MCC is a centrosomal protein that relocalizes to non-centrosomal apical sites during intestinal cell differentiation.

The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.

Using Optogenetics to Model Cellular Effects of Alzheimer's Disease.

Across the world a dementia case is diagnosed every three seconds. Alzheimer's disease (AD) causes 50-60% of these cases. The most prominent theory for AD correlates the deposition of amyloid beta (Aß) with the onset of dementia. Whether Aß is causative remains unclear due to findings such as the recently approved drug Aducanumab showing effective clearance of Aß, but not improving cognition. New approaches for understanding Aß function, are therefore necessary. Here we discuss the application of optogenetic techniques to gain insight into AD. Optogenetics, or genetically encoded, light-dependent on/off switches, provides precise spatiotemporal control to regulate cellular dynamics. This precise control over protein expression and oligomerization or aggregation could provide a better understanding of the etiology of AD.

Optogenetic approaches for understanding homeostatic and degenerative processes in Drosophila.

Many organs and tissues have an intrinsic ability to regenerate from a dedicated, tissue-specific stem cell pool. As organisms age, the process of self-regulation or homeostasis begins to slow down with fewer stem cells available for tissue repair. Tissues become more fragile and organs less efficient. This slowdown of homeostatic processes leads to the development of cellular and neurodegenerative diseases. In this review, we highlight the recent use and future potential of optogenetic approaches to study homeostasis. Optogenetics uses photosensitive molecules and genetic engineering to modulate cellular activity in vivo, allowing precise experiments with spatiotemporal control. We look at applications of this technology for understanding the mechanisms governing homeostasis and degeneration as applied to widely used model organisms, such as Drosophila melanogaster, where other common tools are less effective or unavailable.

PTK7/Otk interacts with Wnts and inhibits canonical Wnt signalling.

Wnt signalling is an evolutionarily conserved pathway that directs cell-fate determination and morphogenesis during metazoan development. Wnt ligands are secreted glycoproteins that act at a distance causing a wide range of cellular responses from stem cell maintenance to cell death and cell proliferation. How Wnt ligands cause such disparate responses is not known, but one possibility is that different outcomes are due to different receptors. Here, we examine PTK7/Otk, a transmembrane receptor that controls a variety of developmental and physiological processes including the regulation of cell polarity, cell migration and invasion. PTK7/Otk co-precipitates canonical Wnt3a and Wnt8, indicating a role in Wnt signalling, but PTK7 inhibits rather than activates canonical Wnt activity in Xenopus, Drosophila and luciferase reporter assays. Loss of PTK7 function activates canonical Wnt signalling and epistasis experiments place PTK7 at the level of the Frizzled receptor. In Drosophila, Otk interacts with Wnt4 and opposes canonical Wnt signalling in embryonic patterning. We propose a model where PTK7/Otk functions in non-canonical Wnt signalling by turning off the canonical signalling branch.

Drosophila-A Model System for Developmental Biology.

In this Special Issue, titled "Drosophila-A Model System for Developmental Biology", we present a series of articles and reviews looking at the diverse ways that researchers are using the humble fruit fly, also known as the vinegar fly, to tackle the many aspects of development and homeostasis [...].

ggbulksurv: An R package for easy Drosophila and C. elegans survival analysis.

Lifespan studies on fast-aging model organisms like C.elegans and D.melanogaster are conducted with multiple organisms per vial. Lifespan data results in a "one row, multiple individuals" format, which is incompatible with R packages that require a "one row, one individual" format. We present ggbulksurv , an R package for user-friendly survival analysis and highlight three key features. (1) pivot_prism converts data for PRISM, allowing biologists to plot survival curves without manually expanding each observation. (2) run_bulksurv() takes in a "one row, multiple individuals" table and plots a customizable survival curve. (3) Advanced users who require custom survival objects can specify a custom formula, facilitating complex survival analysis. We provide a time saving solution for lifespan data analysis.

The Wnt Co-Receptor PTK7/Otk and Its Homolog Otk-2 in Neurogenesis and Patterning.

Wnt signaling is a highly conserved metazoan pathway that plays a crucial role in cell fate determination and morphogenesis during development. Wnt ligands can induce disparate cellular responses. The exact mechanism behind these different outcomes is not fully understood but may be due to interactions with different receptors on the cell membrane. PTK7/Otk is a transmembrane receptor that is implicated in various developmental and physiological processes including cell polarity, cell migration, and invasion. Here, we examine two roles of Otk-1 and Otk-2 in patterning and neurogenesis. We find that Otk-1 is a positive regulator of signaling and Otk-2 functions as its inhibitor. We propose that PTK7/Otk functions in signaling, cell migration, and polarity contributing to the diversity of cellular responses seen in Wnt-mediated processes.

Spatially defined Dsh-Lgl interaction contributes to directional tissue morphogenesis.

The process of epithelial morphogenesis defines the structure of epidermal tissue sheets. One such sheet, the ventral epidermis of the Drosophila embryo, shows both intricate segmental patterning and complex cell organization. Within a segment, cells produce hair-like denticles in a stereotypical and highly organized pattern over the surface of the tissue. To understand the cell biological basis of this process, we examined cell shapes and alignments, and looked for molecules that showed an asymmetric distribution in this tissue. We found that apical polarity determinants and adherens junctions were enriched at the dorsal and ventral borders of cells, whereas basolateral determinants were enriched at the anterior and posterior borders. We report that the basolateral determinant Lgl has a novel function in the planar organization of the embryonic epidermis, and this function depends on Dsh and myosin. We conclude that apical-basal proteins, used to establish polarity within a cell, can be independently co-opted to function in epithelial morphogenesis.

The many roles of PTK7: a versatile regulator of cell-cell communication.

PTK7 (protein tyrosine kinase 7) is an evolutionarily conserved transmembrane receptor with functions in various processes ranging from embryonic morphogenesis to epidermal wound repair. Here, we review recent findings indicating that PTK7 is a versatile co-receptor that functions as a molecular switch in Wnt, Semaphorin/Plexin and VEGF signaling pathways. We focus in particular on the role of PTK7 in Wnt signaling, as recent data indicate that PTK7 acts as a Wnt co-receptor, which activates the planar cell polarity pathway, but inhibits canonical Wnt signaling.

Wnt Signaling Rescues Amyloid Beta-Induced Gut Stem Cell Loss.

Patients with Alzheimer's disease suffer from a decrease in brain mass and a prevalence of amyloid-ß plaques. These plaques are thought to play a role in disease progression, but their exact role is not entirely established. We developed an optogenetic model to induce amyloid-ß intracellular oligomerization to model distinct disease etiologies. Here, we examine the effect of Wnt signaling on amyloid in an optogenetic, Drosophila gut stem cell model. We observe that Wnt activation rescues the detrimental effects of amyloid expression and oligomerization. We analyze the gene expression changes downstream of Wnt that contribute to this rescue and find changes in aging related genes, protein misfolding, metabolism, and inflammation. We propose that Wnt expression reduces inflammation through repression of Toll activating factors. We confirm that chronic Toll activation reduces lifespan, but a decrease in the upstream activator Persephone extends it. We propose that the protective effect observed for lithium treatment functions, at least in part, through Wnt activation and the inhibition of inflammation.

Combining stem cell rejuvenation and senescence targeting to synergistically extend lifespan.

Why biological age is a major risk factor for many of the most important human diseases remains mysterious. We know that as organisms age, stem cell pools are exhausted while senescent cells progressively accumulate. Independently, induction of pluripotency via expression of Yamanaka factors (Oct4, Klf4, Sox2, c-Myc; OKSM) and clearance of senescent cells have each been shown to ameliorate cellular and physiological aspects of aging, suggesting that both processes are drivers of organismal aging. But stem cell exhaustion and cellular senescence likely interact in the etiology and progression of age-dependent diseases because both undermine tissue and organ homeostasis in different if not complementary ways. Here, we combine transient cellular reprogramming (stem cell rejuvenation) with targeted removal of senescent cells to test the hypothesis that simultaneously targeting both cell-fate based aging mechanisms will maximize life and health span benefits. We find that OKSM extends lifespan and show that both interventions protect the intestinal stem cell pool, lower inflammation, activate pro-stem cell signaling pathways, and synergistically improve health and lifespan. Our findings suggest that a combination therapy, simultaneously replacing lost stem cells and removing senescent cells, shows synergistic potential for anti-aging treatments. Our finding that transient expression of both is the most effective suggests that drug-based treatments in non-genetically tractable organisms will likely be the most translatable.

A non-canonical Raf function is required for dorsal-ventral patterning during Drosophila embryogenesis.

Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGFß signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. Here we describe a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NFκB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGFß. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. Our results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.

An insect symbiosis is influenced by bacterium-specific polymorphisms in outer-membrane protein A.

Beneficial bacterial symbioses are ubiquitous in nature. However, the functional and molecular basis of host tolerance to resident symbiotic microbes, in contrast to resistance to closely related bacteria that are recognized as foreign, remain largely unknown. We used the tsetse fly (Glossina morsitans), which depends on symbiotic flora for fecundity and has limited exposure to foreign microbes, to investigate the tolerance phenomenon exhibited during symbiosis. We examined the potential role of bacterium-specific polymorphisms present in the major bacterial surface protein, outer-membrane protein A (OmpA), on host infection outcomes. Tsetse were successfully superinfected with their mutualistic facultative symbiont, Sodalis glossinidius, whereas infections with Escherichia coli K12 were lethal. In contrast, tsetse were resistant to an E. coli OmpA mutant strain, whereas recombinant Sodalis expressing E. coli OmpA became pathogenic. Profiling of tsetse immunity-related gene expression incriminated peptidoglycan recognition protein (pgrp)-lb as a determinant of the infection outcomes we observed. RNAi-induced knockdown of tsetse pgrp-lb significantly reduced host mortality after infection with otherwise lethal E. coli K12. Our results show that polymorphisms in the exposed loop domains of OmpA represent a microbial adaptation that mediates host tolerance of endogenous symbiotic bacteria.

GSK3beta affects apical-basal polarity and cell-cell adhesion by regulating aPKC levels.

The dynamic rearrangement of cell-cell contacts is required for the establishment of functional epithelial cell sheets. However, the signaling pathways and cellular mechanisms that initiate and maintain this polarity are not well understood. We show that loss of the Wnt signaling component GSK3 beta results in increased levels of aPKC and leads to defects in apical-basal polarity. We find that GSK3 beta directly phosphorylates aPKC, which likely promotes its ubiquitin-mediated proteosomal degradation. aPKC increases the levels of Armadillo and stabilizes adherens junctions. These results suggest that the Wnt pathway component GSK3 beta regulates the polarity determinant aPKC, which in turn affects cell-cell contacts during the development of polarized tissues.

Shared signaling pathways in Alzheimer's and metabolic disease may point to new treatment approaches.

'A peculiar severe disease process of the cerebral cortex' are the exact words used by A. Alzheimer in 1906 to describe a patient's increasingly severe condition of memory loss, changes in personality, and sleep disturbance. A century later, this 'peculiar' disease has become widely known as Alzheimer's disease (AD), the world's most common neurodegenerative disease, affecting more than 35 million people globally. At the same time, its pathology remains unclear and no successful treatment exists. Several theories for AD etiology have emerged throughout the past century. In this review, we focus on the metabolic mechanisms that are similar between AD and metabolic diseases, based on the results from genome-wide association studies. We discuss signaling pathways involved in both types of disease and look into new optogenetic methods to study the in vivo mechanisms of AD.

Membrane bound axin is sufficient for Wingless signaling in Drosophila embryos.

The Wingless signaling pathway controls various developmental processes in both vertebrates and invertebrates. Here I probe the requirement for nuclear localization of APC2 and Axin in the Wg signal transduction pathway during embryonic development of Drosophila melanogaster. I find that nuclear localization of APC2 appears to be required, but Axin can block signaling when tethered to the membrane. These results support the model where Axin regulates Armadillo localization and activity in the cytoplasm.

An Optogenetic Method to Study Signal Transduction in Intestinal Stem Cell Homeostasis.

Homeostasis in adult organs involves replacement of cells from a stem cell pool maintained in specialized niches regulated by extracellular signals. This cell-to-cell communication employs signal transduction pathways allowing cells to respond with a variety of behaviors. To study these cellular behaviors, signaling must be perturbed within tissues in precise patterns, a technique recently made possible by the development of optogenetic tools. We developed tools to study signal transduction in vivo in an adult fly midgut stem cell model where signaling was regulated by the application of light. Activation was achieved by clustering of membrane receptors EGFR and Toll, while inactivation was achieved by clustering the downstream activators ERK/Rolled and NFκB/Dorsal in the cytoplasm, preventing nuclear translocation and transcriptional activation. We show that both pathways contribute to stem and transit amplifying cell numbers and affect the lifespan of adult flies. We further present new approaches to overcome overexpression phenotypes and novel methods for the integration of optogenetics into the already-established genetic toolkit of Drosophila.

Application of optogenetic Amyloid-ß distinguishes between metabolic and physical damages in neurodegeneration.

The brains of Alzheimer's disease patients show a decrease in brain mass and a preponderance of extracellular Amyloid-ß plaques. These plaques are formed by aggregation of polypeptides that are derived from the Amyloid Precursor Protein (APP). Amyloid-ß plaques are thought to play either a direct or an indirect role in disease progression, however the exact role of aggregation and plaque formation in the aetiology of Alzheimer's disease (AD) is subject to debate as the biological effects of soluble and aggregated Amyloid-ß peptides are difficult to separate in vivo. To investigate the consequences of formation of Amyloid-ß oligomers in living tissues, we developed a fluorescently tagged, optogenetic Amyloid-ß peptide that oligomerizes rapidly in the presence of blue light. We applied this system to the crucial question of how intracellular Amyloid-ß oligomers underlie the pathologies of A. We use Drosophila, C. elegans and D. rerio to show that, although both expression and induced oligomerization of Amyloid-ß were detrimental to lifespan and healthspan, we were able to separate the metabolic and physical damage caused by light-induced Amyloid-ß oligomerization from Amyloid-ß expression alone. The physical damage caused by Amyloid-ß oligomers also recapitulated the catastrophic tissue loss that is a hallmark of late AD. We show that the lifespan deficit induced by Amyloid-ß oligomers was reduced with Li+ treatment. Our results present the first model to separate different aspects of disease progression.

Alzheimer's disease is a progressive condition that damages the brain over time. The cause is not clear, but a toxic molecule called Amyloid-ß peptide seems to play a part. It builds up in the brains of people with Alzheimer's disease, forming hard clumps called plaques. Yet, though the plaques are a hallmark of the disease, experimental treatments designed to break them down do not seem to help. This raises the question ­ do Amyloid-ß plaques actually cause Alzheimer's disease? Answering this question is not easy. One way to study the effect of amyloid plaques is to inject clumps of Amyloid-ß peptides into model organisms. This triggers Alzheimer's-like brain damage, but it is not clear why. It remains difficult to tell the difference between the damage caused by the injected Amyloid-ß peptides and the damage caused by the solid plaques that they form. For this, researchers need a way to trigger plaque formation directly inside animal brains. This would make it possible to test the effects of plaque-targeting treatments, like the drug lithium. Optogenetics is a technique that uses light to control molecules in living animals. Hsien, Kaur et al. have now used this approach to trigger plaque formation by fusing light-sensitive proteins to Amyloid-ß peptides in worms, fruit flies and zebrafish. This meant that the peptides clumped together to form plaques whenever the animals were exposed to blue light. This revealed that, while both the Amyloid-ß peptides and the plaques caused damage, the plaques were much more toxic. They damaged cell metabolism and caused tissue loss that resembled late Alzheimer's disease in humans. To find out whether it was possible to test Alzheimer's treatments in these animals, Hsien, Kaur et al. treated them with the drug, lithium. This increased their lifespan, reversing some of the damage caused by the plaques. Alzheimer's disease affects more than 46.8 million people worldwide and is the sixth leading cause of death in the USA. But, despite over 50 years of research, there is no cure. This new plaque-formation technique allows researchers to study the effects of amyloid plaques in living animals, providing a new way to test Alzheimer's treatments. This could be of particular help in studies of experimental drugs that aim to reduce plaque formation.

Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity.

Activation of the Wnt signaling cascade provides key signals during development and in disease. Here we provide evidence, by designing a Wnt receptor with ligand-independent signaling activity, that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. We have uncovered a branch of the Wnt pathway in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3beta/Zw3, the kinase normally required for controlling beta-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. We propose that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo.

WNT Signaling in Disease.

Developmental signaling pathways control a vast array of biological processes during embryogenesis and in adult life. The WNT pathway was discovered simultaneously in cancer and development. Recent advances have expanded the role of WNT to a wide range of pathologies in humans. Here, we discuss the WNT pathway and its role in human disease and some of the advances in WNT-related treatments.