We are interested in identifying aspects of neuroblast cellular asymmetry and the control of cell polarity. At mitosis polarity is most prominent at the cell cortex where different protein complex localise to the apical and basal cell cortex defining the neuroblast polarity axis. We are currently disection how the differnt domains are established. The Video shows a neuroblast in culture in metaphase. The apical cortex (green) is labled with Par-3/Bazooka GFP and the basal cortex is decorated with mCHerry-Miranda, an adaptor protein carrying fate determinants such as Prospero.
Larval brain neuroblasts divide strikingly at the same orientation in consecutive divisions, hence daughter cells cluster at the basal pole. Since the cortex depolarises during interphase other mechanisms must ensure the stability of cell division orientation. We are adressing external and intrinsic signals that operate in this polarity memory. One key player is the microtubule cytoskeleton of the neuroblast. During interphase cellular asymmetry is apparent in the microtubule cytoskeleton, that is mainly organised from prominent microtubule organising center at the apical cell pole. The Video shows a neuroblast engineered to express fluorescently labeled Tubulin and centriole markers.
How cells position their proteins is a key problem in cell biology. Targeting mRNAs to distinct regions of the cytoplasm contributes to protein localization by providing local control over translation. We are currently testing the contribution of mRNA localization to asymmetric cell division in neuroblasts of the Drosophila larvae.
In one approach, we tagged endogenous mRNA using CrispR with MS2 stem loops, to be able to trace the mRNA of candidate genes in living neuroblasts since this allows tagging of mRNA with GFP. We then used the subcellular expression GFP-specific nanobodies, to alter the subcellular localization of that GFP tagged mRNA, by using fusions of the nanobodyto subcellular localization domains. In Ramat et alwe report that the mRNA of Miranda, which is asymmetrically localized in neuroblasts and required for cell fate specification localizes like the protein it encodes asymmetrically in mitosis. Altering the localization of the mRNA resulted in mislocalization of the protein. Protein localization defects caused by mislocalization of the cognate mRNA were rescued by introducing untagged mRNA coding for mutant non-localizable protein. Thus the MS2 system and nanobody expression can be combined to redirect subcellular mRNA localization and in the case of Miranda, we reveal that interaction of Miranda protein and its mRNA is required for maintenance of the subcellular localization of Mira protein. We explore how mRNAs can localize proteins throgh otehr means than just serving as a local source for synthesis of teh cognate protein.
In a complementary approach we are looking for new genes required for the establishment of stem cell polarity and cell fate determination in a mRNA localisation screen that we perform in collaboration with the group of Maria Leptin.