Neuron-Subtype-Specific Expression, Interaction Affinities, and Specificity Determinants of DIP/Dpr Cell Recognition Proteins

Abstract

Binding between DIP and Dpr neuronal recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system. Each lamina neuron was previously shown to express many Dprs. Here, we demonstrate, by contrast, that their synaptic partners typically express one or two DIPs, with binding specificities matched to the lamina neuron-expressed Dprs. A deeper understanding of the molecular logic of DIP/Dpr interaction requires quantitative studies on the properties of these proteins. We thus generated a quantitative affinity-based DIP/Dpr interactome for all DIP/Dpr protein family members. This revealed a broad range of affinities and identified homophilic binding for some DIPs and some Dprs. These data, along with full-length ectodomain DIP/Dpr and DIP/DIP crystal structures, led to the identification of molecular determinants of DIP/Dpr specificity. This structural knowledge, along with a comprehensive set of quantitative binding affinities, provides new tools for functional studies in vivo.

Figure 1:. Identification of medulla neuron types expressing different DIPs using MCFO.

(A) Schematic of different classes of medulla neurons, Dm, Mi, Tm and TmY. (B, B’) Cell types express DIP-ε can be identified by MCFO. It is very difficult to identify cell types expressing DIPs by driving membrane-bound GFP using DIP gene traps, as shown in B. In contrast, individual cell types can be identified by morphology using MCFO to generate sparsely labeled cells, as shown in B’. White triangles, Tm4 (green). Scale bar: 10μm. (C-F) Examples of medulla neuron types identified by MCFO for the remaining seven DIPs. Colors for the lettering of cell types are the same as the single cells labeled in the images. (C) DIP-α: arrow Dm1 (green); arrowheads, Lawfl (green); triangles, T1 (red). (D) DIP-β: arrow, Lawfl (green); triangles, Lawf2 (magenta). (E) DIP-γ: Tm9 (green). (F) DIP-δ: Dm6 (Cyan); triangles, Y3 (Cyan). (G) DIP-η: arrows, TmY3 neurons (red) and another TmY3 is in green; triangles, Tm2 (red); (H) DIP-θ: triangles, Tm3 (green) and TmY3 (green) without triangles. (I) DIP-ζ: triangles, Tm20 (green); arrowheads, Tm20 (orange); Arrow, Pm2 (yellow). Pm2 neurons are always labeled in the entire layer in different colors. Scale bar: 10μm. (J, J’) An example of identifying single labeled medulla neuron in a densely labeled environment (J) by comparing its morphology to a single labeled cell in a reference image (J’). A green Tm5c is labeled in J, but it partially overlaps with another cell (described in Figure S1A, A’). By comparing patterns of arborization in specific layers in medulla and lobula between J and J’ (triangles), we can identify the cell in J. Scale bar: 10μm. See also Figures S1-S3.

Figure 2:. Summary of DIP expression in medulla neuron types.

Expression of eight DIPs was assessed in 60 well-characterized cell types (see text). A reference for each cell type is listed in the second column. Ref 1: (Takemura et al., 2013), 2: (Tuthill et al., 2013), 3: (Nern et al., 2015), 4: (Gao et al., 2008), 5: (Takemura et al., 2017), 6: (Mauss et al., 2015). Blue, no labeled cell of the indicated type; orange, labeled cell of the indicated type. Summary of DIP-expression in each medulla neuron type is listed in the last two columns. Note that the cell types from Takemura et al. (2013) are shown in Supplementary Table 2 (see https://media.nature.com/original/nature-assets/nature/journal/v500/n7461/extref/nature12450-s1.pdf) and the Dm and Pm cells are described in Nern et al. (2015). References for a few additional cell types are as indicated.See also Figures S1-S3.

Figure 3:. SPR binding analysis of DIP-Dpr interactions

(A) An example of SPR sensorgrams of 21 Dpr analytes binding over a DIP-λ-immobilized surface and the fit of the binding data to 1:1 binding isotherms to calculate K_Ds. Sensorgrams of Dprs 9, 8, 6 and 10, which bind with K_Ds lower than 200μM are shown individually and sensorgrams for all other Dprs with K_Ds above 200 μM are overlaid in a single panel. The concentrations for each experiment are listed in the Star Methods section. See also Figures S4C and S5 for sensorgrams and binding isotherms for the 21 Dprs binding to the 10 other DIP-immobilized surfaces. (B) Equilibrium-binding K_Ds of DIP-Dpr interactions determined by SPR. The Dprs are tabulated according to their DIP binding preference. “*” indicates apparent K_Ds that are likely to be overestimates due to the presence of some nonspecific binding. The number in brackets represents the error of the fit.

Figure 4:. DIP-Dpr quantitative interactome and phylogeny

(A) Heterophilic and homophilic interaction network according to SPR and AUC experiments, respectively. The interactions highlighted have K_Ds lower than 200μM. Lines are color-coded according to the affinity of the binding pairs while dashed lines correspond to interactions between 150-200μM. Dpr2 interactions with DIP-η, -ι and -κ are represented as estimates in the 40-95 μM range and DIP- κ/Dpr1 binding is represented as an estimate in the 150-200μM range due to some non-specific binding observed in SPR sensorgrams. Color-coded self-pointing arrows highlight DIPs or Dprs that homodimerize. A black self-pointing arrow is used for DIP-θ, which homodimerizes but an accurate affinity could not be determined. (B) Phylogenetic trees of Dprs and DIPs based on Ig1 domain similarity. The scale bar denotes protein distances estimated by Jones-Taylor-Thornton model (Jones et al., 1992). Dprs are colored according to primary DIP binding preference(s). “*” indicate Dprs with binding preferences deviating from group: Dpr1 and Dpr3 do not bind to DIP-θ and Dpr21 does not bind to DIP-λ with affinities lower than 200μM (see also text and Figure 3B).

Figure 5:. Structure of DIP homodimer and DIP-Dpr complexes

(A) Ribbon representation of the full Ig ectodomain of DIP-θ and DIP-α homodimers. Individual protomers of DIP-θ are in blue and light blue, and DIP-α protomers are in pink and purple. N-linked glycans that were visible in electron density maps are represented as colored shaded spheres. (B) Structural details of DIP-α homodimer interface with side-chains contributing to interface shown as sticks. Residues comprising the hydrophobic core are underlined. See also Figure S6A (C) Ribbon representation of DIP-θ/Dpr2 and DIP-η/Dpr4 complexes rotated 30° counter clockwise in relation to structures in (A). (D) Structural details of DIP-η/Dpr4 complex interface. Dashed purple line highlights the salt bridge between Asp74 and Lys94. See also Figures S6B-C. (E) Ig1-Ig1 superposition of DIP homodimers and DIP-Dpr heterophilic complexes reported here and DIP-α/Dpr6 complex (PDB ID: 5E09). Shown as carbon-α traces and superposed on the Ig1 of the left DIP protomer. (F) SPR sensorgrams for Dpr6 and Dpr10 binding over wild-type DIP-α, I83D and A78K N94D point mutants designed to disrupt heterophilic and/or homophilic interactions. Binding of Dpr10 Y103D to wild-type DIP-α is also shown. (G) Binding K_Ds from SPR analysis as well as oligomeric state determined by AUC for DIP-α wild-type and mutants.

Figure 6:. DIP-Dpr binding specificity

(A) DIPs are grouped based on similar binding preference. Residues highlighted show variability among DIPs with different Dpr binding specificity and their positions are denoted S_I1-S_I4. Shaded boxes below alignment indicate interfacial residues. Yellow residues highlight residue positions of the hydrophobic core seen in crystal structures. (B) Dprs are grouped based on binding specificity with specificity residues labeled S_R1-S_R3. P_R labels an additional residue position that is highly conserved among Dpr groups and is potentially involved in binding specificity. (C) and (D) Structural details of DIP-Dpr interaction region with specificity residues in DIP-η/Dpr4 and DIP-α/Dpr6 shown as sticks. The N102 N-glycan present in the DIP-α/Dpr6 structure is shown as grey spheres. (E) and (F) SPR sensorgrams of different Dpr4 and Dpr6 S_R mutants used as analytes over DIP-η and DIP-α immobilized surfaces. Labels indicate which S_R position(s) were mutated for Dpr4 and Dpr6. See also Figure S6D-E.