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.

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.

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.

A Novel Mechanism for Activation of Myosin Regulatory Light Chain by Protein Kinase C-Delta in Drosophila.

Myosin is an essential motor protein, which in muscle is comprised of two molecules each of myosin heavy-chain (MHC), the essential or alkali myosin light-chain 1 (MLC1), and the regulatory myosin light-chain 2 (MLC2). It has been shown previously that MLC2 phosphorylation at two canonical serine residues is essential for proper flight muscle function in Drosophila; however, MLC2 is also phosphorylated at additional residues for which the mechanism and functional significance is not known. We found that a hypomorphic allele of Pkcδ causes a flightless phenotype; therefore, we hypothesized that PKCδ phosphorylates MLC2. We rescued flight disability by duplication of the wild-type Pkcδ gene. Moreover, MLC2 is hypophosphorylated in Pkcδ mutant flies, but it is phosphorylated in rescued animals. Myosin isolated from Pkcδ mutant flies shows a reduced actin-activated ATPase activity, and MLC2 in these myosin preparations can be phosphorylated directly by recombinant human PKCδ. The flightless phenotype is characterized by a shortened and disorganized sarcomere phenotype that becomes apparent following eclosion. We conclude that MLC2 is a direct target of phosphorylation by PKCδ, and that this modification is necessary for flight muscle maturation and function.

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.

Use of Drosophila S2 Cells for Live Imaging of Cell Division.

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:α-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as 'CID' gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

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.

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity.

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial-mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation.

A Kinase Duet Performance in the Asymmetric Division of Drosophila Neuroblasts.

The ability of progenitor stem cells to divide asymmetrically allows for the production of diverse daughter cell fates.[...].

Warts phosphorylates mud to promote pins-mediated mitotic spindle orientation in Drosophila, independent of Yorkie.

Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. Here, we describe a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. Our results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily conserved cell proliferation pathway.

Cell Fate Decision Making through Oriented Cell Division.

The ability to dictate cell fate decisions is critical during animal development. Moreover, faithful execution of this process ensures proper tissue homeostasis throughout adulthood, whereas defects in the molecular machinery involved may contribute to disease. Evolutionarily conserved protein complexes control cell fate decisions across diverse tissues. Maintaining proper daughter cell inheritance patterns of these determinants during mitosis is therefore a fundamental step of the cell fate decision-making process. In this review, we will discuss two key aspects of this fate determinant segregation activity, cortical cell polarity and mitotic spindle orientation, and how they operate together to produce oriented cell divisions that ultimately influence daughter cell fate. Our focus will be directed at the principal underlying molecular mechanisms and the specific cell fate decisions they have been shown to control.

Dishevelled binds the Discs large 'Hook' domain to activate GukHolder-dependent spindle positioning in Drosophila.

Communication between cortical cell polarity cues and the mitotic spindle ensures proper orientation of cell divisions within complex tissues. Defects in mitotic spindle positioning have been linked to various developmental disorders and have recently emerged as a potential contributor to tumorigenesis. Despite the importance of this process to human health, the molecular mechanisms that regulate spindle orientation are not fully understood. Moreover, it remains unclear how diverse cortical polarity complexes might cooperate to influence spindle positioning. We and others have demonstrated spindle orientation roles for Dishevelled (Dsh), a key regulator of planar cell polarity, and Discs large (Dlg), a conserved apico-basal cell polarity regulator, effects which were previously thought to operate within distinct molecular pathways. Here we identify a novel direct interaction between the Dsh-PDZ domain and the alternatively spliced "I3-insert" of the Dlg-Hook domain, thus establishing a potential convergent Dsh/Dlg pathway. Furthermore, we identify a Dlg sequence motif necessary for the Dsh interaction that shares homology to the site of Dsh binding in the Frizzled receptor. Expression of Dsh enhanced Dlg-mediated spindle positioning similar to deletion of the Hook domain. This Dsh-mediated activation was dependent on the Dlg-binding partner, GukHolder (GukH). These results suggest that Dsh binding may regulate core interdomain conformational dynamics previously described for Dlg. Together, our results identify Dlg as an effector of Dsh signaling and demonstrate a Dsh-mediated mechanism for the activation of Dlg/GukH-dependent spindle positioning. Cooperation between these two evolutionarily-conserved cell polarity pathways could have important implications to both the development and maintenance of tissue homeostasis in animals.