Clathrin heavy chain plays multiple roles in polarizing the Drosophila oocyte downstream of Bic-D

Paula Vazquez-Pianzola 1 Jacqueline Adam 1 Dominique Haldemann 1 Daniel Hain 1 Henning Urlaub 0 Beat Suter () 1 0 Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry , 37077 Go ttingen , Germany 1 Institute of Cell Biology, University of Bern , 3012 Berne , Switzerland Bicaudal-D (Bic-D), Egalitarian (Egl), microtubules and their motors form a transport machinery that localizes a remarkable diversity of mRNAs to specific cellular regions during oogenesis and embryogenesis. Bic-D family proteins also promote dynein-dependent transport of Golgi vesicles, lipid droplets, synaptic vesicles and nuclei. However, the transport of these different cargoes is still poorly understood. We searched for novel proteins that either mediate Bic-Ddependent transport processes or are transported by them. Clathrin heavy chain (Chc) co-immunopurifies with Bic-D in embryos and ovaries, and a fraction of Chc colocalizes with Bic-D. Both proteins control posterior patterning of the Drosophila oocyte and endocytosis. Although the role of Chc in endocytosis is well established, our results show that Bic-D is also needed for the elevated endocytic activity at the posterior of the oocyte. Apart from affecting endocytosis indirectly by its role in osk mRNA localization, Bic-D is also required to transport Chc mRNA into the oocyte and for transport and proper localization of Chc protein to the oocyte cortex, pointing to an additional, more direct role of Bic-D in the endocytic pathway. Furthermore, similar to Bic-D, Chc also contributes to proper localization of osk mRNA and to oocyte growth. However, in contrast to other endocytic components and factors of the endocytic recycling pathway, such as Rabenosyn-5 (Rbsn-5) and Rab11, Chc is needed during early stages of oogenesis (from stage 6 onwards) to localize osk mRNA correctly. Moreover, we also uncovered a novel, presumably endocytosis-independent, role of Chc in the establishment of microtubule polarity in stage 6 oocytes. - INTRODUCTION Studies in different organisms portray Bic-D homologs as factors that link the dynein-dynactin minus end-directed microtubule (MT) motor complex to different cargoes, moving them to particular cellular regions (Claussen and Suter, 2005; Vazquez-Pianzola and Suter, 2012). Drosophila Bic-D (also known as BicD) directly binds Egalitarian (Egl), which engages with Dynein light chain (Dlc; also know as Ctp) and specific mRNA localization signals (Navarro et al., 2004; Dienstbier et al., 2009). This complex is thought to associate with further proteins that regulate translation and stability of transported mRNAs, and the resulting larger complex translocates with the help of motors along the MT cytoskeleton. The Bic-D-dependent transport machinery is used repeatedly during Drosophila development. In oogenesis, Bic-D is needed for the transport of osk, bcd and grk mRNAs from the nurse cells into the oocyte and then to specific compartments within the oocyte (Suter and Steward, 1991; Ran et al., 1994; Clark et al., 2007). Correct localization of these factors within the oocyte is crucial for specifying anteroposterior and dorsoventral axes of oocytes and embryos. Later in the life cycle, the mRNA localization machinery delivers specific mRNAs to the apical side of the syncytial embryo (Bullock and Ish-Horowicz, 2001) and promotes localization of inscuteable mRNAs in neuroblasts (Hughes et al., 2004). Drosophila Bic-D binds mRNA cargoes not only through the cargobinding adaptor Egl, but also through the Fragile-X Mental Retardation Protein (Fmr1) (Bianco et al., 2010). Mammalian BicD2 regulates centrosome and nuclear positioning during mitotic entry (Splinter et al., 2010) and mammalian Bic-D isoforms bind Rab6 to control COPI-independent Golgi-endoplasmic reticulum transport (Matanis et al., 2002). Furthermore, Rab6B-BicD1 interaction regulates retrograde membrane transport in human neurites (Wanschers et al., 2007). Fly and worm Bic-D genes are also involved in nuclear migration in photoreceptors, oocytes and hypodermal precursor cells (Swan et al., 1999; Swan and Suter, 1996; Fridolfsson et al., 2010), and Bic-D also dynamically regulates transport of lipid droplets (Larsen et al., 2008). Given the involvement of Bic-D in the localization of surprisingly diverse cargoes, we searched for adaptor proteins that mediate cargo binding as well as for novel cargo molecules. RESULTS Chc associates with Bic-D in ovaries and embryos Using two different monoclonal anti-Bic-D antibodies that recognize different epitopes, we immunopurified Bic-D complexes and identified Chc as a complex component in both approaches through mass spectrometry (Fig. 1A). Beads alone and beads coupled to control antibodies did not precipitate Chc. Myc-tagged Chc expressed under UAS control and a Flag-Tetracysteine (4C)-tagged Chc expressed from its endogenous promoter (Kasprowicz et al., 2008) were also immunoprecipitated with both anti-Bic-D antibodies from embryonic (Fig. 1B) and ovarian (Fig. 1C) extracts. Li et al. showed that Chc interacts directly with Bic-D and is its major interactor in the nervous system (Li et al., 2010). In contrast to this, Bic-D interacts with Egl, Chc, Pabp and others in embryos (Fig. 1A) (VazquezPianzola et al., 2011; P.V.P. and B.S. unpublished data). Thus, in non-neuronal tissues, such as young embryos and ovaries, Chc appears to be one of many partners of Bic-D. A fraction of Chc dynamically colocalizes with Bic-D in cortical regions of the oocyte Anti-Bic-D antibody staining of ovaries that were fixed in a healthy state does not reveal aggregated structures, suggesting that native complexes are small particles that cannot be resolved with normal Fig. 1. Chc forms complexes with Bic-D in ovaries and embryos and is enriched in the oocyte. (A) Coomassie-stained SDS-PAGE showing anti-Bic-D IPs of total embryo extracts. Anti-Cdk7 antibodies were used as controls for nonspecific binding. Gel areas in which Chc, Bic-D and Egl were identified by mass spectrometry are indicated. Chc was identified in IPs performed with both anti-Bic-D antibodies, but was not found in the corresponding gel position after IPs with control antibodies. (B) IP of total embryo extracts expressing a Myc-tagged Chc expressed with the Nullo-Gal 4 driver. Antibodies used for IPs are indicated on top. Bic-D antibodies (4C2 and 1B11), a negative control mouse monoclonal antibody (HTm4) and beads alone were used. Western blots of the precipitated material were tested for the presence of Bic-D, Myc::Chc and Egl. (C) IPs with the antibodies indicated on top and input controls. Extracts were from ovaries of wild-type (wt) flies and from ovaries expressing a Flag-tagged Chc from the 4C-CHC genomic construct. Anti-Bic-D (1B11) and anti-GFP antibodies were used. IP material was analyzed by western blot to check for the presence of Bic-D, Egl and Chc. Chc was detected with anti-Flag and with rabbit anti-mammalian Chc ant (...truncated)