Role of BicDR in bristle shaft construction and support of BicD functions

ABSTRACT Cell polarization requires asymmetric localization of numerous mRNAs, proteins and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors. In the dynein–dynactin–Bicaudal-D transport machinery, Bicaudal-D (BicD) links the cargo to the motor. Here, we focus on the role of Drosophila BicD-related (BicDR, CG32137) in the development of the long bristles. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft. BicD and BicDR also support the stable expression and distribution of Rab6 and Spn-F in the bristle shaft, including the distal tip localization of Spn-F, pointing to the role of microtubule-dependent vesicle trafficking for bristle construction. BicDR supports the function of BicD, and we discuss the hypothesis whereby BicDR might transport cargo more locally, with BicD transporting cargo over long distances, such as to the distal tip. We also identified embryonic proteins that interact with BicDR and appear to be BicDR cargo. For one of them, EF1γ (also known as eEF1γ), we show that the encoding gene EF1γ interacts with BicD and BicDR in the construction of the bristles.


Journal of Cell Science • Supplementary information
Table S1.Proteins that were significantly enriched in the tagged BicDR::GFP IPs in comparison to the wild-type negative control IPs.Genes encoding these proteins are listed according to their level of significance of enrichment over the control.The abbreviations used are "control" for the white control, "BicDR" (BicDR::GFP), and "BicDR K>A" (BicDR K555A ::GFP).Hits enriched in tagged wild-type BicDR compared to BicDR K>A are listed as potential cargo, whereas peptides enriched in tagged wild-type BicDR and BicDR K555A IPs are listed as potential non-cargo interactors.GN is the abbreviation for gene name.The iBAQ equals the sum of all peptide intensities divided by the number of observable peptides of a protein 1 .log 2 FC is the logarithm of the mean ratio between the two groups and the adjusted p-value (adj.pVal) highlights the factor level comparisons within a family that are significantly different 2,3

Fig. S2 .
Fig. S2.Posterior scutellar bristles (pSC): overview (left) and corresponding bristle tips.The genotypes are indicated above the overview and the corresponding tips to the right.The 2 nd chromosomes with the BicD gene and the 3 rd with the BicDR gene are indicated.BicD alleles were BicD PA66 (PA66) and Df7068 (-).BicDR alleles were BicDR 71 and Df4515 (-)."+" indicates a wild-type allele of BicD or BicDR either on an uncharacterized chromosome or a balancer chromosome.AddiKonal examples from these and other BicDR allelles have been deposited at the Dryad Digital Repository (Suter et al., 2024; https://doi.org/10.5061/dryad.dfn2z358t).

Fig. S3 .
Fig. S3.Drosophila BicDR does not interact with EF1g, Egl, or BicD.(A) The activation domain is indicated on the left and the binding domains are on the right side.The latter is drawn where these activation domains were plated out.(-LW): grown on medium selecting for the two plasmids, (-LWH): selective plates on which cells with the activator domain and the DNA binding domain clones can grow if they weakly interact.(-LWH) with 20 mM 3aminotriazole [3-AT]: selective plates on which cells with the activator domain and the DNA binding domain clones can grow if they strongly interact.FL: full length, CTD: C-terminal domain, BD: binding domain, AD: activation domain.BicD and Egl were used as positive controls since their interaction had already been demonstrated 79 .Here, the full-length protein of BicDR, fused to the activation domain, binds to the C-terminal domain of BicDR as well as to the C-terminal domain of BicD.However, this result is not confirmed under stringent conditions, since neither the full-length BicD protein nor the C-terminal domain of BicD seems to bind to BicDR in immunoprecipitations nor according to the yeast 2-hybrid assay.Thus, these results seem to indicate that the direct interaction between BicDR FL (AD) and BicD (BD) resulted from the nonspecific entanglement of the coiled coil domains 80 .(B) Negative controls.

Fig. S4 (
Fig. S4 (Display of signal intensity is normalized for each channel).This shows the distribution of the indicated signals; but levels cannot be compared between channels.The top left picture shows a maximal projection of the region with the pupal posterior Scutellar Bristle (pSC).On top, the F-actin signal and a drawing of the line along which the staining intensities of the three channels were measured.Below, the staining for Spn-F in green and Rab6 in red.Pictures are oriented such that the proximal end of the bristle shaft is to the left side and the distal one to the right.The other three panels show the intensity of the signal in the different z-planes along the drawn line.The approximate position of the bristle shaft was estimated from the Factin (Actin) and the Spn-F signal and is shown with a dashed line in the F-actin panel.The methods are described in the main part of the paper.

Fig. S5 .
Fig. S5.Same data as in Figure S4 but with signal intensities normalized over the 3 channels.Taking the background signals into consideration, this allows to compare Rab6 and Spn-F expression levels along the bristle shaft across genotypes relative to F-actin.The maximal projection image and the approximate outline of the pSC through the z-stack planes are depicted in the corresponding top panels of Figure S4.Graphs are oriented such that the proximal end of the bristle shaft is to the left and the distal one to the right.The three panels show the intensity of the signal in the different z-planes measured along the pupal bristle (see Figure S4).The methods are described in the main part of the paper.