* joined first author # joined corresponding author

2021
Sarita Hebbar, Elisabeth Knust
Reactive oxygen species (ROS) constitute an additional player in regulating epithelial development.
Bioessays, 43(8) Art. No. 2100096 (2021)
Open Access DOI
Reactive oxygen species (ROS) are highly reactive molecules produced in cells. So far, they have mostly been connected to diseases and pathological conditions. More recent results revealed a somewhat unexpected role of ROS in control of developmental processes. In this review, we elaborate on ROS in development, focussing on their connection to epithelial tissue morphogenesis. After briefly summarising unique characteristics of epithelial cells, we present some characteristic features of ROS species, their production and targets, with a focus on proteins important for epithelial development and function. Finally, we provide examples of regulation of epithelial morphogenesis by ROS, and also of developmental genes that regulate the overall redox status. We conclude by discussing future avenues of research that will further elucidate ROS regulation in epithelial development.


Sarita Hebbar✳︎, Malte Lehmann✳︎, Sarah Behrens, Catrin Hälsig, Weihua Leng, Michaela Yuan, Sylke Winkler, Elisabeth Knust
Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila.
Biol Open, 10(1) Art. No. bio052332 (2021)
Open Access DOI
Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, we induced mutations in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented here will also contribute to further unravel molecular mechanisms underlying the human disease.This paper has an associated First Person interview with the co-first authors of the article.


Srija Bhagavatula
First person - Srija Bhagavatula.
J Cell Sci, 134(2) Art. No. jcs258327 (2021)
DOI
First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Srija Bhagavatula is first author on `A putative stem-loop structure in Drosophila crumbs is required for mRNA localisation in epithelia and germline cells', published in JCS. Srija is a post-doc in the lab of Dr Elisabeth Knust at Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, investigating the significance of mRNA localization in epithelia.


Sarita Hebbar, Malte Lehmann
First person - Sarita Hebbar and Malte Lehmann.
Biol Open, 10(1) Art. No. bio058519 (2021)
Open Access DOI

2020
Srija Bhagavatula, Elisabeth Knust
A putative stem-loop structure in Drosophila crumbs is required for mRNA localisation in epithelia and germline cells.
J Cell Sci, 134(2) Art. No. jcs236497 (2020)
DOI
Crumbs (Crb) is an evolutionarily conserved transmembrane protein localised in the apical membrane of epithelial cells. Loss or mis-localisation of Crb is often associated with disruption of apico-basal cell polarity. crb mRNA is also apically enriched in epithelial cells, and, as shown here, accumulates in the oocyte of developing egg chambers. We narrowed down the Localization Element (LE) of crb mRNA to 47 nucleotides forming a putative stem-loop structure, suggesting to be recognised by Egalitarian (Egl). Mutations in conserved nucleotides abrogate apical transport. crb mRNA enrichment in the oocyte is affected in egl mutant egg chambers. A CRISPR based genomic deletion of the crb locus that includes the LE disrupts asymmetric crb mRNA localisation in epithelia and prevents its accumulation in the oocyte during early stages of oogenesis, but does not affect Crb protein localisation in embryonic and follicular epithelia. However, flies lacking the LE show ectopic Crb protein expression in the nurse cells. These data suggest an additional role of the Drosophila 3'-UTR in regulating translation in a tissue specific manner.


Sarita Hebbar, Kai Schuhmann, Andrej Shevchenko, Elisabeth Knust
Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis.
J Cell Biol, 219(12) Art. No. e201911100 (2020)
DOI
Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. Here, we addressed the role of lipids during morphogenesis of the rhabdomere, the microvilli-based photosensitive organelle of Drosophila photoreceptor cells. Shotgun lipidomics analysis performed on mutant alleles of the polarity regulator crumbs, exhibiting varying rhabdomeric growth defects, revealed a correlation between increased abundance of hydroxylated sphingolipids and abnormal rhabdomeric growth. This could be attributed to an up-regulation of fatty acid hydroxylase transcription. Indeed, direct genetic perturbation of the hydroxylated sphingolipid metabolism modulated rhabdomere growth in a crumbs mutant background. One of the pathways targeted by sphingolipid metabolism turned out to be the secretory route of newly synthesized Rhodopsin, a major rhabdomeric protein. In particular, altered biosynthesis of hydroxylated sphingolipids impaired apical trafficking via Rab11, and thus apical membrane growth. The intersection of lipid metabolic pathways with apical domain growth provides a new facet to our understanding of apical growth during morphogenesis.


Bharath Kumar Raghuraman✳︎, Sarita Hebbar✳︎, Mukesh Kumar, HongKee Moon, Ian Henry, Elisabeth Knust, Andrej Shevchenko
Absolute Quantification of Proteins in the Eye of Drosophila melanogaster.
Proteomics, 20(23) Art. No. e1900049 (2020)
Open Access DOI
Absolute (molar) quantification of proteins determines their molar ratios in complexes, networks and metabolic pathways. We employed MS Western workflow to determine molar abundances of proteins potentially critical for morphogenesis and phototransduction (PT) in eyes of Drosophila melanogaster. We used a single chimeric 264 kDa protein standard that covers, in total, 197 peptides from 43 proteins. The majority of proteins were independently quantified with 2 to 4 proteotypic peptides with the coefficient of variation of less than 15%, better than 1000-fold dynamic range and sub-femtomole sensitivity. We determined molar abundances of the components of the PT machinery and the rhabdomere, the photosensitive organelle of the fly eye, and how they changed when rhabdomere morphogenesis is perturbed by genetic manipulation of the evolutionary conserved gene crumbs (crb). Data are available via ProteomeXchange with identifier PXD018001 This article is protected by copyright. All rights reserved.


Satu Kujawski✳︎, Catia Crespo✳︎, Marta Luz, Michaela Yuan, Sylke Winkler, Elisabeth Knust
Loss of Crb2b-lf leads to anterior segment defects in old zebrafish.
Biol Open, 9(2) Art. No. bio.047555 (2020)
Open Access DOI
Defects in the retina or the anterior segment of the eye lead to compromised vision and affect millions of people. Understanding how these ocular structures develop and are maintained is therefore of paramount importance. The maintenance of proper vision depends, among others, on the function of genes controlling apico-basal polarity. In fact, mutations in polarity genes are linked to retinal degeneration in several species, including human. Here we describe a novel zebrafish crb2b allele (crb2b e40 ), which specifically affects the crb2b long isoform. crb2b e40 mutants are viable and display normal ocular development. However, old crb2b e40 mutant fish develop multiple defects in structures of the anterior segment, which includes the cornea, the iris and the lens. Phenotypes are characterised by smaller pupils due to expansion of the iris and tissues of the iridocorneal angle, an increased number of corneal stromal keratocytes, an abnormal corneal endothelium and an expanded lens capsule. These findings illustrate a novel role for crb2b in the maintenance of the anterior segment and hence add an important function to this polarity regulator, which may be conserved in other vertebrates, including humans.


2019
Johanna Lattner, Weihua Leng, Elisabeth Knust, Marko Brankatschk#, David Flores-Benitez#
Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila.
Elife, 8 Art. No. e50900 (2019)
Open Access DOI
An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting βHeavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2 and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.


Elisabeth Knust#, Kai Simons#
Suzanne Eaton (1959-2019): A pioneer in quantitative tissue morphogenesis.
J Cell Biol, 218(9) 2819-2821 (2019)
DOI

Volker Hartenstein, Michaela Yuan, Amelia Younossi-Hartenstein, Aanavi Karandikar, F Javier Bernardo-Garcia, Simon Sprecher, Elisabeth Knust
Serial electron microscopic reconstruction of the drosophila larval eye: Photoreceptors with a rudimentary rhabdomere of microvillar-like processes.
Dev Biol, 453(1) 56-67 (2019)
DOI
Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. In the present paper we have analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3 μm diameter) alternating with thin segments (>0.1 μm). We show that loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.


Anna Bajur, K Venkatesan Iyer, Elisabeth Knust
Cytocortex-dependent dynamics of Drosophila Crumbs controls junctional stability and tension during germ band retraction.
J Cell Sci, 132(15) Art. No. jcs228338 (2019)
DOI
During morphogenesis, epithelia undergo dynamic rearrangements, which requires continuous remodelling of junctions and cell shape, but at the same time mechanisms preserving cell polarity and tissue integrity. Apico-basal polarity is key for the localisation of the machinery that enables cell shape changes. The evolutionarily conserved Drosophila Crumbs protein is critical for maintaining apico-basal polarity and epithelial integrity. How Crumbs is maintained in a dynamically developing embryo remains largely unknown. Here, we applied quantitative fluorescence techniques to show that, during germ band retraction, Crumbs dynamics correlates with the morphogenetic activity of the epithelium. Genetic and pharmacological perturbations revealed that the mobile pool of Crumbs is fine-tuned by the actomyosin cortex in a stage-dependent manner. Stabilisation of Crumbs at the plasma membrane depends on a proper link to the actomyosin cortex via an intact FERM-domain-binding site in its intracellular domain, loss of which leads to increased junctional tension and higher DE-cadherin (also known as Shotgun) turnover, resulting in impaired junctional rearrangements. These data define Crumbs as a mediator between polarity and junctional regulation to orchestrate epithelial remodelling in response to changes in actomyosin activity.This article has an associated First Person interview with the first author of the paper.


Kassiani Skouloudaki#, Ioannis Christodoulou, Dilan Khalili, Vasilios Tsarouhas, Christos Samakovlis, Pavel Tomancak, Elisabeth Knust#, Dimitrios Papadopoulos#
Yorkie controls tube length and apical barrier integrity during airway development.
J Cell Biol, 218(8) 2762-2781 (2019)
DOI
Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the δ-aminolevulinate synthase gene (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.


Satu Kujawski, Mahendra Sonawane, Elisabeth Knust
penner/lgl2 is required for the integrity of the photoreceptor layer in the zebrafish retina.
Biol Open, 8(4) Art. No. bio041830 (2019)
Open Access DOI
The vertebrate retina is a complex tissue built from multiple neuronal cell types, which develop from a pseudostratified neuroepithelium. These cells are arranged into a highly organized and stereotypic pattern formed by nuclear and plexiform layers. The process of lamination as well as the maturation and differentiation of photoreceptor cells rely on the establishment and maintenance of apico-basal cell polarity and formation of adhesive junctions. Defects in any of these processes can result in impaired vision and are causally related to a variety of human diseases leading to blindness. While the importance of apical polarity regulators in retinal stratification and disease is well established, little is known about the function of basal regulators in retinal development. Here, we analyzed the role of Lgl2, a basolateral polarity factor, in the zebrafish retina. Lgl2 is upregulated in photoreceptor cells and in the retinal pigment epithelium by 72 h post fertilization. In both cell types, Lgl2 is localized basolaterally. Loss of zygotic Lgl2 does not interfere with retinal lamination or photoreceptor cell polarity or maturation. However, knockdown of both maternal and zygotic Lgl2 leads to impaired cell adhesion. As a consequence, severe layering defects occur in the distal retina, manifested by a breakdown of the outer plexiform layer and the outer limiting membrane. These results define zebrafish Lgl2 as an important regulator of retinal lamination, which, given the high degree of evolutionary conservation, may be preserved in other vertebrates, including human.


Sara Caviglia, David Flores-Benitez, Johanna Lattner, Stefan Luschnig, Marko Brankatschk
Rabs on the fly: Functions of Rab GTPases during development.
Small GTPases, 10(2) 89-98 (2019)
Open Access DOI
The organization of intracellular transport processes is adapted specifically to different cell types, developmental stages, and physiologic requirements. Some protein traffic routes are universal to all cells and constitutively active, while other routes are cell-type specific, transient, and induced under particular conditions only. Small GTPases of the Rab (Ras related in brain) subfamily are conserved across eukaryotes and regulate most intracellular transit pathways. The complete sets of Rab proteins have been identified in model organisms, and molecular principles underlying Rab functions have been uncovered. Rabs provide intracellular landmarks that define intracellular transport sequences. Nevertheless, it remains a challenge to systematically map the subcellular distribution of all Rabs and their functional interrelations. This task requires novel tools to precisely describe and manipulate the Rab machinery in vivo. Here we discuss recent findings about Rab roles during development and we consider novel approaches to investigate Rab functions in vivo.


Dimitrios Papadopoulos#, Kassiani Skouloudaki, Ylva Engström, Lars Terenius, Rudolf Rigler, Christoph Zechner, Vladana Vukojević, Pavel Tomancak#
Control of Hox transcription factor concentration and cell-to-cell variability by an auto-regulatory switch.
Development, 146(12) Art. No. dev168179 (2019)
Open Access DOI
The variability in transcription factor concentration among cells is an important developmental determinant, yet how variability is controlled remains poorly understood. Studies of variability have focused predominantly on monitoring mRNA production noise. Little information exists about transcription factor protein variability, as this requires the use of quantitative methods with single-molecule sensitivity. Using Fluorescence Correlation Spectroscopy (FCS), we have characterized the concentration and variability of 14 endogenously tagged TFs in live Drosophila imaginal discs. For the Hox TF Antennapedia, we investigated whether protein variability results from random stochastic events or is developmentally regulated. We found that Antennapedia transitioned from low concentration/high variability early, to high concentration/low variability later, in development. FCS and temporally resolved genetic studies uncovered that Antennapedia itself is necessary and sufficient to drive a developmental regulatory switch from auto-activation to auto-repression, thereby reducing variability. This switch is controlled by progressive changes in relative concentrations of preferentially activating and repressing Antennapedia isoforms, which bind chromatin with different affinities. Mathematical modeling demonstrated that the experimentally supported auto-regulatory circuit can explain the increase of Antennapedia concentration and suppression of variability over time.


Kassiani Skouloudaki✳︎#, Dimitrios Papadopoulos✳︎, Pavel Tomancak, Elisabeth Knust#
The apical protein Apnoia interacts with Crumbs to regulate tracheal growth and inflation.
PLoS Genet, 15(1) Art. No. e1007852 (2019)
Open Access DOI
Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.


Joan Sim, Kathleen Amy Osborne, Irene Argudo García, Artur Matysik, Rachel Kraut
The BEACH Domain Is Critical for Blue Cheese Function in a Spatial and Epistatic Autophagy Hierarchy.
Front Cell Dev Biol, 7 Art. No. 129 (2019)
Open Access DOI
Drosophila blue cheese (bchs) encodes a BEACH domain adaptor protein that, like its human homolog ALFY, promotes clearance of aggregated proteins through its interaction with Atg5 and p62. bchs mutations lead to age-dependent accumulation of ubiquitinated inclusions and progressive neurodegeneration in the fly brain, but neither the influence of autophagy on bchs-related degeneration, nor bchs' placement in the autophagic hierarchy have been shown. We present epistatic evidence in a well-defined larval motor neuron paradigm that in bchs mutants, synaptic accumulation of ubiquitinated aggregates and neuronal death can be rescued by pharmacologically amplifying autophagic initiation. Further, pharmacological rescue requires at least one intact BEACH-containing isoform of the two identified in this study. Genetically augmenting a late step in autophagy, however, rescues even a strong mutation which retains only a third, non-BEACH containing isoform. Using living primary larval brain neurons, we elucidate the primary defect in bchs to be an excess of early autophagic compartments and a deficit in mature compartments. Conversely, rescuing the mutants by full-length Bchs over-expression induces mature compartment proliferation and rescues neuronal death. Surprisingly, only the longest Bchs isoform colocalizes well with autophagosomes, and shuttles between different vesicular locations depending on the type of autophagic impetus applied. Our results are consistent with Bchs promoting autophagic maturation, and the BEACH domain being required for this function.


Sarita Hebbar, Elisabeth Knust, Guillaume Thibault#, Rachel Kraut#
Editorial: Connections to Membrane Trafficking Where You Least Expect Them: Diseases, Dynamics, Diet and Distance.
Front Cell Dev Biol, 7 Art. No. 327 (2019)
Open Access DOI

Malte Lehmann, Elisabeth Knust, Sarita Hebbar
Drosophila melanogaster: A Valuable Genetic Model Organism to Elucidate the Biology of Retinitis Pigmentosa.
Methods Mol Biol, 1834 221-249 (2019)
DOI
Retinitis pigmentosa (RP) is a complex inherited disease. It is associated with mutations in a wide variety of genes with many different functions. These mutations impact the integrity of rod photoreceptors and ultimately result in the progressive degeneration of rods and cone photoreceptors in the retina, leading to complete blindness. A hallmark of this disease is the variable degree to which symptoms are manifest in patients. This is indicative of the influence of the environment, and/or of the distinct genetic makeup of the individual.The fruit fly, Drosophila melanogaster, has effectively proven to be a great model system to better understand interconnected genetic networks. Unraveling genetic interactions and thereby different cellular processes is relatively easy because more than a century of research on flies has enabled the creation of sophisticated genetic tools to perturb gene function. A remarkable conservation of disease genes across evolution and the similarity of the general organization of the fly and vertebrate photoreceptor cell had prompted research on fly retinal degeneration. To date six fly models for RP, including RP4, RP11, RP12, RP14, RP25, and RP26, have been established, and have provided useful information on RP disease biology. In this chapter, an outline of approaches and experimental specifications are described to enable utilizing or developing new fly models of RP.


Rachel Kraut, Elisabeth Knust
Changes in endolysosomal organization define a pre-degenerative state in the crumbs mutant Drosophila retina.
PLoS ONE, 14(12) Art. No. e220220 (2019)
Open Access DOI
Mutations in the epithelial polarity gene crumbs (crb) lead to retinal degeneration in Drosophila and in humans. The overall morphology of the retina and its deterioration in Drosophila crb mutants has been well-characterized, but the cell biological origin of the degeneration is not well understood. Degenerative conditions in the retina and elsewhere in the nervous system often involve defects in degradative intracellular trafficking pathways. So far, however, effects of crb on the endolysosomal system, or on the spatial organization of these compartments in photoreceptor cells have not been described. We therefore asked whether photoreceptors in crb mutants exhibit alterations in endolysosomal compartments under pre-degenerative conditions, where the retina is still morphologically intact. Data presented here show that, already well before the onset of degeneration, Arl8, Rab7, and Atg8-carrying endolysosomal and autophagosomal compartments undergo changes in morphology and positioning with respect to each other in crb mutant retinas. We propose that these changes may be early signs of the degeneration-prone condition in crb retinas.


Monalisa Mishra, Elisabeth Knust
Analysis of the Drosophila Compound Eye with Light and Electron Microscopy.
Methods Mol Biol, 1834 345-364 (2019)
DOI
The Drosophila compound eye is composed of about 750 units, called ommatidia, which are arranged in a highly regular pattern. Eye development proceeds in a stereotypical fashion, where epithelial cells of the eye imaginal discs are specified, recruited, and differentiated in a sequential order that leads to the highly precise structure of an adult eye. Even small perturbations, for example in signaling pathways that control proliferation, cell death, or differentiation, can impair the regular structure of the eye, which can be easily detected and analyzed. In addition, the Drosophila eye has proven to be an ideal model for studying the genetic control of neurodegeneration, since the eye is not essential for viability. Several human neurodegeneration diseases have been modeled in the fly, leading to a better understanding of the function/misfunction of the respective gene. In many cases, the genes involved and their functions are conserved between flies and human. More strikingly, when ectopically expressed in the fly eye some human genes, even those without a Drosophila counterpart, can induce neurodegeneration, detectable by aberrant phototaxis, impaired electrophysiology, or defects in eye morphology and retinal histology. These defects are often rather subtle alteration in shape, size, or arrangement of the cells, and can be easily scored at the ultrastructural level. This chapter aims to provide an overview regarding the analysis of the retina by light and electron microscopy.


2018
Catia Crespo, Elisabeth Knust
Characterisation of maturation of photoreceptor cell subtypes during zebrafish retinal development.
Biol Open, 7(11) Art. No. bio036632 (2018)
Open Access DOI
Photoreceptor cells (PRCs) mature from simple epithelial cells, a process characterised by growth and compartmentalisation of the apical membrane into an inner and an outer segment. So far, a PRC subtype-specific description of morphological and cellular changes in the developing zebrafish retina is missing. Here, we performed an in-depth characterisation of four of the five PRC subtypes of the zebrafish retina between 51 and 120 h post fertilisation, including quantification of the size of different compartments, localisation of polarity proteins and positioning of organelles. One of the major findings was the anisotropic and subtype-specific growth of the different PRC compartments. In addition, a transient accumulation of endoplasmic reticulum in rod PRCs, changes in chromatin organisation in UV sensitive cones and differential expression of polarity proteins during the initial stages of PRC maturation were observed. The results obtained provide a developmental timeline that can be used as a platform for future studies on PRC maturation and function. This platform was applied to document that increased exposure to light leads to smaller apical domains of PRCs.


Catia Crespo, Daniele Soroldoni, Elisabeth Knust
A novel transgenic zebrafish line for red opsin expression in outer segments of photoreceptor cells.
Dev Dyn, 247(7) 951-959 (2018)
Open Access DOI
Opsins are a group of light-sensitive proteins present in photoreceptor cells, which convert the energy of photons into electrochemical signals, thus allowing vision. Given their relevance, we aimed to visualize the two red opsins at subcellular scale in photoreceptor cells.


Giorgos Tsoumpekos, Linda Nemetschke, Elisabeth Knust
Drosophila Big bang regulates the apical cytocortex and wing growth through junctional tension.
J Cell Biol, 217(3) 1033-1045 (2018)
DOI
Growth of epithelial tissues is regulated by a plethora of components, including signaling and scaffolding proteins, but also by junctional tension, mediated by the actomyosin cytoskeleton. However, how these players are spatially organized and functionally coordinated is not well understood. Here, we identify theDrosophila melanogasterscaffolding protein Big bang as a novel regulator of growth in epithelial cells of the wing disc by ensuring proper junctional tension. Loss ofbig bangresults in the reduction of the regulatory light chain of nonmuscle myosin, Spaghetti squash. This is associated with an increased apical cell surface, decreased junctional tension, and smaller wings. Strikingly, these phenotypic traits ofbig bangmutant discs can be rescued by expressing constitutively active Spaghetti squash. Big bang colocalizes with Spaghetti squash in the apical cytocortex and is found in the same protein complex. These results suggest that in epithelial cells of developing wings, the scaffolding protein Big bang controls apical cytocortex organization, which is important for regulating cell shape and tissue growth.


Shradha Das, Elisabeth Knust
A dual role of the extracellular domain of DrosophilaCrumbs for morphogenesis of the embryonic neuroectoderm.
Biol Open, 7(1) Art. No. bio031435 (2018)
Open Access DOI
Epithelia are highly polarised tissues and several highly conserved polarity protein complexes serve to establish and maintain polarity. The transmembrane protein Crumbs (Crb), the central component of the Crb protein complex, is required, among others, for the maintenance of polarity in most epithelia in theDrosophilaembryo. However, different epithelia exhibit different phenotypic severity upon loss ofcrbUsing a transgenomic approach allowed us to more accurately define the role ofcrbin different epithelia. In particular, we provide evidence that the loss of epithelial tissue integrity in the ventral epidermis ofcrbmutant embryos is due to impaired actomyosin activity and an excess number of neuroblasts. We demonstrate that the intracellular domain of Crb could only partially rescue this phenotype, while it is able to completely restore tissue integrity in other epithelia. Based on these results we suggest a dual role of the extracellular domain of Crb in the ventral neuroectoderm. First, it is required for apical enrichment of the Crb protein, which in turn regulates actomyosin activity and thereby ensures tissue integrity; and second, the extracellular domain of Crb stabilises the Notch receptor and thereby ensures proper Notch signalling and specification of the correct number of neuroblasts.


Alejandro Vignoni, Anna Bajur, Elisabeth Knust, Ivo F. Sbalzarini
Multi-objective identification from fluorescence recovery after photobleaching experiments: Understanding morphogenetic regulation of epithelial polarity
IFAC-PapersOnLine, 51(19) 8-11 (2018)
Open Access

2017
Arturo Raya-Sandino, Alejandro Castillo-Kauil, Alaide Domínguez-Calderón, Lourdes Alarcón, David Flores-Benitez, Francisco Cuellar-Perez, Bruno López-Bayghen, Bibiana Chávez-Munguía, José Vázquez-Prado, Lorenza González-Mariscal
Zonula occludens-2 regulates Rho proteins activity and the development of epithelial cytoarchitecture and barrier function.
Biochim Biophys Acta, 1864(10) 1714-1733 (2017)
PDF DOI
Silencing Zonula occludens 2 (ZO-2), a tight junctions (TJ) scaffold protein, in epithelial cells (MDCK ZO-2 KD) triggers: 1) Decreased cell to substratum attachment, accompanied by reduced expression of claudin-7 and integrin β1, and increased vinculin recruitment to focal adhesions and stress fibers formation; 2) Lowered cell-cell aggregation and appearance of wider intercellular spaces; 3) Increased RhoA/ROCK activity, mediated by GEF-HI recruitment to cell borders by cingulin; 4) Increased Cdc42 activity, mitotic spindle disorientation and the appearance of cysts with multiple lumens; 5) Increased Rac and cofilin activity, multiple lamellipodia formation and random cell migration but increased wound closure; 6) Diminished cingulin phosphorylation and disappearance of planar network of microtubules at the TJ region; and 7) Increased transepithelial electrical resistance at steady state, coupled to an increased expression of ZO-1 and claudin-4 and a decreased expression of claudin-2 and paracingulin. Hence, ZO-2 is a crucial regulator of Rho proteins activity and the development of epithelial cytoarchitecture and barrier function.


Shradha Das, Elisabeth Knust
Stardust, the Janus-faced partner of Crumbs.
J Cell Biol, 216(5) 1219-1221 (2017)
DOI
The Drosophila melanogaster scaffolding protein Stardust (Sdt) stabilizes the transmembrane protein Crumbs, a conserved regulator of apical-basal epithelial polarity. In this issue, Perez-Mockus et al. (2017. J. Cell Biol https://doi.org/10.1083/jcb.201611196) report that a subset of Sdt isoforms are targeted by the ubiquitin ligase Neuralized, thus fine tuning the endocytosis and activity of this apical determinant.


Stephanie Spannl✳︎, Alexandra Kumichel✳︎, Sarita Hebbar, Katja Kapp, Marcos Gonzalez-Gaitan, Sylke Winkler, Rosana Blawid, Gregor Jessberger, Elisabeth Knust
The Crumbs_C isoform of Drosophila shows tissue- and stage-specific expression and prevents light-dependent retinal degeneration.
Biol Open, 6(2) 165-175 (2017)
Open Access PDF DOI
Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress.


2016
Linda Nemetschke, Elisabeth Knust
Drosophila Crumbs prevents ectopic Notch activation in developing wings by inhibiting ligand-independent endocytosis.
Development, 143(23) 4543-4553 (2016)
DOI
Many signalling components are apically restricted in epithelial cells, and receptor localisation and abundance is key for morphogenesis and tissue homeostasis. Hence, controlling apicobasal epithelial polarity is crucial for proper signalling. Notch is a ubiquitously expressed, apically localised receptor, which performs a plethora of functions; therefore, its activity has to be tightly regulated. Here, we show that Drosophila Crumbs, an evolutionarily conserved polarity determinant, prevents Notch endocytosis in developing wings through direct interaction between the two proteins. Notch endocytosis in the absence of Crumbs results in the activation of the ligand-independent, Deltex-dependent Notch signalling pathway, and does not require the ligands Delta and Serrate or γ-secretase activity. This function of Crumbs is not due to general defects in apicobasal polarity, as localisation of other apical proteins is unaffected. Our data reveal a mechanism to explain how Crumbs directly controls localisation and trafficking of the potent Notch receptor, and adds yet another aspect of Crumbs regulation in Notch pathway activity. Furthermore, our data highlight a close link between the apical determinant Crumbs, receptor trafficking and tissue homeostasis.


Shradha Das
Dissecting Crumbs to understand its function in epithelial development of Drosophila embryos
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2016)


Anne Morbach
The Role of Cdep in the Embryonic Morphogenesis of Drosophila melanogaster
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2016)


David Flores-Benitez, Elisabeth Knust
Dynamics of epithelial cell polarity in Drosophila: how to regulate the regulators?
Curr Opin Cell Biol, 42 13-21 (2016)
PDF DOI
Apico-basal polarity is a hallmark of epithelial tissues. The integrated activity of several evolutionarily conserved protein complexes is essential to control epithelial polarity during development and homeostasis. Many components of these protein complexes were originally identified in genetic screens performed in Drosophila or Caenorhabditis elegans due to defects in cell polarity. With time, it became obvious that these protein complexes not only control various aspects of apico-basal polarity, but also perform a plethora of other functions, such as growth control, organization of endocytic activity, regulation of signaling and asymmetric cell division, to mention just a few. Here we summarize some results mostly obtained from studies in Drosophila to elucidate how variation in protein composition and modification of individual components contribute to make polarity complexes versatile platforms to fulfill a variety of functions.


Giorgos Tsoumpekos
big bang, a novel regulator of tissue growth in Drosophila melanogaster
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2016)


Mihail Sarov#, Christiane Barz, Helena Jambor, Marco Y Hein, Christopher Schmied, Dana Suchold, Bettina Stender, Stephan Janosch, Vinay Vikas Kj, R T Krishnan, Aishwarya Krishnamoorthy, Irene R S Ferreira, Radoslaw K Ejsmont, Katja Finkl, Susanne Hasse, Philipp Kämpfer, Nicole Plewka, Elisabeth Vinis, Siegfried Schloissnig, Elisabeth Knust, Volker Hartenstein, Matthias Mann, Mani Ramaswami, K VijayRaghavan, Pavel Tomancak#, Frank Schnorrer#
A genome-wide resource for the analysis of protein localisation in Drosophila.
Elife, 5 Art. No. e12068 (2016)
Open Access PDF DOI
The Drosophila genome contains >13,000 protein coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here we present a genome-wide fosmid library of 10,000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins we created transgenic lines and for a total of 207 lines we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.


2015
Ya-Huei Lin, Heather Currinn, Shirin Pocha, Alice Rothnie, Thomas Wassmer, Elisabeth Knust
AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust.
J Cell Sci, 128(24) 4538-4549 (2015)
DOI
Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. Here, we show that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The adaptor protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutation of AP-2 causes expansion of the Crumbs-positive plasma membrane domain and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking down both AP-2 and Stardust leads to the retention of Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity.


Sarita Hebbar, Ishtapran Sahoo, Artur Matysik, Irene Argudo Garcia, Kathleen Amy Osborne, Cyrus Papan, Federico Torta, Pradeep Narayanaswamy, Xiu Hui Fun, Markus R Wenk, Andrej Shevchenko, Dominik Schwudke, Rachel Kraut
Ceramides And Stress Signalling Intersect With Autophagic Defects In Neurodegenerative Drosophila blue cheese (bchs) Mutants.
Sci Rep, 5 Art. No. 15926 (2015)
Open Access PDF DOI
Sphingolipid metabolites are involved in the regulation of autophagy, a degradative recycling process that is required to prevent neuronal degeneration. Drosophila blue cheese mutants neurodegenerate due to perturbations in autophagic flux, and consequent accumulation of ubiquitinated aggregates. Here, we demonstrate that blue cheese mutant brains exhibit an elevation in total ceramide levels; surprisingly, however, degeneration is ameliorated when the pool of available ceramides is further increased, and exacerbated when ceramide levels are decreased by altering sphingolipid catabolism or blocking de novo synthesis. Exogenous ceramide is seen to accumulate in autophagosomes, which are fewer in number and show less efficient clearance in blue cheese mutant neurons. Sphingolipid metabolism is also shifted away from salvage toward de novo pathways, while pro-growth Akt and MAP pathways are down-regulated, and ER stress is increased. All these defects are reversed under genetic rescue conditions that increase ceramide generation from salvage pathways. This constellation of effects suggests a possible mechanism whereby the observed deficit in a potentially ceramide-releasing autophagic pathway impedes survival signaling and exacerbates neuronal death.


David Flores-Benitez, Elisabeth Knust
Crumbs is an essential regulator of cytoskeletal dynamics and cell-cell adhesion during dorsal closure in Drosophila.
Elife, 4 Art. No. e07398 (2015)
Open Access PDF DOI
The evolutionarily conserved Crumbs protein is required for epithelial polarity and morphogenesis. Here we identify a novel role of Crumbs as a negative regulator of actomyosin dynamics during dorsal closure in the Drosophila embryo. Embryos carrying a mutation in the FERM (protein 4.1/ezrin/radixin/moesin) domain-binding motif of Crumbs die due to an overactive actomyosin network associated with disrupted adherens junctions. This phenotype is restricted to the amnioserosa and does not affect other embryonic epithelia. This function of Crumbs requires DMoesin, the Rho1-GTPase, class-I p21-activated kinases and the Arp2/3 complex. Data presented here point to a critical role of Crumbs in regulating actomyosin dynamics, cell junctions and morphogenesis.


David J. Jörg, Luis G. Morelli, Daniele Soroldoni, Andrew C. Oates, Frank Jülicher
Continuum theory of gene expression waves during vertebrate segmentation
New J Phys, 17 Art. No. 093042 (2015)
Open Access PDF DOI

Ya-Huei Lin
The AP2 complex regulates epithelial polarity via regulating Crumbs endocytosis
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2015)


David Flores-Benitez, Elisabeth Knust
Ménage a Trois to Form the Tricellular Junction.
Dev Cell, 33(5) 501-503 (2015)
DOI
Tricellular junctions tightly seal epithelia at the corners of three cells. In this issue of Developmental Cell, Byri et al. (2015) show that Anakonda, a novel Drosophila transmembrane protein, contains an unusual tripartite extracellular domain organization, which explains the tripartite septum filling the tricellular junction, previously revealed by ultrastructure analysis.


Alexandra Kumichel, Katja Kapp, Elisabeth Knust
A Conserved Di-Basic Motif of Drosophila Crumbs Contributes to Efficient ER Export.
Traffic, 16(6) 604-616 (2015)
DOI
The Drosophila type I transmembrane protein Crumbs is an apical determinant required for the maintenance of apico-basal epithelial cell polarity. The level of Crumbs at the plasma membrane is crucial, but how it is regulated is poorly understood. In a genetic screen for regulators of Crumbs protein trafficking we identified Sar1, the core component of the coat protein complex II transport vesicles. sar1 mutant embryos show a reduced plasma membrane localization of Crumbs, a defect similar to that observed in haunted and ghost mutant embryos, which lack Sec23 and Sec24CD, respectively. By pulse-chase assays in Drosophila Schneider cells and analysis of protein transport kinetics based on Endoglycosidase H resistance we identified an RNKR motif in Crumbs, which contributes to efficient ER export. The motif identified fits the highly conserved di-basic RxKR motif and mediates interaction with Sar1. The RNKR motif is also required for plasma membrane delivery of transgene-encoded Crumbs in epithelial cells of Drosophila embryos. Our data are the first to show that a di-basic motif acts as a signal for ER exit of a type I plasma membrane protein in a metazoan organism.


2014
Sarita Hebbar, Wolf Dieter Schulz, Ulrich Sauer, Dominik Schwudke
Laser capture microdissection coupled with on-column extraction LC-MS(n) enables lipidomics of fluorescently labeled Drosophila neurons.
Anal Chem, 86(11) 5345-5352 (2014)
Open Access DOI
We have used laser capture microdissection (LCM) and fluorescence microscopy to isolate genetically labeled neurons from the Drosophila melanogaster brain. From native thin sections, regions of interest could be analyzed with a spatial resolution better than 50 μm. To exploit the specificity of LCM for lipidomics, catapulted tissue patches were directly collected on a reversed phase column and analyzed using an on-column extraction (OCE) that was directly coupled with liquid chromatography-multistage mass spectrometry (LC-MS(n)). With this approach, more than 50 membrane lipids belonging to 9 classes were quantified in tissue regions equivalent to a sample amount of 50 cells. Using this method, the limit of quantitation and the extraction efficiency could be estimated enabling a reliable evaluation of acquired lipid profiles. The lipid profiles of cell body- and synapse-enriched regions of the Drosophila brain were determined and found to be distinct. We argue that this workflow represents a tremendous improvement for tissue lipidomics by integrating genetics, fluorescence microscopy, LCM and LC-MS(n).


Nagananda Gurudev, Michaela Yuan, Elisabeth Knust
chaoptin, prominin, eyes shut and crumbs form a genetic network controlling the apical compartment of Drosophila photoreceptor cells.
Biol Open, 3(5) 332-341 (2014)
Open Access PDF DOI
The apical surface of epithelial cells is often highly specialised to fulfil cell type-specific functions. Many epithelial cells expand their apical surface by forming microvilli, actin-based, finger-like membrane protrusions. The apical surface of Drosophila photoreceptor cells (PRCs) forms tightly packed microvilli, which are organised into the photosensitive rhabdomeres. As previously shown, the GPI-anchored adhesion protein Chaoptin is required for the stability of the microvilli, whereas the transmembrane protein Crumbs is essential for proper rhabdomere morphogenesis. Here we show that chaoptin synergises with crumbs to ensure optimal rhabdomere width. In addition, reduction of crumbs ameliorates morphogenetic defects observed in PRCs mutant for prominin and eyes shut, known antagonists of chaoptin. These results suggest that these four genes provide a balance of adhesion and anti-adhesion to maintain microvilli development and maintenance. Similar to crumbs mutant PRCs, PRCs devoid of prominin or eyes shut undergo light-dependent retinal degeneration. Given the observation that human orthologues of crumbs, prominin and eyes shut result in progressive retinal degeneration and blindness, the Drosophila eye is ideally suited to unravel the genetic and cellular mechanisms that ensure morphogenesis of PRCs and their maintenance under light-mediated stress.


Ruth Rincon-Heredia, David Flores-Benitez, Catalina Flores-Maldonado, José Bonilla-Delgado, Vicky García-Hernández, Odette Verdejo-Torres, Aida M Castillo, Isabel Larré, Augusto C Poot-Hernández, Martha Franco, Patricio Gariglio, José L Reyes, Rubén G Contreras
Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways.
Exp Cell Res, 320(1) 108-118 (2014)
DOI
In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.


Gerit Linneweber, Jake Jacobson, Karl Emanuel Busch, Bruno Hudry, Christo P. Christov, Dirk Dormann, Michaela Yuan, Tomoki Otani, Elisabeth Knust, Mario de Bono, Irene Miguel-Aliaga
Neuronal Control of Metabolism through Nutrient-Dependent Modulation of Tracheal Branching.
Cell, 156(1-2) 69-83 (2014)
PDF DOI
During adaptive angiogenesis, a key process in the etiology and treatment of cancer and obesity, the vasculature changes to meet the metabolic needs of its target tissues. Although the cues governing vascular remodeling are not fully understood, target-derived signals are generally believed to underlie this process. Here, we identify an alternative mechanism by characterizing the previously unrecognized nutrient-dependent plasticity of the Drosophila tracheal system: a network of oxygen-delivering tubules developmentally akin to mammalian blood vessels. We find that this plasticity, particularly prominent in the intestine, drives-rather than responds to-metabolic change. Mechanistically, it is regulated by distinct populations of nutrient- and oxygen-responsive neurons that, through delivery of both local and systemic insulin- and VIP-like neuropeptides, sculpt the growth of specific tracheal subsets. Thus, we describe a novel mechanism by which nutritional cues modulate neuronal activity to give rise to organ-specific, long-lasting changes in vascular architecture.


Alexandra Kumichel, Elisabeth Knust
Apical localisation of crumbs in the boundary cells of the Drosophila hindgut is independent of its canonical interaction partner stardust.
PLoS ONE, 9(4) Art. No. e94038 (2014)
Open Access PDF DOI
The transmembrane protein Crumbs/Crb is a key regulator of apico-basal epithelial cell polarity, both in Drosophila and in vertebrates. In most cases studied so far, the apical localisation of Drosophila Crumbs depends on the interaction of its C-terminal amino acids with the scaffolding protein Stardust. Consequently, embryos lacking either Crumbs or Stardust develop a very similar phenotype, characterised by the loss of epithelial tissue integrity and cell polarity in many epithelia. An exception is the hindgut, which is not affected by the loss of either gene. The hindgut is a single layered epithelial tube composed of two cell populations, the boundary cells and the principal cells. Here we show that Crumbs localisation in the principal cells depends on Stardust, similarly to other embryonic epithelia. In contrast, localisation of Crumbs in the boundary cells does not require Stardust and is independent of its PDZ domain- and FERM-domain binding motifs. In line with this, the considerable upregulation of Crumbs in boundary cells is not followed by a corresponding upregulation of its canonical binding partners. Our data are the first to suggest a mechanism controlling apical Crumbs localisation, which is independent of its conserved FERM- and PDZ-domain binding motifs.


2013
Sandra Soukup, Shirin Pocha, Michaela Yuan, Elisabeth Knust
DLin-7 Is Required in Postsynaptic Lamina Neurons to Prevent Light-Induced Photoreceptor Degeneration in Drosophila.
Curr Biol, 23(14) 1349-1354 (2013)
PDF DOI
Inherited retinal degeneration in humans is caused by mutations in a wide spectrum of genes that regulate photoreceptor development and homeostasis. Many of these genes are structurally and functionally conserved in Drosophila, making the fly eye an ideal system in which to study the cellular and molecular basis of blindness [1, 2]. DLin-7, the ortholog of vertebrate MALS/Veli, is a core component of the evolutionarily conserved Crumbs complex [3]. Mutations in any core member of the Crb complex lead to retinal degeneration in Drosophila [4]. Strikingly, mutations in the human ortholog, CRB1, result in retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis, two severe retinal dystrophies [5, 6]. Unlike Crumbs, DLin-7 is expressed not only in photoreceptor cells but also in postsynaptic lamina neurons. Here, we show that DLin-7 is required in postsynaptic neurons, but not in photoreceptors such as Crumbs, to prevent light-dependent retinal degeneration. At the photoreceptor synapse, DLin-7 acts as part of a conserved DLin-7/CASK/DlgS97 complex required to control the number of capitate projections and active zones, important specializations in the photoreceptor synapse that are essential for proper neurotransmission [7]. These results are the first to demonstrate that a postsynaptically acting protein prevents light-dependent photoreceptor degeneration and describe a novel, Crumbs-independent mechanism for photoreceptor degeneration.


Shirin Pocha#, Elisabeth Knust#
Complexities of Crumbs function and regulation in tissue morphogenesis.
Curr Biol, 23(7) 289-293 (2013)
PDF DOI
Establishing and maintaining epithelial polarity is crucial during development and for adult tissue homeostasis. A complex network of evolutionarily conserved proteins regulates this compartmentalization. One such protein is Crumbs, a type I transmembrane protein initially shown to be an important apical determinant in Drosophila. We discuss recent studies that have advanced our understanding of the function and regulation of Crumbs. New findings obtained in flies and fish, reporting homotypic interactions of the extracellular domain and retromer-mediated recycling, shed light on the regulation of Crumbs levels and activity. These results - obtained in different organisms, tissues and developmental stages - point to more complex functions and regulation than previously assumed.


Sven Klose, David Flores-Benitez, Falko Riedel, Elisabeth Knust
Fosmid-based structure-function analysis reveals functionally distinct domains in the cytoplasmic domain of Drosophila crumbs.
G3 (Bethesda), 3(2) 153-165 (2013)
PDF DOI
The evolutionarily conserved transmembrane protein Crumbs is required for epithelial polarity and morphogenesis in the embryo, control of tissue size in imaginal discs and morphogenesis of photoreceptor cells, and prevents light-dependent retinal degeneration. The small cytoplasmic domain contains two highly conserved regions, a FERM (i.e., protein 4.1/ezrin/radixin/moesin)-binding and a PDZ (i.e., postsynaptic density/discs large/ZO-1)-binding domain. Using a fosmid-based transgenomic approach, we analyzed the role of the two domains during invagination of the tracheae and the salivary glands in the Drosophila embryo. We provide data to show that the PDZ-binding domain is essential for the maintenance of cell polarity in both tissues. In contrast, in embryos expressing a Crumbs protein with an exchange of a conserved Tyrosine residue in the FERM-binding domain to an Alanine, both tissues are internalized, despite some initial defects in apical constriction, phospho-Moesin recruitment, and coordinated invagination movements. However, at later stages these embryos fail to undergo dorsal closure, germ band retraction, and head involution. In addition, frequent defects in tracheal fusion were observed. These results suggest stage and/or tissue specific binding partners. We discuss the power of this fosmid-based system for detailed structure-function analyses in comparison to the UAS/Gal4 system.


Nagananda Gurudev, Mareike Florek, Denis Corbeil, Elisabeth Knust
Prominent role of prominin in the retina.
Adv Exp Med Biol, 777 55-71 (2013)
DOI
Prominin molecules represent a new family of pentaspan membrane glycoproteins expressed throughout the animal kingdom. The name originates from its localization on membrane protrusion, such as microvilli, filopodia, lamellipodia, and microspikes. Following the original description in mouse and human, representative prominin members were found in fish (e.g., Danio rerio), amphibian (Ambystoma mexicanum, Xenopus laevis), worm (Caenorhabditis elegans), and flies (Drosophila melanogaster). Mammalian prominin-1 was identified as a marker of somatic and cancer stem cells and plays an essential role in the visual system, which contributed to increased interest of the medical field in this molecule. Here we summarize recent data from various fields, including Drosophila, which will aid to our understanding of its still elusive function.


Marta Luz, Elisabeth Knust
Fluorescently tagged Lin7c is a dynamic marker for polarity maturation in the zebrafish retinal epithelium.
Biol Open, 2(9) 867-871 (2013)
Open Access DOI
Development of epithelial cell polarity is a highly dynamic process, and often established by the sequential recruitment of conserved protein complexes, such as the Par or the Crumbs (Crb) complex. However, detailed insights into the refinement of polarity and the formation of the complexes are still lacking. Here, we established fluorescently tagged Lin7c, a core member of the Crb complex, as an ideal tool to follow development of polarity in zebrafish epithelia. We find that in gastrula stages, RFP-Lin7c is found in the cytosol of the enveloping layer, while Pard3-GFP is already polarized at this stage. During development of the retinal epithelium, RFP-Lin7c localization is refined from being cytosolic at 14 hours post fertilization (hpf) to almost entirely apical in cells of the eye cup at 28 hpf. This apical Lin7c localization depends on the Crb complex members Oko meduzy and Nagie oko. Thus, fluorescently tagged Lin7c can be used in a broad range of epithelia to follow polarity maturation in vivo and specifically to elucidate the sequence of events determining Crb complex-mediated polarity.


Joao Firmino, Jean-Yves Tinevez, Elisabeth Knust
Crumbs affects protein dynamics in anterior regions of the developing Drosophila embryo.
PLoS ONE, 8(3) Art. No. e58839 (2013)
Open Access PDF DOI
Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.


Monalisa Mishra, Elisabeth Knust
Analysis of the Drosophila compound eye with light and electron microscopy.
Methods Mol Biol, 935 161-182 (2013)
PDF DOI
The Drosophila compound eye is a regular structure, in which about 750 units, called ommatidia, are arranged in a highly regular pattern. Eye development proceeds in a stereotypical fashion, where epithelial cells of the eye imaginal discs are specified, recruited, and differentiated in a sequential order that leads to the highly precise structure of an adult eye. Even small perturbations, for example in signaling pathways that control proliferation, cell death, or differentiation, can impair the regular structure of the eye, which can be easily detected and analyzed. In addition, the Drosophila eye has proven to be an ideal model for studying the genetic control of neurodegeneration, since the eye is not essential for viability. Several human neurodegeneration diseases have been modeled in the fly, leading to a better understanding of the function/misfunction of the respective gene. In many cases, the genes involved and their function are conserved between flies and human. More strikingly, when ectopically expressed in the fly eye some human genes without a Drosophila counterpart can induce neurodegeneration, detectable by aberrant phototaxis, impaired electrophysiology, or defects in eye morphology. These defects are often rather subtle alteration in shape, size, or arrangement of the cells, and can be easily scored at the ultrastructural level. This chapter aims to provide an overview regarding the analysis of the retina by various means.


2012
Monalisa Mishra, Michaela Rentsch, Elisabeth Knust
Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli
Eur J Cell Biol, 91(9) 706-716 (2012)
PDF DOI
The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. Here we examine the role of Crb in the ocelli, the simple eyes of Drosophila. We show that Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. Our data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila. The data will be discussed with respect to the difference in apico-basal organisation of these two cell types.


Katja Kapp✳︎, Johannes Siemens✳︎, Hans-Ulrich Häring, Reiner Lammers
Proteolytic processing of the protein tyrosine phosphatase α extracellular domain is mediated by ADAM17/TACE.
Eur J Cell Biol, 91(9) 687-693 (2012)
PDF DOI
The receptor protein tyrosine phosphatase alpha (PTPα) is involved in the regulation of tyrosine kinases like the Src kinase and the insulin receptor. As with other PTPs, its function is determined by alternative splicing, dimerisation, phosphorylation and proteolytical processing. PTPα is cleaved by calpain in its intracellular domain, which decreases its potential to dephosphorylate Src kinase. Here, we demonstrate that PTPα is also processed in the extracellular domain. Extracellular processing was exclusively found for a splice variant containing an extra nine amino acid insert three residues amino-terminal from the transmembrane domain. Processing was sensitive to the metalloprotease-inhibitor Batimastat, and CHO-M2 cells lacking a disintegrin and metalloproteinase 17 (ADAM17; tumor-necrosis-factor α converting enzyme) activity were not able to cleave PTPα. After transient overexpression of ADAM17 and PTPα in these cells, processing was restored, proving that ADAM17 is involved in this process. Further characterization of the consequences of processing revealed that dephosphorylation of the insulin receptor or activation of Src was not affected but focus formation was reduced. We conclude that extracellular proteolytic processing is a novel mechanism for PTPα regulation.


2011
Nadine Muschalik, Elisabeth Knust
Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors
J Cell Sci, 124(21) 3715-3725 (2011)
PDF DOI
Photoreceptor morphogenesis in Drosophila requires remodelling of apico-basal polarity and adherens junctions (AJs), and includes cell shape changes, as well as differentiation and expansion of the apical membrane. The evolutionarily conserved transmembrane protein Crumbs (Crb) organises an apical membrane-associated protein complex that controls photoreceptor morphogenesis. Expression of the small cytoplasmic domain of Crb in crb mutant photoreceptor cells (PRCs) rescues the crb mutant phenotype to the same extent as the full-length protein. Here, we show that overexpression of the membrane-tethered cytoplasmic domain of Crb in otherwise wild-type photoreceptor cells has major effects on polarity and morphogenesis. Whereas early expression causes severe abnormalities in apico-basal polarity and ommatidial integrity, expression at later stages affects the shape and positioning of AJs. This result supports the importance of Crb for junctional remodelling during morphogenetic changes. The most pronounced phenotype observed upon early expression is the formation of ectopic apical membrane domains, which often develop into a complete second apical pole, including ectopic AJs. Induction of this phenotype requires members of the Par protein network. These data point to a close integration of the Crb complex and Par proteins during photoreceptor morphogenesis and underscore the role of Crb as an apical determinant.


Shirin Pocha, Anna Shevchenko, Elisabeth Knust
Crumbs regulates rhodopsin transport by interacting with and stabilizing myosin V
J Cell Biol, 195(5) 827-838 (2011)
PDF DOI
The evolutionarily conserved Crumbs (Crb) complex is crucial for photoreceptor morphogenesis and homeostasis. Loss of Crb results in light-dependent retinal degeneration, which is prevented by feeding mutant flies carotenoid-deficient medium. This suggests a defect in rhodopsin 1 (Rh1) processing, transport, and/or signaling, causing degeneration; however, the molecular mechanism of this remained elusive. In this paper, we show that myosin V (MyoV) coimmunoprecipitated with the Crb complex and that loss of crb led to severe reduction in MyoV levels, which could be rescued by proteasomal inhibition. Loss of MyoV in crb mutant photoreceptors was accompanied by defective transport of the MyoV cargo Rh1 to the light-sensing organelle, the rhabdomere. This resulted in an age-dependent accumulation of Rh1 in the photoreceptor cell (PRC) body, a well-documented trigger of degeneration. We conclude that Crb protects against degeneration by interacting with and stabilizing MyoV, thereby ensuring correct Rh1 trafficking. Our data provide, for the first time, a molecular mechanism for the light-dependent degeneration of PRCs observed in crb mutant retinas.


Sven Klose
Investigation of the signal peptide and establishment of a novel structure-function analysis of crumbs in drosophila melanogaster
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2011)


Shirin Pocha, Thomas Wassmer#, Christian Niehage, Bernard Hoflack, Elisabeth Knust#
Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs
Curr Biol, 21(13) 1111-1117 (2011)
PDF DOI
The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. We also show a novel function for retromer in maintaining epithelial cell polarity.


Goran Stjepanovic, Katja Kapp, Gert Bange, Christian Graf, Richard Parlitz, Klemens Wild, Matthias P. Mayer, Irmgard Sinning
Lipids trigger a conformational switch that regulates signal recognition particle (SRP)-mediated protein targeting.
J Biol Chem, 286(26) 23489-23497 (2011)
PDF DOI
Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.


Elisabeth Knust
An interview with Elisabeth Knust: President of the German Society for Developmental Biology
Development, 138(12) 2399-2400 (2011)
PDF DOI

2010
Natalia Bulgakova, Michaela Rentsch, Elisabeth Knust
Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.
Mol Biol Cell, 21(22) 3915-3925 (2010)
PDF DOI
Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.


Annett Kilic✳︎, Sven Klose✳︎, Bernhard Dobberstein, Elisabeth Knust, Katja Kapp
The Drosophila Crumbs signal peptide is unusually long and is a substrate for signal peptide peptidase.
Eur J Cell Biol, 89(6) 449-461 (2010)
PDF DOI
N-terminal signal sequences mediate nascent protein targeting to and protein insertion into the membrane of the endoplasmic reticulum. They are typically 15-30 amino acid residues long with a core hydrophobic region flanked by an N-terminal (n-) and a C-terminal region. Following cleavage by signal peptidase, some of the resulting signal peptides are further processed by signal peptide peptidase (SPP) and fragments are liberated into the cytosol. Such fragments can have independent, post-targeting functions affecting diverse cellular processes. We show that Drosophila melanogaster Crumbs, a transmembrane protein controlling cell polarity and morphogenesis, is synthesized with an 83 residues-long signal sequence. To our knowledge, this is currently the longest signal sequence described for an eukaryotic protein. The unusual length is caused by an extended n-region, but the extension does neither affect protein targeting nor signal sequence cleavage. The signal sequence is cleaved off and the resulting signal peptide, SP(Crb), is proteolytically processed by SPP, thus representing the first substrate described for the Drosophila enzyme. We further show that signal peptide fragments can be degraded by the proteasome. Expression of transgenes encoding tagged variants of Crumbs in Drosophila embryos suggests that the signal peptide is short-lived in vivo. Our findings support a model suggesting that besides generating fragments with post-targeting functions, SPP-mediated processing is the first step in the degradation of signal peptides.


André Bachmann✳︎, Oliver Kobler✳︎, Robert J Kittel, Carolin Wichmann, Jimena Sierralta, Stephan J Sigrist, Eckart D Gundelfinger, Elisabeth Knust, Ulrich Thomas
A perisynaptic ménage à trois between Dlg, DLin-7, and Metro controls proper organization of Drosophila synaptic junctions.
J Neurosci, 30(17) 5811-5824 (2010)
PDF DOI
Structural plasticity of synaptic junctions is a prerequisite to achieve and modulate connectivity within nervous systems, e.g., during learning and memory formation. It demands adequate backup systems that allow remodeling while retaining sufficient stability to prevent unwanted synaptic disintegration. The strength of submembranous scaffold complexes, which are fundamental to the architecture of synaptic junctions, likely constitutes a crucial determinant of synaptic stability. Postsynaptic density protein-95 (PSD-95)/ Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) are principal scaffold proteins at both vertebrate and invertebrate synapses. At Drosophila larval glutamatergic neuromuscular junctions (NMJs) DlgA and DlgS97 exert pleiotropic functions, probably reflecting a few known and a number of yet-unknown binding partners. In this study we have identified Metro, a novel p55/MPP-like Drosophila MAGUK as a major binding partner of perisynaptic DlgS97 at larval NMJs. Based on homotypic LIN-2,-7 (L27) domain interactions, Metro stabilizes junctional DlgS97 in a complex with the highly conserved adaptor protein DLin-7. In a remarkably interdependent manner, Metro and DLin-7 act downstream of DlgS97 to control NMJ expansion and proper establishment of synaptic boutons. Using quantitative 3D-imaging we further demonstrate that the complex controls the size of postsynaptic glutamate receptor fields. Our findings accentuate the importance of perisynaptic scaffold complexes for synaptic stabilization and organization.


Nadine Muschalik
Epithelial cell polarity and photoreceptor morphogensis in Drosophila
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)


2009
Mélisande Richard✳︎, Nadine Muschalik✳︎, Ferdi Grawe, Susann Ozüyaman, Elisabeth Knust
A role for the extracellular domain of Crumbs in morphogenesis of Drosophila photoreceptor cells.
Eur J Cell Biol, 88(12) 765-777 (2009)
PDF DOI
Morphogenesis of Drosophila photoreceptor cells includes the subdivision of the apical membrane into the photosensitive rhabdomere and the associated stalk membrane, as well as a considerable elongation of the cell. Drosophila Crumbs (Crb), an evolutionarily conserved transmembrane protein, organizes an apical protein scaffold, which is required for elongation of the photoreceptor cell and extension of the stalk membrane. To further elucidate the role played by different Crb domains during eye morphogenesis, we performed a structure-function analysis in the eye. The analysis showed that the three variants tested, namely full-length Crb, the membrane-bound intracellular domain and the extracellular domain were able to rescue the elongation defects of crb mutant rhabdomeres. However, only full-length Crb and the membrane-bound intracellular domain could partially restore the length of the stalk membrane, while the extracellular domain failed to do so. This failure was associated with the inability of the extracellular domain to recruit beta(Heavy)-spectrin to the stalk membrane. These results highlight the functional importance of the extracellular domain of Crb in the Drosophila eye. They are in line with previous observations, which showed that mutations in the extracellular domain of human CRB1 are associated with retinitis pigmentosa 12 and Leber congenital amaurosis, two severe forms of retinal dystrophy.


Natalia Bulgakova, Elisabeth Knust
The Crumbs complex: from epithelial-cell polarity to retinal degeneration.
J Cell Sci, 122(Pt 15) 2587-2596 (2009)
PDF DOI
The evolutionarily conserved Crumbs protein complex is a key regulator of cell polarity and cell shape in both invertebrates and vertebrates. The important role of this complex in normal cell function is illustrated by the finding that mutations in one of its components, Crumbs, are associated with retinal degeneration in humans, mice and flies. Recent results suggest that the Crumbs complex plays a role in the development of other disease processes that are based on epithelial dysfunction, such as tumorigenesis or the formation of cystic kidneys. Localisation of the complex is restricted to a distinct region of the apical plasma membrane that abuts the zonula adherens in epithelia and photoreceptor cells of invertebrates and vertebrates, including humans. In addition to the core components, a variety of other proteins can be recruited to the complex, depending on the cell type and/or developmental stage. Together with diverse post-transcriptional and post-translational mechanisms that regulate the individual components, this provides an enormous functional diversity and flexibility of the complex. In this Commentary, we summarise findings concerning the organisation and modification of the Crumbs complex, and the conservation of its constituents from flies to mammals. In addition, we discuss recent results that suggest its participation in various human diseases, including blindness and tumour formation.


Kyra Campbell, Elisabeth Knust, Helen Skaer
Crumbs stabilises epithelial polarity during tissue remodelling.
J Cell Sci, 122(Pt 15) 2604-2612 (2009)
PDF DOI
The apicobasal polarity of epithelia depends on the integrated activity of apical and basolateral proteins, and is essential for tissue integrity and body homeostasis. Yet these tissues are frequently on the move as they are sculpted by active morphogenetic cell rearrangements. How does cell polarity survive these stresses? We analyse this question in the renal tubules of Drosophila, a tissue that undergoes dramatic morphogenetic change as it develops. Here we show that, whereas the Bazooka and Scribble protein groups are required for the establishment of tubule cell polarity, the key apical determinant, Crumbs, is required for cell polarity in the tubules only from the time when morphogenetic movements start. Strikingly, if these movements are stalled, polarity persists in the absence of Crumbs. Similar rescue of the ectodermal phenotype of the crumbs mutant when germ-band extension is reduced suggests that Crumbs has a specific, conserved function in stabilising cell polarity during tissue remodelling rather than in its initial stabilisation. We also identify a requirement for the exocyst component Exo84 during tissue morphogenesis, which suggests that Crumbs-dependent stability of epithelial polarity is correlated with a requirement for membrane recycling and targeted vesicle delivery.


Elisabeth Knust, Rainer Hertel
Jose-Antonio Campos-Ortega (1940-2004) and his scientific work - a personal perspective.
Int J Dev Biol, 53(8-10) 1193-1203 (2009)
PDF DOI
Jose Antonio Campos-Ortega (1940-2004), a Spanish scientist who became a leading figure in the developmental genetics of the nervous system, spent most of his scientific life in Germany. Nevertheless, he remained deeply rooted in his native country. His thinking, his ambition and his work were driven by scientific, philosophical and historical questions. He started as a neuroanatomist, working first in Valencia, then in Gottingen, Tubingen and Freiburg. He used primates, reptiles, then the house fly and finally Drosophila to address the question How is the brain or the eye structured in order to function?. While in Freiburg, the problem shifted to How does the nervous system come into being, into form? Campos-Ortega tried to understand early neurogenesis in Drosophila through formal genetics, by identifying relevant genes and studying their genetic interactions. Since he was convinced that not only a single experimental approach could solve a problem as complex as the development of the nervous system, he also included the molecular biological approach when he moved to Cologne, while maintaining a strong focus on anatomy, embryology and genetics. There, he also started to work on the neurogenesis of the zebrafish, using similar concepts and approaches. Throughout his scientific career, he thought, wrote and taught about the evolution of methods and ideas in his field of research. At Campos-Ortegas early death, an unfinished book manuscript was left, entitled Developmental Genetics. The Path to the Biological Synthesis. Some parts of his introductory overview are included here.


Ines Kock, Natalia Bulgakova, Elisabeth Knust, Irmgard Sinning, Valérie Panneels
Targeting of Drosophila rhodopsin requires helix 8 but not the distal C-terminus.
PLoS ONE, 4(7) Art. No. e6101 (2009)
Open Access PDF DOI
BACKGROUND: The fundamental role of the light receptor rhodopsin in visual function and photoreceptor cell development has been widely studied. Proper trafficking of rhodopsin to the photoreceptor membrane is of great importance. In human, mutations in rhodopsin involving its intracellular mislocalization, are the most frequent cause of autosomal dominant Retinitis Pigmentosa, a degenerative retinal pathology characterized by progressive blindness. Drosophila is widely used as an animal model in visual and retinal degeneration research. So far, little is known about the requirements for proper rhodopsin targeting in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: Different truncated fly-rhodopsin Rh1 variants were expressed in the eyes of Drosophila and their localization was analyzed in vivo or by immunofluorescence. A mutant lacking the last 23 amino acids was found to properly localize in the rhabdomeres, the light-sensing organelle of the photoreceptor cells. This constitutes a major difference to trafficking in vertebrates, which involves a conserved QVxPA motif at the very C-terminus. Further truncations of Rh1 indicated that proper localization requires the last amino acid residues of a region called helix 8 following directly the last transmembrane domain. Interestingly, the very C-terminus of invertebrate visual rhodopsins is extremely variable but helix 8 shows conserved amino acid residues that are not conserved in vertebrate homologs. CONCLUSIONS/SIGNIFICANCE: Despite impressive similarities in the folding and photoactivation of vertebrate and invertebrate visual rhodopsins, a striking difference exists between mammalian and fly rhodopsins in their requirements for proper targeting. Most importantly, the distal part of helix 8 plays a central role in invertebrates. Since the last amino acid residues of helix 8 are dispensable for rhodopsin folding and function, we propose that this domain participates in the recognition of targeting factors involved in transport to the rhabdomeres.


2008
Natalia Bulgakova, Ozlem Kempkens, Elisabeth Knust
Multiple domains of Stardust differentially mediate localisation of the Crumbs-Stardust complex during photoreceptor development in Drosophila.
J Cell Sci, 121(Pt 12) 2018-2026 (2008)
PDF DOI
Drosophila Stardust (Sdt), a member of the MAGUK family of scaffolding proteins, is a constituent of the evolutionarily conserved Crumbs-Stardust (Crb-Sdt) complex that controls epithelial cell polarity in the embryo and morphogenesis of photoreceptor cells. Although apical localisation is a hallmark of the complex in all cell types and in all organisms analysed, only little is known about how individual components are targeted to the apical membrane. We have performed a structure-function analysis of Sdt by constructing transgenic flies that express altered forms of Sdt to determine the roles of individual domains for localisation and function in photoreceptor cells. The results corroborate the observation that the organisation of the Crb-Sdt complex is differentially regulated in pupal and adult photoreceptors. In pupal photoreceptors, only the PDZ domain of Sdt - the binding site of Crb - is required for apical targeting. In adult photoreceptors, by contrast, targeting of Sdt to the stalk membrane, a distinct compartment of the apical membrane between the rhabdomere and the zonula adherens, depends on several domains, and seems to be a two-step process. The N-terminus, including the two ECR domains and a divergent N-terminal L27 domain that binds the multi-PDZ domain protein PATJ in vitro, is necessary for targeting the protein to the apical pole of the cell. The PDZ-, the SH3- and the GUK-domains are required to restrict the protein to the stalk membrane. Drosophila PATJ or Drosophila Lin-7 are stabilised whenever a Sdt variant that contains the respective binding site is present, independently of where the variant is localised. By contrast, only full-length Sdt, confined to the stalk membrane, stabilises and localises Crb, although only in reduced amounts. The amount of Crumbs recruited to the stalk membrane correlates with its length. Our results highlight the importance of the different Sdt domains and point to a more intricate regulation of the Crb-Sdt complex in adult photoreceptor cells.


André Bachmann, Ferdi Grawe, Kevin Johnson, Elisabeth Knust
Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration.
Eur J Cell Biol, 87(3) 123-136 (2008)
PDF DOI
The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin-7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin-7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.


André Bachmann, Elisabeth Knust
The use of P-Element Transposons to Generate Transgenic Flies
In: Drosophila: Methods and Protocols. (Eds.) Christian Dahmann Methods in molecular biology, 420.,Totowa, USA,Humana Press (2008),61-77 Ch. 4
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André Bachmann, Margarete Draga, Ferdi Grawe, Elisabeth Knust
On the role of the MAGUK proteins encoded by Drosophila varicose during embryonic and postembryonic development.
BMC Dev Biol, 8 55-55 (2008)
Open Access PDF DOI
BACKGROUND: Membrane-associated guanylate kinases (MAGUKs) form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the Drosophila septate junction or the neuromuscular junction. Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. They often appear in different variants suggesting that they also mediate dynamic changes in the composition of the complexes. RESULTS: Here we show by electron microscopic analysis that Drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. In the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the MAGUK family. The two isoforms can be distinguished by the presence or absence of two L27 domains and are differentially affected in different varicose alleles. While the short isoform is essential for viability, the long isoform seems to have a supportive function. Varicose proteins co-localise with Neurexin IV in pleated septate junctions and are necessary, but not sufficient for its recruitment. The two proteins interact in vitro by the PDZ domain of Varicose and the four C-terminal amino acids of Neurexin IV. Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes. CONCLUSION: Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.


Elisabeth Knust, Wilfried Janning
Genetik : allgemeine Genetik, molekulare Genetik, Entwicklungsgenetik
Stuttgart, Thieme (2008), 515 S.


2007
Elisabeth Knust
Photoreceptor morphogenesis and retinal degeneration: lessons from Drosophila.
Curr Opin Neurobiol, 17(5) 541-547 (2007)
PDF DOI
Cells exhibit an amazingly wide range of different forms, and in most cases the shape of a cell is crucial for performing its specific function(s). But how does a cell obtain its particular shape during development, how can the shape be adapted to different environmental conditions, and what are the consequences if morphogenesis is impaired? An ideal cell type to study these questions is the photoreceptor cell, a photosensitive cell present in most metazoa, highly specialised to transform the energy from the light into a visual response. In the last few years, studies in the Drosophila eye have led to a considerable increase in understanding of the genetic control of photoreceptor morphogenesis; lessons, which may apply to other cell types as well. Most of the genes involved have been conserved during evolution, and mutations in several of them result in retinal degeneration, both in flies and humans. This makes the fly eye an attractive model to unravel the genetic, molecular and cell biological basis of the mechanisms that prevent retinal dystrophies.


Sandra Berger✳︎, Natalia Bulgakova✳︎, Ferdi Grawe, Kevin Johnson, Elisabeth Knust
Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration.
Genetics, 176(4) 2189-2200 (2007)
PDF DOI
Drosophila Stardust, a membrane-associated guanylate kinase (MAGUK), recruits the transmembrane protein Crumbs and the cytoplasmic proteins DPATJ and DLin-7 into an apically localized protein scaffold. This evolutionarily conserved complex is required for epithelial cell polarity in Drosophila embryos and mammalian cells in culture. In addition, mutations in Drosophila crumbs and DPATJ impair morphogenesis of photoreceptor cells (PRCs) and result in light-dependent retinal degeneration. Here we show that stardust is a genetically complex locus. While all alleles tested perturb epithelial cell polarity in the embryo, only a subset of them affects morphogenesis of PRCs or induces light-dependent retinal degeneration. Alleles retaining particular postembryonic functions still express some Stardust protein in pupal and/or adult eyes. The phenotypic complexity is reflected by the expression of distinct splice variants at different developmental stages. All proteins expressed in the retina contain the PSD95, Discs Large, ZO-1 (PDZ), Src homology 3 (SH3), and guanylate kinase (GUK) domain, but lack a large region in the N terminus encoded by one exon. These results suggest that Stardust-based protein scaffolds are dynamic, which is not only mediated by multiple interaction partners, but in addition by various forms of the Stardust protein itself.


Elisabeth Knust, Wieland B. Huttner
Cell polarity from cell division.
Dev Cell, 12(5) 664-666 (2007)
PDF DOI
Apical-basal polarity of epithelial cells is critical for their symmetric versus asymmetric division and commonly thought to be established in interphase. In a novel type of cell division termed "mirror-symmetric", apical cell constituents accumulate during M-phase at the cleavage furrow, resulting in epithelial daughter cells with opposite apical-basal polarity.


Isabel Wasserscheid, Ulrich Thomas, Elisabeth Knust
Isoform-specific interaction of Flamingo/Starry Night with excess Bazooka affects planar cell polarity in the Drosophila wing.
Dev Dyn, 236(4) 1064-1071 (2007)
PDF DOI
Epithelia display two types of polarity, apical-basal and planar cell polarity (PCP), and both are crucial for morphogenesis and organogenesis. PCP signaling pathways comprise transmembrane proteins, such as Flamingo/Starry Night, and cytoplasmic, membrane-associated proteins such as Dishevelled. During establishment of PCP in the Drosophila wing, PCP proteins accumulate apically in distinct "cortical domains" on proximal and distal plasma membranes. This finding suggests that their localized function depends on prior definition of apicobasal polarity. Here, we show that overexpression of Bazooka, a PDZ-domain protein essential for apicobasal polarity in the embryo, perturbs development of PCP, but has no effect on apicobasal polarity. The PCP phenotype is associated with a failure to restrict Flamingo/Starry night to the proximal and distal plasma membranes of the wing epithelium. We further demonstrate that flamingo expresses two differentially spliced RNAs in wing imaginal discs, which encode two isoforms of the atypical cadherin Flamingo. The predominant Starry night-type form contains a PDZ-binding motif, which mediates binding to Bazooka in vitro. Pull-down assays support the occurrence of such an interaction in wing imaginal discs. The results suggest that interaction between the apicobasal and planar cell polarity systems has to be tightly coordinated to ensure proper morphogenesis of the wing disc epithelium.


2002
Kevin Johnson, Ferdi Grawe, Nicola Grzeschik, Elisabeth Knust
Drosophila crumbs is required to inhibit light-induced photoreceptor degeneration.
Curr Biol, 12(19) 1675-1680 (2002)
PDF
Mutations in the human transmembrane protein CRB1 are associated with severe forms of retinal dystrophy, retinitis pigmentosa 12 (RP12), and Leber's congenital amaurosis (LCA). The Drosophila homolog, crumbs, is required for polarity and adhesion in embryonic epithelia and for correct formation of adherens junctions and proper morphogenesis of photoreceptor cells. Here, we show that mutations in Drosophila crumbs result in progressive, light-induced retinal degeneration. Degeneration is prevented by expression of p35, an inhibitor of apoptosis, or by reduction of rhodopsin levels through a vitamin A-deficient diet. In the dark, rhabdomeres survive but exhibit morphogenetic defects. We demonstrate that it is the extracellular portion of the Crumbs protein that is essential to suppress light-induced programmed cell death, while proper morphogenesis depends on the intracellular part. We conclude that human and Drosophila Crumbs proteins are functionally conserved to prevent light-dependent photoreceptor degeneration. This experimental system is now ideally suited to study the genetic and molecular basis of RP12- and LCA-related retinal degeneration.


2001
André Bachmann, M Schneider, Eva Theilenberg, Ferdi Grawe, Elisabeth Knust
Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.
Nature, 414(6864) 638-643 (2001)
PDF DOI
The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.