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.
running title: Function of Drosophila Spindly key words: Drosophila, mitosis, cell migration, mitotic spindle, Dynein not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/199414 doi: bioRxiv preprint first posted online Oct. 6, 2017; 2 AbstractSpindly is a mitotic checkpoint protein originally identified as a specific regulator of Dynein activity at the kinetochore. In metaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable
Spindly was originally identified as a specific regulator of Dynein activity at the kinetochore. In early prometaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. Here, we present loss- and gain-of-function studies of Spindly using transgenic RNAi in Drosophila. Knock-down of Spindly in the female germ line results in mitotic arrest during embryonic cleavage divisions. We investigated the requirements of Spindly protein domains for its localisation and function, and found that the carboxy-terminal region controls Spindly localisation in a cell-type specific manner. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells, we altered Spindly protein levels in migrating cells and found that ovarian border cell migration is sensitive to the levels of Spindly protein. Our study uncovers novel functions of Spindly and a differential, functional requirement for its carboxy-terminal region in Drosophila.
Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosome nucleated microtubules with motor proteins. However, it remains unknown how centrioles migrate in cellular contexts in which centrioles do not nucleate microtubules. Here, we demonstrate that during interphase inactive centrioles move directly along the noncentrosomal microtubule network as cargo for the motor protein Kinesin-1. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. PLP directly interacts with the cargo binding domain of Kinesin-1 and they comigrate on microtubules in vitro. Finally, we demonstrate that PLP-Kinesin-1 dependent transport is essential for centrosome asymmetry age dependent centrosome inheritance in asymmetric stem cell division.
SUMMARY 10Cell fate generation can rely on the unequal distribution of molecules during 11 progenitor cell division in the nervous system of vertebrates and 12invertebrates. Here we address asymmetric fate determinant localization in 13 the developing Drosophila nervous system, focussing on the control of 14 asymmetric Miranda distribution in larval neuroblasts. We used live imaging of 15 neuroblast polarity reporters at endogenous levels of expression to address 16Miranda localization during the cell cycle. We reveal that the regulation and 17 dynamics of cortical association of Miranda in interphase and mitosis are 18 different. In interphase Miranda binds directly to the plasma membrane. At 19 the onset of mitosis, Miranda is phosphorylated by aPKC and displaced from 20 the PM. After nuclear envelope breakdown asymmetric localization of Miranda 21 requires actomyosin activity. Therefore, Miranda phosphorylation by aPKC and 22 differential binding to the actomyosin network are required at distinct phases 23 of the cell cycle to polarize fate determinant localization. 24
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