Lethal (2) giant discs (Lgd)/CC2D1 is required for the full activity of the ESCRT machinery.

BACKGROUND: A major task of the endosomal sorting complex required for transport (ESCRT) machinery is the pinching off of cargo-loaded intraluminal vesicles (ILVs) into the lumen of maturing endosomes (MEs), which is essential for the complete degradation of transmembrane proteins in the lysosome. The ESCRT machinery is also required for the termination of signalling through activated signalling receptors, as it separates their intracellular domains from the cytosol. At the heart of the machinery lies the ESCRT-III complex, which is required for an increasing number of processes where membrane regions are abscised away from the cytosol. The core of ESCRT-III, comprising four members of the CHMP protein family, organises the assembly of a homopolymer of CHMP4, Shrub in Drosophila, that is essential for abscission. We and others identified the tumour-suppressor lethal (2) giant discs (Lgd)/CC2D1 as a physical interactor of Shrub/CHMP4 in Drosophila and mammals, respectively. RESULTS: Here, we show that the loss of function of lgd constitutes a state of reduced activity of Shrub/CHMP4/ESCRT-III. This hypomorphic shrub mutant situation causes a slight decrease in the rate of ILV formation that appears to result in incomplete incorporation of Notch into ILVs. We found that the forced incorporation in ILVs of lgd mutant MEs suppresses the uncontrolled and ligand-independent activation of Notch. Moreover, the analysis of Su(dx) lgd double mutants clarifies their relationship and suggests that they are not operating in a linear pathway. We could show that, despite prolonged lifetime, the MEs of lgd mutants have a similar ILV density as wild-type but less than rab7 mutant MEs, suggesting the rate in lgd mutants is slightly reduced. The analysis of the MEs of wild-type and mutant cells in the electron microscope revealed that the ESCRT-containing electron-dense microdomains of ILV formation at the limiting membrane are elongated, indicating a change in ESCRT activity. Since lgd mutants can be rescued to normal adult flies if extra copies of shrub (or its mammalian ortholog CHMP4B) are added into the genome, we conclude that the net activity of Shrub is reduced upon loss of lgd function. Finally, we show that, in solution, CHMP4B/Shrub exists in two conformations. LGD1/Lgd binding does not affect the conformational state of Shrub, suggesting that Lgd is not a chaperone for Shrub/CHMP4B. CONCLUSION: Our results suggest that Lgd is required for the full activity of Shrub/ESCRT-III. In its absence, the activity of the ESCRT machinery is reduced. This reduction causes the escape of a fraction of cargo, among it Notch, from incorporation into ILVs, which in turn leads to an activation of this fraction of Notch after fusion of the ME with the lysosome. Our results highlight the importance of the incorporation of Notch into ILV not only to assure complete degradation, but also to avoid uncontrolled activation of the pathway.

The ESCRT machinery regulates retromer-dependent transcytosis of septate junction components in Drosophila.

Loss of ESCRT function in Drosophila imaginal discs is known to cause neoplastic overgrowth fueled by mis-regulation of signaling pathways. Its impact on junctional integrity, however, remains obscure. To dissect the events leading to neoplasia, we used transmission electron microscopy (TEM) on wing imaginal discs temporally depleted of the ESCRT-III core component Shrub. We find a specific requirement for Shrub in maintaining septate junction (SJ) integrity by transporting the claudin Megatrachea (Mega) to the SJ. In absence of Shrub function, Mega is lost from the SJ and becomes trapped on endosomes coated with the endosomal retrieval machinery retromer. We show that ESCRT function is required for apical localization and mobility of retromer positive carrier vesicles, which mediate the biosynthetic delivery of Mega to the SJ. Accordingly, loss of retromer function impairs the anterograde transport of several SJ core components, revealing a novel physiological role for this ancient endosomal agent.

Proteins are large molecules responsible for a variety of activities that cells needs to perform to survive; from respiration to copying DNA before cells divide. To perform these roles proteins need to be transported to the correct cell compartment, or to the cell membrane. This protein trafficking depends on the endosomal system, a set of membrane compartments that can travel within the cell and act as a protein sorting hub. This system needs its own proteins to work properly. In particular, there are two sets of proteins that are crucial for the endosomal systems activity: a group of proteins known as the ESCRT (endosomal sorting complex required for transport) machinery and a complex called retromer. The retromer complex regulates recycling of receptor proteins so they can be reused, while the ESCRT machinery mediates degradation of proteins that the cell does not require anymore. In the epithelia of fruit fly larvae ­ the tissues that form layers of cells, usually covering an organ but also making structures like wings ­ defects in ESCRT activity lead to a loss of tissue integrity. This loss of tissue integrity suggests that the endosomal system might be involved in transporting proteins that form cellular junctions, the multiprotein complexes that establish contacts between cells or between a cell and the extracellular space. In arthropods such as the fruit fly, the adherens junction and the septate junction are two types of cellular junctions important for the integrity of epithelia integrity. Adherens junctions allow cells to adhere to each other, while septate junctions stop nutrient molecules, ions and water from leaking into the tissue. The role of the endosomal system in trafficking the proteins that form septate junctions remains a mystery. To better understand the role of the endosomal system in regulating cell junctions and tissue integrity, Pannen et al. blocked the activity of either the ESCRT or retromer in wing imaginal discs ­ the future wings ­ of fruit fly larvae. Pannen et al. then analyzed the effects of these endosomal defects on cellular junctions using an imaging technique called transmission electron microscopy. The results showed that both ESCRT and retromer activities are necessary for the correct delivery of septate junction components to the cell membrane. However, neither retromer nor ESCRT were required for the delivery of adherens junction proteins. These findings shed light on how retromer and the ESCRT machinery are involved in the epithelial tissue integrity of fruit fly larvae through their effects on cell junctions. Humans have their own versions of the ESCRT, retromer, and cell junction proteins, all of which are very similar to their fly counterparts. Since defects in the human versions of these proteins have been associated with a variety of diseases, from infections to cancer, these results may have implications for research into treating those diseases.

The auxiliary ESCRT complexes provide robustness to cold in poikilothermic organisms.

The ESCRT pathway, comprising the in sequence acting ESCRT-0, -I, -II, -III and Vps4 complexes, conducts the abscission of membranes away from the cytosol. Whereas the components of the central ESCRT-III core complex have been thoroughly investigated, the function of the components of the associated two auxiliary ESCRT sub-complexes are not well-understood in metazoans, especially at the organismal level. We here present the developmental analysis of the Drosophila orthologs of the auxiliary ESCRTs Chmp5 and Ist1, DChmp5 and DIst1, which belong to the two auxiliary sub-complexes. While each single null mutant displayed mild defects in development, the Dist1 Dchmp5 double mutant displayed a severe defect, indicating that the two genes act synergistically, but in separate pathways. Moreover, the presented results indicate that the auxiliary ESCRTs provide robustness against cold during development of diverse poikilothermic organisms, probably by preventing the accumulation of the ESCRT-III core component Shrub on the endosomal membrane.