Applications of Immobilization of <em>Drosophila</em> Tissues with Fibrin Clots for Live Imaging

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of ...

University of Dundee

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  nervous system, specifically the control of the polarized distribution of the cell fate adapter protein Miranda. We reveal a step-wise 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.

aPKC-mediated displacement and actomyosin-mediated retention polarize Miranda in  neuroblasts.

Controlling the orientation of cell division is important in the context of cell fate choices and tissue morphogenesis. However, the mechanisms providing the required positional information remain incompletely understood. Here we use stem cells of the Drosophila larval brain that stably maintain their axis of polarity and division between cell cycles to identify cues that orient cell division. Using live cell imaging of cultured brains, laser ablation and genetics, we reveal that division axis maintenance relies on their last-born daughter cell. We propose that, in addition to known intrinsic cues, stem cells in the developing fly brain are polarized by an extrinsic signal. We further find that division axis maintenance allows neuroblasts to maximize their contact area with glial cells known to provide protective and proliferative signals to neuroblasts.

The last-born daughter cell contributes to division orientation of Drosophila larval 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.  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  atypical Protein Kinase C (aPKC). We demonstrate that the resulting mutant aPKC kinase can be specifically inhibited  and  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.

A chemical-genetics approach to study the role of atypical Protein Kinase C in .

 () is the most frequently mutated gene in colorectal cancer. APC negatively regulates the Wnt signaling pathway by promoting the degradation of β-catenin, but the extent to which APC exerts Wnt/β-catenin-independent tumor-suppressive activity is unclear. To identify interaction partners and β-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed tandem mass tag-based mass spectrometry. Affinity enrichment-mass spectrometry identified more than 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of β-catenin. Combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an APC-containing destruction complex. We validated the interaction between endogenous MINK1 and APC and further confirmed the negative, and β-catenin-independent, regulation of MINK1 by APC. Increased Mink1/Msn levels were also observed in mouse intestinal tissue and  follicular cells expressing mutant Apc/APC when compared with wild-type tissue/cells. Collectively, our results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease. IMPLICATIONS: The tumor-suppressive function of APC, the most frequently mutated gene in colorectal cancer, is mainly attributed to its role in β-catenin/Wnt signaling. Our study substantially expands the list of APC interaction partners and reveals that approximately half of the changes in the cellular proteome induced by loss of APC function are mediated by β-catenin-independent mechanisms.

Identification of Endogenous Adenomatous Polyposis Coli Interaction Partners and β-Catenin-Independent Targets by Proteomics.

Asymmetric cell division (ACD) is the fundamental process through which one cell divides into two cells with different fates. In animals, it is crucial for the generation of cell-type diversity and for stem cells, which use ACD both to self-renew and produce one differentiating daughter cell. One of the most prominent model systems of ACD, Drosophila neuroblasts, relies on the PAR complex, a conserved set of proteins governing cell polarity in animals. Here, we focus on recent advances in our understanding of the mechanisms that control the orientation of the neuroblast polarity axis, how the PAR complex is positioned, and how its activity may regulate division orientation and cell fate determinant localization and discuss how important findings about the composition polarity complexes in other models may apply to neuroblasts.

Nicolas Loyer

Cell & Developmental Biology,
School of Life Sciences,
Cell & Developmental Biology, School of Life Sciences

University of Dundee

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

Nicolas Loyer

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University of Dundee

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

Cell & Developmental Biology,
School of Life Sciences,
Cell & Developmental Biology, School of Life Sciences