Drosophila Adducin facilitates phase separation and function of a conserved spindle orientation complex

Asymmetric cell division (ACD) allows stem cells to generate differentiating progeny while simultaneously maintaining their own pluripotent state. ACD involves coupling mitotic spindle orientation with cortical polarity cues to direct unequal segregation of cell fate determinants. In Drosophila neural stem cells (neuroblasts; NBs), spindles orient along an apical-basal polarity axis through a conserved complex of Partner of Inscuteable (Pins; human LGN) and Mushroom body defect (Mud; human NuMA). While many details of its function are well known, the molecular mechanics that drive assembly of the cortical Pins/Mud complex remain unclear, particularly with respect to the mutually exclusive Pins complex formed with the apical scaffold protein Inscuteable (Insc). Here we identify Hu li tai shao (Hts; human Adducin) as a direct Mud-binding protein, using an aldolase fold within its head domain (HtsHEAD) to bind a short Mud coiled-coil domain (MudCC) that is adjacent to the Pins-binding domain (MudPBD). Hts is expressed throughout the larval central brain and apically polarizes in mitotic NBs where it is required for Mud-dependent spindle orientation. In vitro analyses reveal that Pins undergoes liquid-liquid phase separation with Mud, but not with Insc, suggesting a potential molecular basis for differential assembly mechanics between these two competing apical protein complexes. Furthermore, we find that Hts binds an intact Pins/Mud complex, reduces the concentration threshold for its phase separation, and alters the liquid-like property of the resulting phase separated droplets. Domain mapping and mutational analyses implicate critical roles for both multivalent interactions (via MudCC oligomerization) and protein disorder (via an intrinsically disordered region in Hts; HtsIDR) in phase separation of the Hts/Mud/Pins complex. Our study identifies a new component of the spindle positioning machinery in NBs and suggests that phase separation of specific protein complexes might regulate ordered assembly within the apical domain to ensure proper signaling output

Mud Loss Restricts Yki-Dependent Hyperplasia in Drosophila Epithelia

Tissue development demands precise control of cell proliferation and organization, which is achieved through multiple conserved signaling pathways and protein complexes in multicellular animals. Epithelia are a ubiquitous tissue type that provide diverse functions including physical protection, barrier formation, chemical exchange, and secretory activity. However, epithelial cells are also a common driver of tumorigenesis; thus, understanding the molecular mechanisms that control their growth dynamics is important in understanding not only developmental mechanisms but also disease. One prominent pathway that regulates epithelial growth is the conserved Hippo/Warts/Yorkie network. Hippo/Warts inactivation, or activating mutations in Yorkie that prevent its phosphorylation (e.g., YkiS168A), drive hyperplastic tissue growth. We recently reported that loss of Mushroom body defect (Mud), a microtubule-associated protein that contributes to mitotic spindle function, restricts YkiS168A-mediated growth in Drosophila imaginal wing disc epithelia. Here we show that Mud loss alters cell cycle progression and triggers apoptosis with accompanying Jun kinase (JNK) activation in YkiS168A-expressing discs. To identify additional molecular insights, we performed RNAseq and differential gene expression profiling. This analysis revealed that Mud knockdown in YkiS168A-expressing discs resulted in a significant downregulation in expression of core basement membrane (BM) and extracellular matrix (ECM) genes, including the type IV collagen gene viking. Furthermore, we found that YkiS168A-expressing discs accumulated increased collagen protein, which was reduced following Mud knockdown. Our results suggest that ECM/BM remodeling can limit untoward growth initiated by an important driver of tumor growth and highlight a potential regulatory link with cytoskeleton-associated genes

Emerging Roles of RNA-Binding Proteins in Neurodevelopment

Diverse cell types in the central nervous system (CNS) are generated by a relatively small pool of neural stem cells during early development. Spatial and temporal regulation of stem cell behavior relies on precise coordination of gene expression. Well-studied mechanisms include hormone signaling, transcription factor activity, and chromatin remodeling processes. Much less is known about downstream RNA-dependent mechanisms including posttranscriptional regulation, nuclear export, alternative splicing, and transcript stability. These important functions are carried out by RNA-binding proteins (RBPs). Recent work has begun to explore how RBPs contribute to stem cell function and homeostasis, including their role in metabolism, transport, epigenetic regulation, and turnover of target transcripts. Additional layers of complexity are provided by the different target recognition mechanisms of each RBP as well as the posttranslational modifications of the RBPs themselves that alter function. Altogether, these functions allow RBPs to influence various aspects of RNA metabolism to regulate numerous cellular processes. Here we compile advances in RNA biology that have added to our still limited understanding of the role of RBPs in neurodevelopment

Effect of praziquantel on the differential expression of mouse hepatic genes and parasite ATP binding cassette transporter gene family members during <i>Schistosoma mansoni</i> infection

<div><p>Schistosomiasis is a chronic parasitic disease caused by sexually dimorphic blood flukes of the genus <i>Schistosoma</i>. Praziquantel (PZQ) is the only drug widely available to treat the disease but does not kill juvenile parasites. Here we report the use of next generation sequencing to study the transcriptional effect of PZQ on murine hepatic inflammatory, immune and fibrotic responses to <i>Schistosoma mansoni</i> worms and eggs. An initial T helper cell 1 (Th1) response is induced against schistosomes in mice treated with drug vehicle (Vh) around the time egg laying begins, followed by a T helper cell 2 (Th2) response and the induction of genes whose action leads to granuloma formation and fibrosis. When PZQ is administered at this time, there is a significant reduction in egg burden yet the hepatic Th1, Th2 and fibrotic responses are still observed in the absence of granuloma formation suggesting some degree of gene regulation may be induced by antigens released from the dying adult worms. Quantitative real-time PCR was used to examine the relative expression of 16 juvenile and adult <i>S</i>. <i>mansoni</i> genes during infection and their response to Vh and PZQ treatment <i>in vivo</i>. While the response of stress genes in adult parasites suggests the worms were alive immediately following exposure to PZQ, they were unable to induce transcription of any of the 9 genes encoding ATP-binding cassette (ABC) transporters tested. In contrast, juvenile schistosomes were able to significantly induce the activities of ABCB, C and G family members, underscoring the possibility that these efflux systems play a major role in drug resistance.</p></div

The effects of PZQ on infected host liver.

<p>(A) Effect of treatment with Vh and PZQ on the number of parasites present in host livers at days 32, 35 and 46 post infection, n = 5 per treatment group. (B) Number of parasite eggs per gram of liver at days 32, 35, 39 and 46 post infection after treatment with Vh and PZQ (n = 4). (C) Weight of liver at days 32, 35, 39 and 46 post infection after treatment with Vh and PZQ (n = 4). Error bars represent mean with standard deviation. * p <0.05 and *** p < 0.001.</p

Canonical pathway analysis of T helper cell maturation and differentiation in PZQ treated infected mice.

<p>Signaling events in the T cell maturation and differentiation pathway at (A) 32, (B) 35, (C) 39 and (D) 46 days in infected PZQ (Sm_PZQ) treated mice. Increasing expression in infected mice relative to uninfected mice is indicated by deeper blue shading. None of the genes indicated were down-regulated. Non-expression and non-differential expression is indicated by a lack of shading.</p

Canonical pathway analysis of hepatic fibrosis and stellate cell activation in vehicle treated infected mice.

<p>Signaling events in the fibrotic and stellate cell activation pathway at (A) 32, (B) 35, (C) 39 and (D) 46 days post infection in infected Vh treated (Sm_Vh) mice. Increasing expression in infected mice relative to uninfected mice is indicated by deeper blue shading. Decreased expression is shown in dark red. Non-expression and non-differential expression is indicated by a lack of shading.</p

Temporal expression of mouse hepatic genes during infection with <i>S</i>. <i>mansoni</i>.

<p>Heat maps of (A) immune and (B) fibrotic gene markers depicting differentially expressed genes in infected Vh and PZQ treated mice relative to uninfected mice. Gray map sections represent genes not expressed at that time point or in that treatment group. Regions of blue and red indicate, relative to uninfected Vh treated controls, increased and decreased gene expression respectively. The color scale indicates log₂ fold change (FC) and the profile of each group is the average of three biological replicates. Gene names associated with the figure differ slightly from those used in the text. Relevant differences include: Ccl (rather than CCL as it appears in the main body of text), Cxc (CXC), Cxcl (CXCL), Il1b (IL1β), Tnfa (TNFα), Tgfb (TGFβ), Ifng (IFNγ), Cd (CD) and Timp (TIMP).</p