Identification of an Aurora-A/PinsLINKER/Dlg spindle orientation pathway using induced cell polarity in S2 cells.

Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. Here, we develop a method to construct cortical polarity in a normally unpolarized cell line and use this method to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals) -dependent spindle orientation. We identify a previously unrecognized evolutionarily conserved Pins domain (Pins(LINKER)) that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized Pins(TPR) domain has no function alone, but placing the Pins(TPR) in cis to the Pins(LINKER) gives dynein-dependent precise spindle orientation. This "induced cortical polarity" assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity.

Olfactory sensory neurons mediate ultrarapid antiviral immune responses in a TrkA-dependent manner.

The nervous system regulates host immunity in complex ways. Vertebrate olfactory sensory neurons (OSNs) are located in direct contact with pathogens; however, OSNs' ability to detect danger and initiate immune responses is unclear. We report that nasal delivery of rhabdoviruses induces apoptosis in crypt OSNs via the interaction of the OSN TrkA receptor with the viral glycoprotein in teleost fish. This signal results in electrical activation of neurons and very rapid proinflammatory responses in the olfactory organ (OO), but dampened inflammation in the olfactory bulb (OB). CD8α+ cells infiltrate the OO within minutes of nasal viral delivery, and TrkA blocking, but not caspase-3 blocking, abrogates this response. Infiltrating CD8α+ cells were TCRαß T cells with a nonconventional phenotype that originated from the microvasculature surrounding the OB and not the periphery. Nasal delivery of viral glycoprotein (G protein) recapitulated the immune responses observed with the whole virus, and antibody blocking of viral G protein abrogated these responses. Ablation of crypt neurons in zebrafish resulted in increased susceptibility to rhabdoviruses. These results indicate a function for OSNs as a first layer of pathogen detection in vertebrates and as orchestrators of nasal-CNS antiviral immune responses.

Addressing Ligand-Based Redox in Molybdenum-Dependent Methionine Sulfoxide Reductase.

A combination of pulsed EPR, CW EPR, and X-ray absorption spectroscopies has been employed to probe the geometric and electronic structure of the E. coli periplasmic molybdenum-dependent methionine sulfoxide reductase (MsrP). 17O and 1H pulsed EPR spectra show that the as-isolated Mo(V) enzyme form does not possess an exchangeable H2O/OH- ligand bound to Mo as found in the sulfite oxidizing enzymes of the same family. The nature of the unusual CW EPR spectrum has been re-evaluated in light of new data on the MsrP-N45R variant and related small-molecule analogues of the active site. These data point to a novel "thiol-blocked" [(PDT)MoVO(SCys)(thiolate)]- structure, which is supported by new EXAFS data. We discuss these new results in the context of ligand-based and metal-based redox chemistry in the enzymatic oxygen atom transfer reaction.

Molecular pathways regulating mitotic spindle orientation in animal cells.

Orientation of the cell division axis is essential for the correct development and maintenance of tissue morphology, both for symmetric cell divisions and for the asymmetric distribution of fate determinants during, for example, stem cell divisions. Oriented cell division depends on the positioning of the mitotic spindle relative to an axis of polarity. Recent studies have illuminated an expanding list of spindle orientation regulators, and a molecular model for how cells couple cortical polarity with spindle positioning has begun to emerge. Here, we review both the well-established spindle orientation pathways and recently identified regulators, focusing on how communication between the cell cortex and the spindle is achieved, to provide a contemporary view of how positioning of the mitotic spindle occurs.

Formin-mediated actin polymerization cooperates with Mushroom body defect (Mud)-Dynein during Frizzled-Dishevelled spindle orientation.

To position the mitotic spindle, cytoskeletal components must be coordinated to generate cortical forces on astral microtubules. Although the dynein motor is common to many spindle orientation systems, 'accessory pathways' are often also required. In this work, we identified an accessory spindle orientation pathway in Drosophila that functions with Dynein during planar cell polarity, downstream of the Frizzled (Fz) effector Dishevelled (Dsh). Dsh contains a PDZ ligand and a Dynein-recruiting DEP domain that are both required for spindle orientation. The Dsh PDZ ligand recruits Canoe/Afadin and ultimately leads to Rho GTPase signaling mediated through RhoGEF2. The formin Diaphanous (Dia) functions as the Rho effector in this pathway, inducing F-actin enrichment at sites of cortical Dsh. Chimeric protein experiments show that the Dia-actin accessory pathway can be replaced by an independent kinesin (Khc73) accessory pathway for Dsh-mediated spindle orientation. Our results define two 'modular' spindle orientation pathways and show an essential role for actin regulation in Dsh-mediated spindle orientation.

Conversion of the enzyme guanylate kinase into a mitotic-spindle orienting protein by a single mutation that inhibits GMP-induced closing.

New protein functions can require complex sequence changes, but the minimal path is not well understood. The guanylate kinase enzyme (GK(enz)), which catalyzes phosphotransfer from ATP to GMP, evolved into the GK domain (GK(dom)), a protein-binding domain found in membrane associate guanylate kinases that function in mitotic spindle orientation and cell adhesion. Using an induced polarity assay for GK(dom) function, we show that a single serine to proline mutation is sufficient to switch extant GK(enz) into a functional GK(dom). The mutation blocks catalysis (GK(enz) function) but allows protein binding and spindle orientation (GK(dom) function). Furthermore, whereas the GK(enz) undergoes a large closing motion upon GMP binding, fluorescence quenching and NMR demonstrate that the S â†’ P mutation inhibits GMP-induced GK movements. Disrupting GK closing with a mutation at a different position also leads to GK(dom) function, suggesting that blocking the GK(enz) closing motion is sufficient for functional conversion of GK(enz) to GK(dom). Although subtle changes in protein function can require complex sequence paths, our work shows that entirely new functions can arise from single mutations that alter protein dynamics.

Multiple tail domain interactions stabilize nonmuscle myosin II bipolar filaments.

Contractile force transduction by myosin II derives from its assembly into bipolar filaments. The coiled-coil tail domain of the myosin II heavy chain mediates filament assembly, although the mechanism is poorly understood. Tail domains contain an alternating electrostatic repeat, yet only a small region of the tail (termed the "assembly domain") is typically required for assembly. Using computational analysis, mutagenesis, and electron microscopy we discovered that the assembly domain does not function through self-interaction as previously thought. Rather, the assembly domain acts as a unique, positively charged interaction surface that can stably contact multiple complementary, negatively charged surfaces in the upstream tail domain. The relative affinities of the assembly domain to each complementary interaction surface sets the characteristic molecular staggers observed in myosin II filaments. Together these results explain the relationship between the charge repeat and assembly domain in stabilizing myosin bipolar filaments.

Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity.

G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G betagamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of "regulator of G-protein signaling" (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a G alpha switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP x AlF(4)(-)-bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A) x GDP assumed a divergent conformation more closely resembling the GDP x AlF(4)(-)-bound state. When placed within Saccharomyces cerevisiae G alpha subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context.

Phase Separation as a Driver of Stem Cell Organization and Function during Development.

A properly organized subcellular composition is essential to cell function. The canonical organizing principle within eukaryotic cells involves membrane-bound organelles; yet, such structures do not fully explain cellular complexity. Furthermore, discrete non-membrane-bound structures have been known for over a century. Liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mode of cellular organization without the need for formal lipid membranes, with an ever-expanding and diverse list of cellular functions that appear to be regulated by this process. In comparison to traditional organelles, LLPS can occur across wider spatial and temporal scales and involves more distinct protein and RNA complexes. In this review, we discuss the impacts of LLPS on the organization of stem cells and their function during development. Specifically, the roles of LLPS in developmental signaling pathways, chromatin organization, and gene expression will be detailed, as well as its impacts on essential processes of asymmetric cell division. We will also discuss how the dynamic and regulated nature of LLPS may afford stem cells an adaptable mode of organization throughout the developmental time to control cell fate. Finally, we will discuss how aberrant LLPS in these processes may contribute to developmental defects and disease.

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.

Modeling a variant of unknown significance in the Drosophila ortholog of the human cardiogenic gene NKX2.5.

Sequencing of human genome samples has unearthed genetic variants for which functional testing is necessary to validate their clinical significance. We used the Drosophila system to analyze a variant of unknown significance in the human congenital heart disease gene NKX2.5 (also known as NKX2-5). We generated an R321N allele of the NKX2.5 ortholog tinman (tin) to model a human K158N variant and tested its function in vitro and in vivo. The R321N Tin isoform bound poorly to DNA in vitro and was deficient in activating a Tin-dependent enhancer in tissue culture. Mutant Tin also showed a significantly reduced interaction with a Drosophila T-box cardiac factor named Dorsocross1. We generated a tinR321N allele using CRISPR/Cas9, for which homozygotes were viable and had normal heart specification, but showed defects in the differentiation of the adult heart that were exacerbated by further loss of tin function. We propose that the human K158N variant is pathogenic through causing a deficiency in DNA binding and a reduced ability to interact with a cardiac co-factor, and that cardiac defects might arise later in development or adult life.

Using Drosophila to model a variant of unknown significance in the human cardiogenic gene Nkx2.5.

Sequencing of human genome samples has unearthed genetic variants for which functional testing is necessary to validate their clinical significance. We used the Drosophila system to analyze a variant of unknown significance in the human congenital heart disease gene, Nkx2 . 5 . We generated an R321N allele of the Nkx2 . 5 ortholog tinman ( tin ) to model a human K158N variant and tested its function in vitro and in vivo. The R321N Tin isoform bound poorly to DNA in vitro and was deficient in activating a Tin-dependent enhancer in tissue culture. Mutant Tin also showed a significantly reduced interaction with a Drosophila Tbox cardiac factor named Dorsocross1. We generated a tin R321N allele using CRISPR/Cas9, for which homozygotes were viable and had normal heart specification, but showed defects in the differentiation of the adult heart that were exacerbated by further loss of tin function. We conclude that the human K158N mutation is likely pathogenic through causing both a deficiency in DNA binding and a reduced ability to interact with a cardiac cofactor, and that cardiac defects might arise later in development or adult life.

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.

Corrigendum: A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity.

[This corrects the article DOI: 10.3389/fcell.2020.598492.].

Mud binds the kinesin-14 Ncd in Drosophila.

Maintenance of proper mitotic spindle structure is necessary for error-free chromosome segregation and cell division. Spindle assembly is controlled by force-generating kinesin motors that contribute to its geometry and bipolarity, and balancing motor-dependent forces between opposing kinesins is critical to the integrity of this process. Non-claret dysjunctional (Ncd), a Drosophila kinesin-14 member, crosslinks and slides microtubule minus-ends to focus spindle poles and sustain bipolarity. However, mechanisms that regulate Ncd activity during mitosis are underappreciated. Here, we identify Mushroom body defect (Mud), the fly ortholog of human NuMA, as a direct Ncd binding partner. We demonstrate this interaction involves a short coiled-coil domain within Mud (MudCC) binding the N-terminal, non-motor microtubule-binding domain of Ncd (NcdnMBD). We further show that the C-terminal ATPase motor domain of Ncd (NcdCTm) directly interacts with NcdnMBD as well. Mud binding competes against this self-association and also increases NcdnMBD microtubule binding in vitro. Our results describe an interaction between two spindle-associated proteins and suggest a potentially new mode of minus-end motor protein regulation at mitotic spindle poles.

Structure of the parathyroid hormone receptor C terminus bound to the G-protein dimer Gbeta1gamma2.

A critical role of the Gbetagamma dimer in heterotrimeric G-protein signaling is to facilitate the engagement and activation of the Galpha subunit by cell-surface G-protein-coupled receptors. However, high-resolution structural information of the connectivity between receptor and the Gbetagamma dimer has not previously been available. Here, we describe the structural determinants of Gbeta1gamma2 in complex with a C-terminal region of the parathyroid hormone receptor-1 (PTH1R) as obtained by X-ray crystallography. The structure reveals that several critical residues within PTH1R contact only Gbeta residues located within the outer edge of WD1- and WD7-repeat segments of the Gbeta toroid structure. These regions encompass a predicted membrane-facing region of Gbeta thought to be oriented in a fashion that is accessible to the membrane-spanning receptor. Mutation of key receptor contact residues on Gbeta1 leads to a selective loss of function in receptor/heterotrimer coupling while preserving Gbeta1gamma2 activation of the effector phospholipase-C beta.

A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity.

Cell polarity is an evolutionarily conserved process of asymmetric spatial organization within cells and is essential to tissue structure, signal transduction, cell migration, and cell division. The establishment and maintenance of polarity typically involves extensive protein-protein interactions that can be made further intricate by cell cycle-dependent regulation. These aspects can make interpreting phenotypes within traditional in vivo genetic systems challenging due to pleiotropic effects in loss-of-function experiments. Minimal reconstitution methods offer investigators the advantage of stricter control of otherwise complex systems and allow for more direct assessment of the role of individual components to the process of interest. Here I provide a detailed protocol for a cell adhesion-based method of inducing cell polarity within non-polarized Drosophila S2 cells. This technique is simple, cost effective, moderate throughput, and amenable to RNAi-based loss-of-function studies. The ability to "plug-and-play" genes of interest allows investigators to easily assess the contribution of individual protein domains and post-translational modifications to their function. The system is ideally suited to test not only the requirement of individual components but also their sufficiency, and can provide important insight into the epistatic relationship among multiple components in a protein complex. Although designed for use within Drosophila cells, the general premise and protocol should be easily adapted to mammalian cell culture or other systems that may better suit the interests of potential users.

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.

Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia.

Epithelia are an eminent tissue type and a common driver of tumorigenesis, requiring continual precision in cell division to maintain tissue structure and genome integrity. Mitotic defects often trigger apoptosis, impairing cell viability as a tradeoff for tumor suppression. Identifying conditions that lead to cell death and understanding the mechanisms behind this response are therefore of considerable importance. Here we investigated how epithelia of the Drosophila wing disc respond to loss of Short stop (Shot), a cytoskeletal crosslinking spectraplakin protein that we previously found to control mitotic spindle assembly and chromosome dynamics. In contrast to other known spindle-regulating genes, Shot knockdown induces apoptosis in the absence of Jun kinase (JNK) activation, but instead leads to elevated levels of active p38 kinase. Shot loss leads to double-strand break (DSB) DNA damage, and the apoptotic response is exacerbated by concomitant loss of p53. DSB accumulation is increased by suppression of the spindle assembly checkpoint, suggesting this effect results from chromosome damage during error-prone mitoses. Consistent with DSB induction, we found that the DNA damage and stress response genes, Growth arrest and DNA damage (GADD45) and Apoptosis signal-regulating kinase 1 (Ask1), are transcriptionally upregulated as part of the shot-induced apoptotic response. Finally, co-depletion of Shot and GADD45 induced significantly higher rates of chromosome segregation errors in cultured cells and suppressed shot-induced mitotic arrest. Our results demonstrate that epithelia are capable of mounting molecularly distinct responses to loss of different spindle-associated genes and underscore the importance of proper cytoskeletal organization in tissue homeostasis.