Acute manipulation and real-time visualization of membrane trafficking and exocytosis in Drosophila.

Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but so far, tools for investigating the dynamics of membrane trafficking have been limited to cultured cells. Here, we present a system that enables acute manipulation and real-time visualization of membrane trafficking through the reversible retention of proteins in the endoplasmic reticulum (ER) in living multicellular organisms. By adapting the "retention using selective hooks" (RUSH) approach to Drosophila, we show that trafficking of GPI-linked, secreted, and transmembrane proteins can be controlled with high temporal precision in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and apical secretion and the spatiotemporal dynamics of tricellular junction assembly in epithelia of living embryos. Furthermore, we show that controllable ER retention enables tissue-specific depletion of secretory protein function. The system is broadly applicable to visualizing and manipulating membrane trafficking in diverse cell types in vivo.

Myosin V facilitates polarised E-cadherin secretion.

E-cadherin has a fundamental role in epithelial tissues by providing cell-cell adhesion. Polarised E-cadherin exocytosis to the lateral plasma membrane is central for cell polarity and epithelial homeostasis. Loss of E-cadherin secretion compromises tissue integrity and is a prerequisite for metastasis. Despite this pivotal role of E-cadherin secretion, the transport mechanism is still unknown. Here we identify Myosin V as the motor for E-cadherin secretion. Our data reveal that Myosin V and F-actin are required for the formation of a continuous apicolateral E-cadherin belt, the zonula adherens. We show by live imaging how Myosin V transports E-cadherin vesicles to the plasma membrane, and distinguish two distinct transport tracks: an apical actin network leading to the zonula adherens and parallel actin bundles leading to the basal-most region of the lateral membrane. E-cadherin secretion starts in endosomes, where Rab11 and Sec15 recruit Myosin V for transport to the zonula adherens. We also shed light on the endosomal sorting of E-cadherin by showing how Rab7 and Snx16 cooperate in moving E-cadherin into the Rab11 compartment. Thus, our data help to understand how polarised E-cadherin secretion maintains epithelial architecture and prevents metastasis.

RabX1 Organizes a Late Endosomal Compartment that Forms Tubular Connections to Lysosomes Consistent with a "Kiss and Run" Mechanism.

Degradation of endocytosed proteins involves the formation of transient connections between late endosomes and lysosomes in a process called "kiss and run." Genes and proteins controlling this mechanism are unknown. Here, we identify the small guanosine triphosphatase (GTPase) RabX1 as an organizer of a late endosomal compartment that forms dynamic tubular connections to lysosomes. By analyzing trafficking of the adhesion protein Fasciclin2 in the Drosophila follicular epithelium, we show that a reduction of RabX1 function leads to defects in Fasciclin2 degradation. RabX1 mutants fail to form normal lysosomes and accumulate Fasciclin2 in a swelling late-endosomal compartment. RabX1 protein localizes to late endosomes, where it induces the formation of tubular connections to lysosomes. We propose that these tubules facilitate influx of lysosomal content into late endosomes and that this influx leads to the formation of endolysosomes, in which Fasciclin2 is degraded. We show that the formation of RabX1 tubules is dependent on the V-ATPase proton pump. Moreover, we provide evidence that V-ATPase activity is upregulated during epithelial differentiation. This upregulation intensifies RabX1 tubulation and thereby boosts the capacity of the endolysosomal pathway. Enhanced endolysosomal capacity is required for the removal of Fasciclin2 from the epithelium, which is part of a developmental program promoting epithelial morphogenesis.

DLIC1, but not DLIC2, is upregulated in colon cancer and this contributes to proliferative overgrowth and migratory characteristics of cancer cells.

Cytoplasmic dynein-1 is a large minus-end-directed microtubule motor complex involved in membrane trafficking, organelle positioning, and microtubule organization. The roles of dynein light intermediate chains (DLICs; DLIC1 and DLIC2) within the complex are, however, still largely undefined. In this study, we investigated the possible roles of DLICs in epithelial homeostasis and colon cancer development. Mutant clonal analysis of Drosophila Dlic in the follicular epithelium of Drosophila ovary showed defects in nuclear positioning, epithelial integrity, and apical cell polarity. Consistently, knockdown of human DLIC1 and DLIC2 in colon carcinoma cells resulted in damaged epithelial organization, disturbed lumen formation, and impaired apical polarity establishment in three-dimensional cell culture. Depletion of DLIC1 and DLIC2 led to reduced proliferation, enhanced apoptosis rates, disrupted mitotic spindle assembly, and induction of G2/M arrest in cell cycle progression. Moreover, reduced levels of DLIC1 in contrast to DLIC2 impaired the migratory ability. On the other hand, immunohistochemical examination of human colorectal tissue samples and further colorectal cancer dataset analysis showed a significant upregulation for DLIC1 in tumors, whereas DLIC2 expression was unchanged. In addition, the overexpression of DLIC1 caused increased proliferation, decreased apoptosis and enhanced migration, whereas DLIC2 overexpression did not result in any significant changes. Together, these results indicate that DLIC1 and DLIC2 contribute to the establishment and maintenance of epithelial homeostasis. Furthermore, these findings present the first evidence that DLIC1 and DLIC2 have distinct roles in colon cancer development and that DLIC1 may contribute to proliferative overgrowth and migratory characteristics.

Three mechanisms control E-cadherin localization to the zonula adherens.

E-cadherin localization to the zonula adherens is fundamental for epithelial differentiation but the mechanisms controlling localization are unclear. Using the Drosophila follicular epithelium we genetically dissect E-cadherin transport in an in vivo model. We distinguish three mechanisms mediating E-cadherin accumulation at the zonula adherens. Two membrane trafficking pathways deliver newly synthesized E-cadherin to the plasma membrane. One is Rab11 dependent and targets E-cadherin directly to the zonula adherens, while the other transports E-cadherin to the lateral membrane. Lateral E-cadherin reaches the zonula adherens by endocytosis and targeted recycling. We show that this pathway is dependent on RabX1, which provides a functional link between early and recycling endosomes. Moreover, we show that lateral E-cadherin is transported to the zonula adherens by an apically directed flow within the plasma membrane. Differential activation of these pathways could facilitate cell shape changes during morphogenesis, while their misregulation compromises cell adhesion and tissue architecture in differentiated epithelia.

A genome-scale in vivo RNAi analysis of epithelial development in Drosophila identifies new proliferation domains outside of the stem cell niche.

The Drosophila oogenesis system provides an excellent model to study the development of epithelial tissues. Here, we report the first genome-scale in vivo RNA interference (RNAi) screen for genes controlling epithelial development. By directly analysing cell and tissue architecture we identified 1125 genes, which we assigned to seven different functions in epithelial formation and homeostasis. We validated the significance of our screen by generating mutants for Vps60, a component of the endosomal sorting complexes required for transport (ESCRT) machinery. This analysis provided new insights into spatiotemporal control of cell proliferation in the follicular epithelium. Previous studies have identified signals controlling divisions in the follicle stem cell niche. However, 99% of cell divisions occur outside of the niche and it is unclear how these divisions are controlled. Our data distinguish two new domains outside of the stem cell niche where there are differing controls on proliferation. One domain abuts the niche and is characterised by ESCRT, Notch and JAK/STAT-mediated control of proliferation. Adjacent to this domain, another domain is defined by loss of the impact of ESCRT on cell division. Thus, during development epithelial cells pass through a variety of microenvironments that exert different modes of proliferation control. The switch between these modes might reflect a decrease in the 'stemness' of epithelial cells over time.

"Vacuum-assisted staining": a simple and efficient method for screening in Drosophila.

The constantly growing number of genetic tools rapidly increases possibilities for various screens in different model organisms and calls for new methods facilitating screen performance. In particular, screening procedures involving fixation and staining of samples are difficult to perform at a genome-wide scale. The time-consuming task to generate these samples makes such screens less attractive. Here, we describe the use of multi-well filter plates for high throughput labellings of different Drosophila organs and zebrafish embryos. Our inexpensive vacuum-assisted staining protocol minimises the risk of sample loss, reduces the amount of staining reagents and drastically decreases labour and repetitive work. The simple handling of the system and the commercial availability of its components makes this method easily applicable to every laboratory.

Expression, function and regulation of Brachyenteron in the short germband insect Tribolium castaneum.

T-domain transcription factors are involved in many different processes during embryogenesis, such as mesoderm, heart or gut development in vertebrates and in invertebrates. In insects, the following five types of T-box genes are known: brachyenteron (byn), optomotor-blind (omb), optomotor-blind-related-gene-1 (org-1), dorsocross (doc) and H15. As all these classes are present in the genome of the fruit fly Drosophila melanogaster and the flour beetle Tribolium, the multiplicity of the five types of genes varies from dipterans to the beetle. In higher dipterans, a small cluster of three doc genes (doc1-doc3) exists, while the Tribolium genome contains a single Tc-doc gene only. Two H15 genes, Tc-H15a and Tc-H15b, are present in the Tribolium genome compared to a single H15 gene in Drosophila. We have analysed the expression and function of the Tribolium brachyenteron ortholog (Tc-byn). During embryogenesis, Tc-byn is exclusively expressed in the growth zone of the extending germband and later becomes confined to the distal proctodeum and the hindgut, a situation that parallels the expression pattern of byn in Drosophila. Tc-byn-RNAi treated embryos phenocopy Drosophila byn mutants and form no hindgut. In addition, we have characterised a regulatory element upstream of the Tc-byn transcription start site that confers specific gene expression in the developing hindgut of the Drosophila embryo. Our results demonstrate a highly conserved role for Brachyury-type transcriptional regulators in posterior gut development of insects at the level of expression, function and regulation.

Distinct functions of the Tribolium zerknüllt genes in serosa specification and dorsal closure.

BACKGROUND: In the long-germ insect Drosophila, a single extraembryonic membrane, the amnioserosa, covers the embryo at the dorsal side. In ancestral short-germ insects, an inner membrane, the amnion, covers the embryo ventrally, and an outer membrane, the serosa, completely surrounds the embryo. An early differentiation step partitions the uniform blastoderm into the anterior-dorsal serosa and the posterior-ventral germ rudiment giving rise to amnion and embryo proper. In Drosophila, amnioserosa formation depends on the dorsoventral patterning gene zerknüllt (zen), a derived Hox3 gene. RESULTS: The short-germ beetle Tribolium castaneum possesses two zen homologs, Tc-zen1 and Tc-zen2. Tc-zen1 acts early and specifies the serosa. The loss of the serosa after Tc-zen1 RNAi is compensated by an expansion of the entire germ rudiment toward the anterior. Instead of the serosa, the amnion covers the embryo at the dorsal side, and later size regulation normalizes the early fate shifts, revealing a high degree of plasticity of short-germ development. Tc-zen2 acts later and initiates the amnion and serosa fusion required for dorsal closure. After Tc-zen2 RNAi, the amnion and serosa stay apart, and the embryo closes ventrally, assuming a completely everted (inside-out) topology. CONCLUSIONS: In Tribolium, the duplication of the zen genes was accompanied by subfunctionalization. One of the paralogues, Tc-zen1, acts as an early anterior-posterior patterning gene by specifying the serosa. In absence of the serosa, Tribolium embryogenesis acquires features of long-germ development with a single extraembryonic membrane. We discuss implications for the evolution of insect development including the origin of the zen-derived anterior determinant bicoid.