Cell fate assignment in the nervous system of vertebrates and invertebrates often hinges on the unequal distribution of molecules during progenitor cell division. We address asymmetric fate determinant localization in the developing Drosophila nervous system, specifically the control of the polarized distribution of the cell fate adapter protein Miranda. We reveal a stepwise polarization of Miranda in larval neuroblasts and find that Miranda's dynamics and cortical association are differently regulated between interphase and mitosis. In interphase, Miranda binds to the plasma membrane. Then, before nuclear envelope breakdown, Miranda is phosphorylated by aPKC and displaced into the cytoplasm. This clearance is necessary for the subsequent establishment of asymmetric Miranda localization. After nuclear envelope breakdown, actomyosin activity is required to maintain Miranda asymmetry. Therefore, phosphorylation by aPKC and differential binding to the actomyosin network are required at distinct phases of the cell cycle to polarize fate determinant localization in neuroblasts.
Studying the function of proteins using genetics in cycling cells is complicated by the fact that there is often a delay between gene inactivation and the time point of phenotypic analysis. This is particularly true when studying kinases that have pleiotropic functions and multiple substrates. Drosophila neuroblasts (NBs) are rapidly dividing stem cells and an important model system for the study of cell polarity. Mutations in multiple kinases cause NB polarity defects, but their precise functions at particular time points in the cell cycle are unknown. Here, we use chemical genetics and report the generation of an analogue-sensitive allele of Drosophila atypical Protein Kinase C (aPKC). We demonstrate that the resulting mutant aPKC kinase can be specifically inhibited in vitro and in vivo. Acute inhibition of aPKC during NB polarity establishment abolishes asymmetric localization of Miranda, whereas its inhibition during NB polarity maintenance does not in the time frame of normal mitosis. However, aPKC helps to sharpen the pattern of Miranda, by keeping it off the apical and lateral cortex after nuclear envelope breakdown.
SummaryHow 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. Here, we reveal that an interdependence of a protein and cognate mRNA maintains asymmetric protein distribution in mitotic Drosophila neural stem cells. We tagged endogenous mRNA or protein products of the gene miranda that is required for fate determination with GFP. We find that the mRNA localizes like the protein it encodes in a basal crescent in mitosis. We then used GFP-specific nanobodies fused to localization domains to alter the subcellular distribution of the GFP-tagged mRNA or protein. Altering the localization of the mRNA resulted in mislocalization of the protein and vice versa. Protein localization defects caused by mislocalization of the cognate mRNA were rescued by introducing untagged mRNA coding for mutant non-localizable protein. Therefore, by combining the MS2 system and subcellular nanobody expression, we uncovered that maintenance of Mira asymmetric localization requires interaction with the cognate mRNA.
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