excluded). While injection of FlAsH (and not HL-3) specifically leads to loss of ‘on’ and ‘off ’ transients upon light inactivation, injection of eyes with FlAsH or with HL-3 also leads to a somewhat smaller depolarization of the photoreceptor layer in response to a light pulse. As the ERG depolarization of flies with HL-3 or FlAsH injection is not significantly different; t-test >0.05, the data indicate that injection per se leads to a depolarization defect. (Average: 2.8 mV ± 0.53 mV for HL-3 injected flies versus 4.2 mV ± 0.8 mV for FlAsH injected flies; 7.1 mV ± 0.9 mV for uninjected flies).