InteractiveFly: GeneBrief

shrub: Biological Overview | References

Gene name - shrub

Synonyms - Vps32, ESCRT-III, snf7

Cytological map position - 45A12-45A12

Function - signaling

Keywords - Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, ESCRT III component, regulation of dendritic branching, completion of membrane abscission during Drosophila female germline stem cell division

Symbol - shrb

FlyBase ID: FBgn0086656

Genetic map position - chr2R:9,142,146-9,143,914

Classification - vacuolar sorting protein Snf7 conserved protein domain

Cellular location - cytoplasmic

NCBI link: EntrezGene
shrb orthologs: Biolitmine
Recent literature
Loncle, N., Agromayor, M., Martin-Serrano, J. and Williams, D. W. (2015). An ESCRT module is required for neuron pruning. Sci Rep 5: 8461. PubMed ID: 25676218
Neural circuits are refined by both functional and structural changes. Structural remodeling by large-scale pruning occurs where relatively long neuronal branches are cut away from their parent neuron and removed by local degeneration. Until now, the molecular mechanisms executing such branch severing events have remained poorly understood. This study reveal a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery during neuronal remodeling. The data show that a specific ESCRT pruning module, including members of the ESCRT-I and ESCRT-III complexes, but not ESCRT-0 or ESCRT-II, are required for the neurite scission event during pruning. Furthermore it was shown that this ESCRT module requires a direct, in vivo, interaction between Shrub/CHMP4B and the accessory protein Myopic/HD-PTP.
McMillan, B. J., Tibbe, C., Jeon, H., Drabek, A. A., Klein, T. and Blacklow, S. C. (2016). Electrostatic interactions between elongated monomers drive filamentation of Drosophila Shrub, a metazoan ESCRT-III protein. Cell Rep 16: 1211-1217. PubMed ID: 27452459
The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. This study reports the structural basis for polymerization of Shrub and defines a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins.
Bulgari, D., Jha, A., Deitcher, D. L. and Levitan, E. S. (2018). Myopic (HD-PTP, PTPN23) selectively regulates synaptic neuropeptide release. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 29378961
Neurotransmission is mediated by synaptic exocytosis of neuropeptide-containing dense-core vesicles (DCVs) and small-molecule transmitter-containing small synaptic vesicles (SSVs). Exocytosis of both vesicle types depends on Ca(2+) and shared secretory proteins. This study shows that increasing or decreasing expression of Myopic (mop, HD-PTP, PTPN23), a Bro1 domain-containing pseudophosphatase implicated in neuronal development and neuropeptide gene expression, increases synaptic neuropeptide stores at the Drosophila neuromuscular junction (NMJ). This occurs without altering DCV content or transport, but synaptic DCV number and age are increased. The effect on synaptic neuropeptide stores is accounted for by inhibition of activity-induced Ca(2+)-dependent neuropeptide release. cAMP-evoked Ca(2+)-independent synaptic neuropeptide release also requires optimal Myopic expression, showing that Myopic affects the DCV secretory machinery shared by cAMP and Ca(2+) pathways. Presynaptic Myopic is abundant at early endosomes, but interaction with the endosomal sorting complex required for transport III (ESCRT III) protein (CHMP4/Shrub) that mediates Myopic's effect on neuron pruning is not required for control of neuropeptide release. Remarkably, in contrast to the effect on DCVs, Myopic does not affect release from SSVs. Therefore, Myopic selectively regulates synaptic DCV exocytosis that mediates peptidergic transmission at the NMJ.
Baumers, M., Klose, S., Bruser, C., Haag, C., Hansch, S., Pannen, H., Weidtkamp-Peters, S., Feldbrugge, M. and Klein, T. (2019). The auxiliary ESCRT complexes provide robustness to cold in poikilothermic organisms. Biol Open 8(9). PubMed ID: 31412999
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. This paper presents 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.
Warecki, B., Ling, X., Bast, I. and Sullivan, W. (2020). ESCRT-III-mediated membrane fusion drives chromosome fragments through nuclear envelope channels. J Cell Biol 219(3). PubMed ID: 32032426
Mitotic cells must form a single nucleus during telophase or exclude part of their genome as damage-prone micronuclei. While research has detailed how micronuclei arise from cells entering anaphase with lagging chromosomes, cellular mechanisms allowing late-segregating chromosomes to rejoin daughter nuclei remain underexplored. This study finds that late-segregating acentric chromosome fragments that rejoin daughter nuclei are associated with nuclear membrane but devoid of lamin and nuclear pore complexes in Drosophila melanogaster. Acentrics pass through membrane-, lamin-, and nuclear pore-based channels in the nuclear envelope that extend and retract as acentrics enter nuclei. Membrane encompassing the acentrics fuses with the nuclear membrane, facilitating integration of the acentrics into newly formed nuclei. Fusion, mediated by the membrane fusion protein Comt/NSF and ESCRT-III components Shrub/CHMP4B and CHMP2B, facilitates reintegration of acentrics into nuclei. These results suggest a previously unsuspected role for membrane fusion, similar to nuclear repair, in the formation of a single nucleus during mitotic exit and the maintenance of genomic integrity.
Chen, M. Y., Tayyeb, A. and Wang, Y. F. (2021). shrub is required for spermatogenesis of Drosophila melanogaster. Arch Insect Biochem Physiol: e21779. PubMed ID: 33660341
Shrub (CG8055) encodes the vps32/snf7 protein, a filament-forming subunit of the ESCRT (endosomal sorting complexes required for transport)-III complex involved in inward membrane budding. It was reported that shrub was required for abscission in female germline stem cells. This study shows that the expression level of shrub in the testis was significantly higher than that in the ovary of 1-day-old Drosophila melanogaster, suggesting a role in male reproduction. nosGal4 driver was used to knockdown shrub specifically in the fly testis and found that this resulted in a significantly lower paternal effect egg hatch rate relative to the control group. Immunofluorescence staining showed that shrub knockdown in fly testes caused an accumulation of early-stage germ cells and lack of spectrin caps. In the late stages (spermiogenesis), the control testis contained multiple compacted spermatid bundles and individualization complexes (ICs) consisting of actin cones, whereas there were scattered spermatid nuclei and only a few ICs with disorganized actin cones in the shrub knockdown testis. Finally, the control seminal vesicle was full of mature sperms with needle-like heads, but in shrub knockdown testis 75% of seminal vesicles had no mature sperms. Knockdown of shrub in fly testes led to upregulated expression of several cytoskeleton-associated genes, and an accumulation of ubiquitylated proteins. These results suggest that knockdown of shrub in fly testes might damage spermatogenesis by affecting transportability.
Baeumers, M., Ruhnau, K., Breuer, T., Pannen, H., Goerlich, B., Kniebel, A., Haensch, S., Weidtkamp-Peters, S., Schmitt, L. and Klein, T. (2020). Lethal (2) giant discs (Lgd)/CC2D1 is required for the full activity of the ESCRT machinery. BMC Biol 18(1): 200. PubMed ID: 33349255
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. The ESCRT-III complex 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. The tumour-suppressor lethal (2) giant discs (Lgd)/CC2D1 is a physical interactor of Shrub/CHMP4 in Drosophila and mammals, respectively. This study shows that the loss of function of lgd constitutes a state of reduced activity of Shrub/CHMP4/ESCRT-III. 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. 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, it is concluded that the net activity of Shrub is reduced upon loss of lgd function. 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. These 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. These 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.
Vita, D. J., Meier, C. J. and Broadie, K. (2021). Neuronal fragile X mental retardation protein activates glial insulin receptor mediated PDF-Tri neuron developmental clearance. Nat Commun 12(1): 1160. PubMed ID: 33608547
Glia engulf and phagocytose neurons during neural circuit developmental remodeling. Disrupting this pruning process contributes to Fragile X syndrome (FXS), a leading cause of intellectual disability and autism spectrum disorder in mammals. Utilizing a Drosophila FXS model central brain circuit, two glial classes responsible for Draper-dependent elimination of developmentally transient PDF-Tri neurons were identified. Neuronal Fragile X Mental Retardation Protein (FMRP) was found to drive insulin receptor activation in glia, promotes glial Draper engulfment receptor expression, and negatively regulates membrane-molding ESCRT-III Shrub function during PDF-Tri neuron clearance during neurodevelopment in Drosophila. In this context, this study demonstrated genetic interactions between FMRP and insulin receptor signaling, FMRP and Draper, and FMRP and Shrub in PDF-Tri (a transient pair of PDF positive tritocerebral neurons) elimination. FMRP is required within neurons, not glia, for glial engulfment, indicating FMRP-dependent neuron-to-glia signaling mediates neuronal clearance. It is conclude neuronal FMRP drives glial insulin receptor activation to facilitate Draper- and Shrub-dependent neuronal clearance during neurodevelopment in Drosophila.
Mathieu, J., Michel-Hissier, P., Boucherit, V. and Huynh, J. R. (2022). The deubiquitinase USP8 targets ESCRT-III to promote incomplete cell division. Science 376(6595): 818-823. PubMed ID: 35587967
In many vertebrate and invertebrate organisms, gametes develop within groups of interconnected cells called germline cysts formed by several rounds of incomplete divisions. This study found that loss of the deubiquitinase USP8 gene in Drosophila can transform incomplete divisions of germline cells into complete divisions. Conversely, overexpression of USP8 in germline stem cells is sufficient for the reverse transformation from complete to incomplete cytokinesis. The ESCRT-III proteins CHMP2B and Shrub/CHMP4 are targets of USP8 deubiquitinating activity. In Usp8 mutant sister cells, ectopic recruitment of ESCRT proteins at intercellular bridges causes cysts to break apart. A Shrub/CHMP4 variant that cannot be ubiquitinated does not localize at abscission bridges and cannot complete abscission. These results uncover ubiquitination of ESCRT-III as a major switch between two types of cell division.


Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. This study shows that ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, this study shows that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. It is concluded that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo (Eikenes, 2015).

Cytokinesis is the final step of cell division that leads to the physical separation of the two daughter cells. It is tightly controlled in space and time and proceeds in multiple steps via sequential specification of the cleavage plane, assembly and constriction of the actomyosin-based contractile ring (CR), formation of a thin intercellular bridge and finally abscission that separates the two daughter cells. Studies in a variety of model organisms and systems have elucidated key machineries and signals governing early events of cytokinesis. However, the mechanisms of the final abscission step of cytokinesis are less understood, especially in vivo in the context of different cell types in a multi-cellular organism (Eikenes, 2015).

During the recent years key insights into the molecular mechanisms and spatiotemporal control of abscission have been gained using a combination of advanced molecular biological and imaging technologies. At late stages of cytokinesis the spindle midzone transforms to densely packed anti-parallel microtubules (MTs) that make up the midbody (MB) and the CR transforms into the midbody ring (MR, diameter of ~1-2 μm). The MR is located at the site of MT overlap and retains several CR components including Anillin, septins (Septins 1, 2 and Peanut in Drosophila melanogaster), myosin-II, Citron kinase (Sticky in Drosophila) and RhoA (Rho1 in Drosophila) and eventually also acquires the centralspindlin component MKLP1 (Pavarotti in Drosophila). In C. elegans embryos the MR plays an important role in scaffolding the abscission machinery even in the absence of MB MTs (Eikenes, 2015).

Studies in human cell lines, predominantly in HeLa and MDCK cells, have shown that components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins play important roles in mediating abscission. Abscission occurs at the thin membrane neck that forms at the constriction zone located adjacent to the MR. An important signal for initiation of abscission is the degradation of the mitotic kinase PLK1 (Polo-like kinase 1) that triggers the targeting of CEP55 (centrosomal protein of 55 kDa) to the MR. CEP55 interacts directly with GPP(3x)Y motifs in the ESCRT-associated protein ALIX (ALG-2-interacting protein X) and in the ESCRT-I component TSG101, thereby recruiting them to the MR. ALIX and TSG101 in turn recruit the ESCRT-III component CHMP4B, which is followed by ESCRT-III polymerization into helical filaments that spiral/slide to the site of abscission. The VPS4 ATPase is thought to promote ESCRT-III redistribution toward the abscission site. Prior to abscission ESCRT-III/CHMP1B recruits Spastin that mediates MT depolymerization at the abscission site. ESCRT-III then facilitates membrane scission of the thin membrane neck, thereby mediating abscission (Eikenes, 2015).

Cytokinesis is tightly controlled by the activation and inactivation of mitotic kinases at several steps to ensure its faithful spatiotemporal progression. Cytokinesis conventionally proceeds to completion via abscission, but is differentially controlled depending on the cell type during the development of metazoan tissues. For example, germ cells in species ranging from insects to humans undergo incomplete cytokinesis leading to the formation of germline cysts in which cells are interconnected via stable intercellular bridges. How cytokinesis is modified to achieve different abscission timing in different cell types is not well understood, but molecular understanding of the regulation of the abscission machinery has started giving some mechanistic insight (Eikenes, 2015).

The Drosophila female germline represents a powerful system to address mechanisms controlling cytokinesis and abscission in vivo. Each Drosophila female germline stem cell (fGSC) divides asymmetrically with complete cytokinesis to give rise to another fGSC and a daughter cell cystoblast (CB). Cytokinesis during fGSC division is delayed so that abscission takes place during the G2 phase of the following cell cycle (about 24 hours later). The CB in turn undergoes four mitotic divisions with incomplete cytokinesis giving rise to a 16-cell cyst in which the cells remain interconnected by stable intercellular bridges called ring canals (RCs). One of the 16 cells with four RCs will become specified as the oocyte and the cyst becomes encapsulated by a single layer follicle cell epithelium to form an egg chamber. Drosophila male GSCs (mGSCs) also divide asymmetrically with complete cytokinesis to give rise to another mGSC and a daughter cell gonialblast (GB). Anillin, Pavarotti, Cindr, Cyclin B and Orbit are known factors localizing at RCs/MRs and/or MBs during complete cytokinesis in fGSCs and/or mGSCs. It has been recently reported that Aurora B delays abscission and that Cyclin B promotes abscission in Drosophila germ cells and that mutual inhibitions between Aurora B and Cyclin B/Cdk-1 control the timing of abscission in Drosophila fGSCs and germline cysts. However, little is known about further molecular mechanisms controlling cytokinesis and abscission in Drosophila fGSCs (Eikenes, 2015).

This study has characterize the roles of ALIX and the ESCRT-III component Shrub during cytokinesis in Drosophila fGSCs. ALIX and Shrub are required for completion of abscission in fGSCs. They co-localize during this process, and their direct interaction is required for abscission with normal kinetics. This study thus shows that a complex between ALIX and Shrub is required for abscission in fGSCs and provide evidence of an evolutionarily conserved functional role of the ALIX/ESCRT-III pathway in mediating cytokinetic abscission in the context of a multi-cellular organism (Eikenes, 2015).

Loss of ALIX or/and Shrub function or inhibition of their interaction delays abscission in fGSCs leading to the formation of stem cysts in which the fGSC remains interconnected to chains of daughter cells via MRs. As abscission eventually takes place a cyst of e.g. 2 germ cells may pinch off and subsequently undergo four mitotic divisions to give rise to a germline cyst with 32 germ cells. Consistently, loss of ALIX or/and Shrub or interference with their interaction caused a high frequency of egg chambers with 32 germ cells during Drosophila oogenesis. It was also found that ALIX controls cytokinetic abscission in both fGSCs and mGSCs and thus that ALIX plays a universal role in cytokinesis during asymmetric GSC division in Drosophila. Taken together this study provides evidence that the ALIX/ESCRT-III pathway is required for normal abscission timing in a living metazoan tissue (Eikenes, 2015).

The results together with findings in other models underline the evolutionary conservation of the ESCRT system and associated proteins in cytokinetic abscission. Specifically, ESCRT-I or ESCRT-III have been implicated in abscission in a subset of Archaea (ESCRT-III), in A. thaliana (elch/tsg101/ESCRT-I) and in C. elegans (tsg101/ESCRT-I). In S. cerevisiae, Bro1 (ALIX) and Snf7 (CHMP4/ESCRT-III) have also been suggested to facilitate cytokinesis. In cultured Drosophila cells, Shrub/ESCRT-III mediates abscission and in human cells in culture ALIX, TSG101/ESCRT-I and CHMP4B/ESCRT-III promote abscission. ALIX and the ESCRT system thus act in an ancient pathway to mediate cytokinetic abscission (Eikenes, 2015).

Despite the fact that an essential role of ALIX in promoting cytokinetic abscission during asymmetric GSC division was found in the Drosophila female and male germlines, strong bi-nucleation directly attributed to cytokinesis failure was found in Drosophila alix mutants in the somatic cell types that were examined. This might have multiple explanations. One possibility is that maternally contributed alix mRNA may support normal cytokinesis and development. Whereas ALIX and CHMP4B depletion in cultured mammalian cells causes a high frequency of bi- and multi-nucleation it is also possible that cells do not readily become bi-nucleate upon failure of the final step of cytokinetic abscission in the context of a multi-cellular organism. Consistent with the observations of a high frequency of stem cysts upon loss of ALIX and Shrub in the germline, Shrub depletion in cultured Drosophila cells resulted in chains of cells interconnected via intercellular bridges/MRs due to multiple rounds of cell division with failed abscission (Steigemann, 2009). Moreover, loss of ESCRT-I/tsg101 function in the C. elegans embryo did not cause furrow regression. These and the current observations suggest that ALIX- and Shrub/ESCRT-depleted cells can halt and are stable at the MR stage for long periods of time and from which cleavage furrows may not easily regress, at least not in these cell types and in the context of a multi-cellular organism. It is also possible that redundant mechanisms contribute to abscission during symmetric cytokinesis in somatic Drosophila cells. Further studies should address the general involvement of ALIX and ESCRT-III in cytokinetic abscission in somatic cells in vivo (Eikenes, 2015).

Different cell types display different abscission timing, intercellular bridge morphologies and spatiotemporal control of cytokinesis. In fGSCs it was found that ALIX and Shrub co-localize throughout late stages of cytokinesis and abscission. In human cells ALIX localizes in the central region of the MB, whereas CHMP4B at first localizes at two cortical ring-like structures adjacent to the central MB region and then progressively distributes also at the constriction zone where it promotes abscission. ALIX and CHMP4B are thus found at discrete locations within the intercellular bridge as cells approach abscission in human cultured cells. In contrast, ESCRT-III localizes to a ring-like structure during cytokinesis in Archaea, resembling the Shrub localization at MRs was observed in Drosophila fGSCs. Moreover, ALIX and Shrub are present at MRs for a much longer time (from G1/S) prior to abscission (in G2) in fGSCs than in human cultured cells. Here, ALIX and CHMP4B are increasingly recruited about an hour before abscission and then CHMP4B acutely increases at the constriction zones shortly (~30 min) before the abscission event (Eikenes, 2015).

How may ALIX and Shrub be recruited to the MR/MB in Drosophila cells in the absence of CEP55 that is a major recruiter of ALIX and ultimately CHMP4/ESCRT-III in human cells? Curiously, a GPP(3x)Y consensus motif was detected within the Drosophila ALIX sequence (GPPPGHY, aa 808-814) resembling the CEP55-interacting motif in human ALIX (GPPYPTY, aa 800-806). Whether Drosophila ALIX is recruited to the MR/MB via a protein(s) interacting with this motif or other domains is presently uncharacterized. Accordingly, alternative pathways of ALIX and ESCRT recruitment have been reported, as well as suggested in C. elegans, where CEP55 is also missing. Further studies are needed to elucidate mechanisms of recruitment and spatiotemporal control of ALIX and ESCRT-III during cytokinesis in fGSCs and different cell types in vivo (Eikenes, 2015).

This study found that the direct interaction between ALIX and Shrub is required for completion of abscission with normal kinetics in fGSCs. This is consistent with findings in human cells in which loss of the interaction between ALIX and CHMP4B causes abnormal midbody morphology and multi-nucleation. Following ALIX-mediated recruitment of CHMP4B/ESCRT-III to cortical rings adjacent to the MR in human cells, ESCRT-III extends in spiral-like filaments to promote membrane scission. Due to the discrete localizations of ALIX and CHMP4B during abscission in human cells ALIX has been proposed to contribute to ESCRT-III filament nucleation. In vitro studies have shown that the interaction between ALIX and CHMP4B may release autoinhibitory intermolecular interactions within both proteins and promote CHMP4B polymerization. Specifically, ALIX dimers can bundle pairs of CHMP4B filaments in vitro. Moreover, in yeast, the interaction of the ALIX homologue Bro1 with Snf7 (CHMP4 homologue) enhances the stability of ESCRT-III polymers. There is a high degree of evolutionary conservation of ALIX and ESCRT-III proteins and because ALIX and Shrub co-localize and interact to promote abscission in fGSCs it is possible that ALIX can facilitate Shrub filament nucleation and/or polymerization during this process (Eikenes, 2015).

The current findings indicate that accurate control of the levels and interaction of ALIX and Shrub ensure proper abscission timing in fGSCs. Their reduced levels or interfering with their complex formation caused delayed abscission kinetics. How cytokinesis is modified to achieve a delay in abscission in Drosophila fGSCs and incomplete cytokinesis in germline cysts is not well understood. Aurora B plays an important role in controlling abscission timing both in human cells and the Drosophila female germline. During Drosophila germ cell development Aurora B contributes to mediating a delay of abscission in fGSCs and a block in cytokinesis in germline cysts. Bam expression has also been proposed to block abscission in germline cysts. It will be interesting to investigate mechanisms regulating the levels, activity and complex assembly of ALIX and Shrub and other abscission regulators at MRs/MBs to gain insight into how the abscission machinery is modified to control abscission timing in fGSCs (Eikenes, 2015).

Intercellular bridge MTs in fGSC-CB pairs were degraded in G1/S when the fusome adopted bar morphology. Abscission in G2 thus appears to occur independently of intercellular bridge MTs in Drosophila fGSCs. This has also been described in C. elegans embryonic cells where the MR scaffolds the abscission machinery as well as in Archaea that lack the MT cytoskeleton]. In mammalian and Drosophila S2 cells in culture, on the other hand, intercellular bridge MTs are present until just prior to abscission (Eikenes, 2015).

It is interesting to note a resemblance of the stem cysts that appeared upon loss of ALIX and Shrub function to germline cysts in that the MRs remained open for long periods of time similar to RCs. Some modification of ALIX and Shrub levels/recruitment may thus contribute to incomplete cytokinesis in Drosophila germline cysts under normal conditions. Because stem cysts were detected in the case when ALIX weakly interacted with Shrub it is also possible that inhibition of their complex assembly/activity may contribute to incomplete cytokinesis in germline cysts. Abscission factors, such as ALIX and Shrub, may thus be modified and/or inhibited during incomplete cytokinesis in germline cysts. Such a scenario has been shown in the mouse male germline where abscission is blocked by inhibition of CEP55-mediated recruitment of the abscission machinery, including ALIX, to stable intercellular bridges. Altogether these data thus suggest that ALIX and Shrub are essential components of the abscission machinery in Drosophila GSCs, and it is speculated that their absence or inactivation may contribute to incomplete cytokinesis. More insight into molecular mechanisms controlling abscission timing and how the abscission machinery is modified in different cellular contexts will give valuable information about mechanisms controlling complete versus incomplete cytokinesis in vivo (Eikenes, 2015).

In summary, this study reports that a complex between ALIX and Shrub is required for completion of cytokinetic abscission with normal kinetics during asymmetric Drosophila GSC division, giving molecular insight into the mechanics of abscission in a developing tissue in vivo (Eikenes, 2015).

Fragile X Mental Retardation Protein, a Shrub interactor, regulates activity-dependent membrane trafficking and trans-synaptic signaling mediating synaptic remodeling

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. This study focusses on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the activity-dependent repression of translation. In the well-characterized Drosophila neuromuscular junction (NMJ) model, developmental synaptogenesis and activity-dependent synaptic remodeling both require extracellular matrix metalloproteinase (MMP) enzymes interacting with the heparan sulfate proteoglycan (HSPG) glypican dally-like protein (Dlp) to restrict trans-synaptic Wnt signaling, with FXS synaptogenic defects alleviated by both MMP and HSPG reduction. This new mechanistic axis spanning from activity to FMRP to HSPG-dependent MMP regulation modulates activity-dependent synaptogenesis (Sears, 2017).

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 fuelled by mis-regulation of signalling pathways. Its impact on junctional integrity, however, remains obscure. To dissect the events leading to neoplasia, transmission electron microscopy (TEM)was used on wing imaginal discs temporally depleted of the ESCRT-III core component Shrub. A specific requirement for Shrub was found 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. 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 (Pannen, 2020).

Developmental and physiological functions of epithelia rely on a set of cellular junctions, linking cells within the tissue to a functional unit. While E-cadherin-based adherens junctions (AJs) provide adhesion and mechanical properties, formation of the paracellular diffusion barrier depends on tight junctions (TJs). Proteins of the conserved claudin family play a key role in establishing and regulating TJ permeability in the intercellular space by homo- and heterophilic interactions with Claudins of neighboring cells. Arthropods, such as Drosophila, do not possess TJs but a functionally similar structure in ectoderm-derived epithelia termed pleated septate junction (pSJ, SJ hereafter), characterized by protein dense septa lining the intercellular space in electron micrographs. Structure and function of Drosophila SJs depend on a convoluted multiprotein complex containing at least a dozen components. Three claudins, among them Megatrachea (Mega), have been shown to be required for SJ formation and barrier function in flies. Besides claudins, several transmembrane proteins (TMPs) such as Neurexin-IV (NrxIV), Neuroglian (Nrg) or ATPα contribute to the formation of the stable SJ core complex, which is characterized by low mobility within the membrane. At the intracellular side of the junction, cytoplasmic proteins such as Coracle (Cora), Varicose (Vari), and Discs large (Dlg) associate with the transmembrane components, contributing to the formation of a stable fence-like scaffold . While junction formation during embryogenesis requires the SJ localized cytoplasmic protein Dlg, this basolateral cell polarity factor is not a structural part of the immobile junction core comple. This explains the functional separation of barrier formation and apicobasal polarity despite the close association of Dlg-complex components with the SJ. Albeit growing knowledge about the structural composition of SJs, the intracellular events required for assembly and maintenance of SJ complexes remain largely unknown. Specifically, how proliferative tissues, such as the imaginal disc epithelium, maintain SJ integrity is not well established (Pannen, 2020).

It was recently shown that newly synthesized SJ components integrate into the junction from the apical side (in between AJ and SJ) in a 'conveyor belt-like' fashion (Babatz, 2018; Daniel, 2018). In addition, SJ components are frequently associated with endosomal compartments, suggesting a role for the endosomal system in coordinating transport and turnover of SJ complexes. Consistently, endocytosis is required to concentrate SJ components at the junctional region during embryogenesis. This suggests that passage of SJ TMP components (or the whole SJ protein complex) through the endosomal system may be a requirement for SJ formation, with the underlying mechanisms remaining poorly characterized (Pannen, 2020).

The endosomal system fulfils a plethora of physiological functions by tightly regulating the intracellular transport of TMPs and membranes within the cell. Following endocytosis from the plasma membrane, TMPs enter the endosomal system where they undergo cargo specific sorting. This process provides separation of proteins destined for degradation from those that exit the endosomal system to be recycled. Two evolutionary conserved endosomal sorting machineries, the endosomal sorting complex required for transport (ESCRT) and the retromer complex, mediate cargo sorting into the degradative and recycling pathway, respectively. To coordinate these opposing transport activities, the endosomal system comprises a highly dynamic membrane network governing retromer-dependent tubulation for recycling and ESCRT-mediated generation of intraluminal vesicles (ILV) for degradation (Pannen, 2020).

Endocytosed proteins can evade ESCRT-dependent packaging into ILVs by exiting the maturing endosome (ME) through tubular retrieval domains induced by specialized recycling machineries such as retromer. Initially characterized as a regulator of endosome-to-Golgi cargo retrieval in yeast, this endosomal agent comprises two subcomplexes that cooperatively drive cargo sorting into tubular recycling carriers. Similar to the ESCRT machinery, cargo clustering and membrane deformation is performed by distinct functional units within the retromer pathway. Motif-based cargo recognition and aggregation is mediated by the endosomally localized Vps26:Vps29:Vps35 complex, which has been termed cargo-selective complex, considered to constitute the core functional component of retromer. Since the ancient CSC does not possess membrane bending activity, cooperation with tubulating factors such as proteins of the SNX-BAR (Sorting Nexin-Bin/Amphiphysin/Rvs) family is required for recycling carrier generation. Proteins containing the curved BAR-domain can assemble into regular helical coats on endosomes, thereby inducing cytoplasm faced tubulation. Concerted action of CSC stably complexed with SNX-BAR proteins to retrieve endosomal cargo was initially characterized as the classical retromer pathway in yeast. In metazoans however, retromer function is not restricted to SNX-BAR-dependent pathways. Specifically, cooperations of CSC with SNX3 or SNX27 (both lacking BAR-domains) emerged as alternative routes for endosomal retrieval. Proteomic data from mammalian cells suggest that surface levels of well over 100 TMPs depend on retromer and many of these proteins seemingly interact with CSC or SNX27. Recently, Drosophila has proven invaluable for assessing and confirming the physiological relevance of some of these putative retromer cargos in vivo (Pannen, 2020).

Cargo proteins within the endosomal system that do not undergo recycling can enter the degradative trafficking route starting with their sorting into ILVs. Generation of ILVs at the limiting membrane of MEs requires the canonical ESCRT function, which is performed by four in sequence acting complexes (ESCRT-0, -I, -II, III) and the ATPase Vps4. Ubiquitination of TMPs serves as the primary degradative sorting signal and sequestration of TMPs into ILVs is an essential prerequisite to complete lysosomal degradation. Several ESCRT components such as Vps27/Hrs (ESCRT-0) and Vps23/TSG101 (ESCRT-I) possess ubiquitin interacting motifs, which allow them to bind and cluster ubiquitinated TMPs. Consequently, local concentration of ubiquitinated cargo by ESCRT complexes establishes a degradative subdomain at the endosomal membrane that is spatially separated from the retrieval subdomain. While ESCRT-0-II complexes provide cargo recognition and clustering, the membrane-deforming activity required to bud and abscise ILVs into the endosomal lumen depends on ESCRT-III components, which polymerize into helical arrays at the endosomal membrane. The most abundant ESCRT-III component is the highly conserved yeast Snf7/Vps32, encoded by the gene shrub (shrb) in Drosophila. Unlike upstream ESCRT components, ESCRT-III proteins only transiently assemble into a heterooligomeric complex at the endosomal membrane. In consequence of ESCRT activity, the maturing endosome accumulates cargo-containing ILVs and is recognized in electron micrographs as a multi-vesicular body (MVB). The ESCRT/MVB pathway ends with Vps4-dependent dissociation of ESCRT-III components from the endosomal membrane. This step is required for the release of the nascent ILV and subsequent rounds of ILV formation. Loss of ESCRT function was initially studied in yeast cells in which it led to the emergence of an aberrant pre-vacuolar endosomal organelle, termed class E compartment. This defective endosomal structure is characterized by accumulation of degradative cargo and a failure to fuse with the vacuole/lysosome (Pannen, 2020).

The physiological relevance of ESCRT-mediated degradative TMP trafficking is particularly evident in Drosophila imaginal disc tissue. Here, loss of ESCRT function induces severe overgrowth, multilayering, apoptosis, and invasive behavior of the tissue; a phenotype attributed to mis-regulation of cellular signaling pathways, such as the Jak/Stat-, Jun-Kinase-, and Notch pathways. Consequently, ESCRT components were classified as endocytic neoplastic tumor suppressor genes (nTSG) in Drosophila. While induction of over-proliferation and apoptosis in nTSG mutants have been extensively characterized, the events leading to loss of cell polarity and ultimately neoplastic transformation of the tissue remain poorly understood (Pannen, 2020).

This study has analyzed the integrity of cellular junctions in an ESCRT-depleted wing imaginal disc epithelium to gain insight into the initial events leading to neoplastic transformation. Surprisingly, preceding neoplastic overgrowth, this study found a strong and specific reduction in the density of SJ. ESCRT and retromer functions are required for anterograde transport of SJ components. By dissecting the intracellular trafficking itinerary of the claudin Megatrachea, this study revealed that biosynthetic delivery of this core SJ component depends on a complex basal to apical transcytosis route relying on ESCRT and retromer functions (Pannen, 2020).

While transcytosis of SJ components has been shown to occur during the initial establishment of the SJ in the embryo, this study revealed that this mechanism is also continuously required during maintenance of the SJs in a rapidly proliferating epithelium. The data reveal that the retromer CSC functions downstream of ESCRT to export Mega from the endosome. A novel physiological role is proposed for the retromer CSC in regulating membrane levels of several SJ core components. While the data suggest that retromer fails to export Mega from aberrant endosomes induced by ESCRT depletion, the exact mechanism behind this remains to be determined (Pannen, 2020).

The data reveal a critical requirement for ESCRT in a transport pathway that depends on retromer-mediated transcytosis to deliver newly synthesized Mega to its apical destination. Defects in endosomal retrieval upon ESCRT inactivation have been previously described in other systems, such as yeast or mammalian cells and, thus, appear to represent a common feature of the pleiotropic ESCRT deficient phenotype. In yeast, the endosome-to-Golgi retrieval of the sorting receptor Vps10p and its cargo carboxypeptidase Y (CPY) depends on retromer function. ESCRT mutant strains accumulate CPY in class E compartments from which retrieval to the Golgi is blocked. Similarly, the mammalian retromer cargo mannose 6-phosphate receptor (M6PR) also failed to recycle from endosomes to the Golgi in HeLa cells depleted of TSG101/ESCRT-I function. In this study, it is suggested that generation of class E compartments occurs at the expense of endosomal tubules. Consistently, the retromer-associated tubulation factor SNX1 and its yeast homolog Vps5p were found on the rims of mammalian and yeast class E compartments, respectively. Together with the finding of CSC accumulation on Drosophila class E-like compartments, this suggests that ESCRT deficient endosomes remain coated with retromer components but fail to export specific cargo. (Pannen, 2020).

While the possibility cannot be ruled out of ESCRT components directly cooperating with retromer to form recycling tubules (note that SNX-BAR, Snx3, and Snx27 are not required for Mega transport), an indirect mechanism is favored linking ESCRT and retromer in this transport pathway. Analysis of the aberrant endosomal compartments induced upon Shrub depletion revealed that they are enriched in endosomal organizers such as Rab5 and Rab7, which could potentially interfere with retromer-dependent export when their activity at the limiting membrane is unrestrained. While the role of Rab7 in endosomal recruitment of the CSC is well established, the necessity for Rab7 GDP/GTP cycling during retromer-dependent carrier generation is still under debate. Rab7 and its GTPase-activating protein (GAP) Tbc1d5 are interaction partners of the CSC and can modulate its capability to retrieve endosomal cargo. For example, interfering with Rab7-GTP hydrolysis by Tbc1d5 depletion yielded defects in retromer-dependent transport in HeLa cells. Strikingly, under these conditions, retromer cargo was trapped in CSC-coated endosomes, paralleling the current observation of Mega subcellular localization upon ESCRT depletion. Similarly, by exposing the interplay between the CSC component Vps29, Tbc1d5, and Rab7 in adult Drosophila brains, the authors of a recent study reported the capability of endosomal Rab7 to interfere with retromer CSC function in vivo (Pannen, 2020).

While the exact mechanism rendering retromer dysfunctional at Drosophila class E compartments remains to be determined, the data support the mounting pool of evidence that ESCRT is required for multiple endosomal retrieval pathways. It is therefore likely that aspects of the pleiotropic ESCRT phenotype in metazoans stem from defective export of proteins from the endosomal system. For example, in Drosophila, leaky SJ could support ESCRT-mediated neoplastic transformation by permitting diffusion of signaling molecules within the imaginal disc tissue (Pannen, 2020).

This study found that biosynthetic delivery of Mega depends on a transcytosis-like mechanism from the basodistal to the apical plasma membrane. This long-distance transport required sequential action of endocytic (clathrin, dynamin, Rab5) and endosomal (ESCRT, retromer CSC) machineries. Importantly, the finding that overexpressed HA-Mega is unable to reach the SJ in absence of retromer and ESCRT function is in agreement with biosynthetic delivery of Mega relying on endosomal function. Therefore, although the possibility that Mega transiently passes the Golgi after endocytosis at the basodistal membrane cannot be excluded, the unconventional transcytosis model is favored. Strikingly, while retromer-dependent endosomal recycling has been extensively documented, only one mammalian cell culture study implicated retromer in transcytosis from one membrane domain to another. Thus, SJ delivery of Mega in imaginal discs represents a novel physiological role of retromer to study this process in vivo. The finding that CSC-mediated anterograde transport of Mega is independent of retromer-associated sorting nexins indicates that this transcytosis pathway is distinct from many established CSC-dependent routes and suggests that it may require unknown cofactors (or does not require endosomal tubulation) (Pannen, 2020).

Analysis of Vps35 clones in pupal wings or leg imaginal discs revealed that in these tissues, clones completely devoid of the SJ core component NrxIV occur frequently. Similarly, shrub mutant clones in the pupal notum were entirely lacking junctional ATPα (Roland Le Borgne, personal communication to Pannen, July 2020). This is in contrast to surface levels of SJ components in Vps35 mutant wing discs, which were consistently reduced by about 50%. This provokes the hypothesis that a parallel endosomal export pathway for SJ components may exist in wing discs that could partially compensate for loss of retromer. However, this is thought unlikely since overexpressed HA-Mega fails to reach the SJ not only upon Shrub but also upon Vps26 depletion. One has to keep in mind that this experiment specifically monitors delivery of newly synthesized HA-Mega while the Vps35 clonal analysis assesses the impact of retromer loss of function on pre-existing SJ. Thus, in a clonal situation, a 'thinning out' of junctions is expected with consecutive rounds of cell division, which could explain incomplete phenotypic expressivity in wing disc Vps35 clones. Nevertheless, it remains to be determined why in leg discs and pupal wings, SJ appear to be more sensitive toward CSC loss. During metamorphosis, wing imaginal disc cells undergo drastic morphogenetic changes to form the pupal wing epithelium, a process known to require AJ remodeling. It is therefore possible that analogously to AJ, SJ may also be actively remodeled during pupal wing formation. This could explain the strong requirement for retromer function in maintaining SJ integrity in a tissue undergoing morphogenetic changes (Pannen, 2020).

Paralleling the findings described in this paper, a previous study showed that embryonic SJ formation depends on endocytosis and subsequent redistribution of junction components from the lateral membrane to the SJ. Thus, transcytosis of SJ components is a mechanism likely required for both initial SJ formation as well as maintenance of SJ integrity in non-embryonic tissues in Drosophila. While the roles of ESCRT and retromer CSC in initial SJ formation were not assessed, it is conceivable that they are already required for transporting SJ components during embryogenesis. Consistently, shrub mutant embryos display a defective epithelial barrier function, suggesting they fail to form functional SJx (Pannen, 2020).

The reason for SJ maintenance to rely on such an elaborate trafficking of its components remains to be determined. It has been suggested that SJ components form stable complexes prior to integration into the junction. Potentially, essential post-translational modifications of certain SJ components required for complex formation may occur exclusively at the basodistal membrane or during the passage through the endosomal system. If transient localization of SJ components at the basodistal membrane is a prerequisite for efficient SJ core complex formation, depositing transcripts for structural components such as Mega in the basal cytoplasm would shorten the route individual SJ components need to pass prior to SJ complex formation at the basodistal membrane domain. Alternatively, the finding that Mega mRNA predominantly localizes in the basal cytoplasm provides the foundation for another hypothesis: It is widely accepted that apical and basolateral cargos undergo motif-based sorting leading to secretion toward the respective membrane domains. Basal subcellular localization of Mega transcripts could potentially reflect an apical/basolateral sorting divergence at the mRNA level. Accordingly, basal translation and exocytosis of Mega (induced by a putative basal/basolateral sorting signal) may lead to its targeting toward the basodistal membrane, despite the fact that the SJ resides apically in wing disc cells. Thus, transcytosis may serve as an adaptation for redistribution of cargos to their destined membrane domain when they are initially secreted to a different one due to early sorting signals. Since the columnar wing imaginal disc cells have a very elongated shape and possess Golgi stacks all along the apicobasal axis, it is conceivable that certain Golgi stacks residing at the apical and basal poles are specialized in secreting apical and basolateral cargos, respectively. Although this is highly hypothetical, systematic analysis of transcript localization for apical (e.g., E-cad) and basolateral (e.g., SJ components) cargos could reveal a potential spatial separation of distinct secretory routes already at the mRNA level (Pannen, 2020).

In wing imaginal discs, a similarly complex transcytosis route (albeit with an apical to basal direction) has been described for the signaling molecule Wingless, which is translated apically, transiently presented at the apical membrane and finally transcytosed toward the basal membrane where it is secreted (Yamazaki, 2016). Thus, distinct transcytosis pathways in the wing disc epithelium provide a mechanism for targeting certain proteins to their site of action, specifically when the protein is translated far away from its terminal destination (Pannen, 2020).

This study has unravel a novel physiological retromer function in regulating surface levels of a claudin and other structural SJ components (e.g., Nrg, ATPα, Lac, NrxIV, Cont) in several Drosophila tissues. Currently, it is not known how the SJ components are selected for this retromer-dependent pathway, and whether it requires physical interaction with CSC components. Since SJ proteins may traverse the endosomal system in complex, the vast number of different components brings about a plethora of possible interaction sites. Importantly, a mass-spectrometry-based study of the Mega interactome did not detect any retromer CSC components or associated factors but confidently found SJ core components as well as clathrin. While the interaction mode of CSC and SJ proteins remains to be determined, the data reflect the assumption that several SJ core components represent novel putative retromer cargos in Drosophila (Pannen, 2020).

Strikingly, among the proteins affected by retromer loss of function, many possess mammalian homologs (e.g., NrxIV/CNTNAP2, ATPα/ATP1A1, Nrg/NRCAM). This suggests they could represent a novel set of conserved retromer cargos. Indeed, several lines of evidence suggest that ESCRT/retromer-mediated transport of SJ components may be evolutionary conserved from Drosophila to mammals. Depletion of ESCRT-I component TSG101 in mammalian epithelial cells led to a reduction of trans-epithelial resistance (TER), indicating defects in TJ-mediated barrier function. Additionally, Claudin-1, an essential TJ component, continuously underwent endocytosis and recycling back to the plasma membrane in several mammalian cell lines in a process requiring ESCRT function (Pannen, 2020).

Importantly, the mechanism behind reduced recycling and entrapment of Claudin-1 in ubiquitin-positive aberrant endosomes upon interference with ESCRT function remained elusive. Thus, it is unknown how the export of Claudin-1 from the endosomal system in mammalian cells is achieved. By revealing an ESCRT-dependent function of the retromer CSC in claudin endosomal export in Drosophila, the data may provide an explanation for a possibly conserved trafficking pathway of claudins. In support of this, Claudin-1 and Claudin-4 membrane levels were significantly reduced in a mass spectrometry-based surface proteome study of Vps35-depleted human cells. Furthermore, the TJ protein Zonula occludens-2 (ZO-2) was strongly enriched in a Vps26 interactome, suggesting that a presumptive retromer function in TJ maintenance in mammalian cells may not be limited to claudins, similar to the findings in Drosophila presented in this study. It remains to be determined whether a physiological role of retromer in mammalian TJ maintenance occurs also in vivo. An increasing amount of tools, such as conditional Vps35 knockout mice, will enable analysis of this putatively conserved retromer function in mammalian systems and reveal any possible implications in development and/or disease (Pannen, 2020).

The tumour suppressor Lethal (2) giant discs is required for the function of the ESCRT-III component Shrub/CHMP4

Recent work indicates that defects in late phases of the endosomal pathway caused by loss of function of the tumour suppressor gene lethal (2) giant discs (lgd) or the function of the ESCRT complexes I-III result in the ligand-independent activation of the Notch pathway in all imaginal disc cells in Drosophila. lgd encodes a member of an uncharacterised protein family, whose members contain one C2 domain and four repeats of the DM14 domain. The function of the DM14 domain is unknown. This study reports a detailed structure-function analysis of Lgd protein, which reveals that the DM14 domains are essential for the function of Lgd and act in a redundant manner. Moreover, this analysis indicates that the DM14 domain provides the specific function, whereas the C2 domain is required for the subcellular location of Lgd. Lgd was found to interact directly with the ESCRT-III subunit Shrub through the DM14 domains. The interaction is required for the function of Shrub, indicating that Lgd contributes to the function of the ESCRT-III complex. Furthermore, genetic studies indicate that the activation of Notch in ESCRT and lgd mutant cells occurs in a different manner and that the activity of Shrub and other ESCRT components are required for the activation of Notch in lgd mutant cells (Troost, 2012).

This study reports the results of a detailed structure-function analysis of Lgd, a member of a recently discovered protein family whose hallmark is the possession of four tandem repeats of the uncharacterised DM14 domain. Although a recent study has reported a similar analysis for human Lgd2 in cell culture (Zhao, 2010), this is the first comprehensive analysis of a member of this uncharacterised protein family in an animal model. For the analysis a new assay system was developed that assured expression of the constructs at the level of endogenous lgd. This was necessary because it was found that the process of protein trafficking is very sensitive to overexpression of Lgd. Thus, data obtained by overexpression of Lgd proteins (e.g., in cell culture) must be interpreted with great caution. This notion can probably be extended to other elements of the endosomal pathway, because dramatic changes have be observed in endosome morphology if other endosomal proteins, such as FYVE-GFP, Rab5-GFP or Rab7-GFP, are expressed with the Gal4 system. Moreover, this study found that overexpression of these proteins suppresses the activation of Notch in lgd cells. These findings indicate that the overexpression of endosomal proteins induces significant changes in protein trafficking through the endosomal pathway (Troost, 2012).

This study found that the DM14 domains are important for the function of Lgd and that they constitute novel modules for direct interaction with a core member of the ESCRT-III complex during protein trafficking. Moreover, this analysis reveals that the DM14 domains provide the specific function of Lgd and function in a redundant manner. Using cell culture, Nakamura (2008) provided evidence that the fourth DM14 domain of Lgd2 is especially important for its function as a scaffold protein that is required for PDK1/Akt signalling activated by the EGF. However, no specific importance of the fourth DM14 domain could be detected in Drosophila. In the assay conditions used, any combination of two of the four domains appears to be sufficient for Lgd function and can rescue the lgd mutant phenotype (Troost, 2012).

However, this notion holds true only if the concentration of Shrub is normal. In situations where the activity of Shrub is reduced (shrub4-1/+), variants with four domains can provide more activity and assure sufficient interaction to maintain correct endosomal trafficking. This was already observed in animals that are hypomorphic for lgd (lgdd7/lgdSH495 shrub4-1). In other words, four DM14 copies enable the organism to tolerate the lgd shrub double heterozygous situation. Because almost all Lgd-like proteins discovered so far have four copies, it is likely that this ability endows members of the family with a functional robustness that is evolutionarily advantageous. The rescue experiments in the sensitized lgd +/lgd shrub4-1 backgrounds also suggest that the second DM14 domain is of greatest importance for the function of Lgd in Drosophila. This is in contrast to results of cell culture experiments for human Lgd2 (Nakamura, 2008). However, it is important to point out that most of the evidence for function in mammals is obtained with cell culture experiments, which often involve the overexpression of Lgd orthologues at levels way above endogenous levels. Given the great difficulties in gaining sensible results using the Gal4 system, these data should be interpreted carefully (Troost, 2012).

It has been previously shown that the C2 domain of Lgd can bind to certain phospholipids, such as phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, in an in vitro assay (Gallagher, 2006). Furthermore, cell fractionation experiments using cytosolic extracts from wild-type and the lgd08 mutant animals that encode a variant lacking the C2 domain, suggest that a small fraction is associated with the membrane in a C2-dependent manner. These biochemical data are contrasted by microscopy studies, which reported a cytosolic distribution of Lgd without any obvious association with membrane structures. In agreement, this study found that tagged Lgd variants expressed at the endogenous level are localised within the cytosol. Moreover, it was found that a lgd construct, encoding little more than the C2 domain and virtually identical to the Lgd fragment used in the in vitro phospholipid binding assay (Gallagher, 2006), is located in the cytosol similarly to Lgd. The discrepancy between the biochemical and microscopy data might be explained by the possibility that only a small fraction of Lgd (which cannot be detected in antibody staining) is associated with membranes. However, knowing that Lgd interacts with Shrub, it is surprising that no obvious association of Lgd was found even upon depletion of Vps4, although the ESCRT-III complex is locked on the endosomal membrane in this situation. One would expect that the membrane-associated fraction of Lgd should be increased in this situation. Thus, it is believed that Lgd is located within the cytosol. This notion is further supported by the fact that variants of Lgd that lack the C2 domain can rescue the lgd mutant phenotype to a high degree, although they are produced at a much lower level than the other constructs tested and than endogenous Lgd (Troost, 2012).

Three distinct functions were determined for the C2 domain. The first function is that it provides protein stability, because it was found that the constructs encoding variants without the C2 domain give rise to significantly lower amounts of protein than variants with the domain. The second function is the localisation of Lgd within the cytosol. This function provides an explanation for the discrepancy between the in vivo and biochemical studies, because variants without the C2 domains were found to be located in the nucleus. The reason for the mis-localisation of Lgd variants that lack the C2 domain is unclear at the moment. No cryptic nuclear localisation sequence (NLS) has been found within Lgd. Thus, it is possible that it is transported in the nucleus in complex with another protein that contains an NLS (Troost, 2012).

The presented results suggest a third function for the C2 domain, because it was found that LgdδDM14, which cannot provide any specific function in the rescue assay, can out-compete NESLgdδC2 in a C2-dependent manner and thereby prevent the partial rescue of lgd mutants. A likely possibility is that the C2 domain mediates an interaction with other proteins that results in concentration of Lgd at the site of action within the cytosol. In agreement with this possibility, recent reports have shown that the C2 domains of Nedd4L, PKC and PKCe mediate protein-protein interactions. Furthermore, human Lgd2/CC2D1A appears to interact via its C2 domain with the E2 enzyme Ubc13 during NF-kappaB signalling (Zhao, 2010). Therefore, the possibility is favored that the C2 domain of Lgd mediates protein-protein interactions instead of localising Lgd to a distinct membrane. It is possible that the cytosolic interaction prevents Lgd from migrating into the nucleus (Troost, 2012).

Recent results obtained in mammalian cell culture experiments suggest that human Lgd1 and Lgd2 might also act as transcriptional repressors (Hadjighassem, 2009; Ou, 2003). This study found that Lgd requires location within the cytosol for its function. Hence, the current results are not easily compatible with a function as a transcription factor, as suggested for human Lgd1 and Lgd2, and it is believed that a gene regulatory function for Lgd inDrosophila is unlikely (Troost, 2012).

Previous work has established that loss of function of ESCRT-I-ESCRT-III complexes results in non-autonomous and autonomous cell proliferation and activation of the Notch pathway. In addition, the mutant cells lose their epithelial organisation and eventually die. Although loss of function of lgd results in activation of the Notch pathway and overproliferation, these effects are cell-autonomous, and the mutant cells do not lose their polarity and survive well. Thus, the phenotypes of the two groups overlap, but are not identical. Nevertheless, this study has found an intimate relationship between the ESCRT-III component Shrub and Lgd. Both proteins physically interact and this direct interaction is important in vivo, as indicated by the strong genetic interactions uncovered between the two genes. Importantly, it was observed that the time of death for a hypomorphic allelic combination of lgd, which normally results in pharate adults, is earlier than that of lgd null mutants if the activity of shrub is reduced by half. The earlier time of death suggests that the function of shrub is impaired upon loss of lgd function. Thus, it appears that the physical interaction with Lgd is required for the proper function of Shrub. Because the loss-of-function phenotype of shrub is more deleterious and includes more aspects than that of lgd, it is likely that lgd contributes to, but is not absolutely required for, the function of shrub. Either loss of lgd results in the loss of one distinct aspect of Shrub function or it reduces its activity beyond a threshold that is required for complete function. The finding that overexpression of Shrub can rescue the lgd phenotype supports the second possibility. Recent work suggests that Shrub forms long homopolymers on the cytosolic surface of the endosomal membrane. This polymerisation is required for the abscission of vesicles into the lumen of the maturing endosome (Saksena, 2009). In order to polymerise, Shrub has to be converted from a closed cytosolic into the open form. After intraluminal vesicle (ILV) formation, Shrub becomes converted into the closed form by Vps4, with consumption of ATP. Because the data suggest that Shrub and Lgd interact in the cytosol, it is possible that Lgd somehow helps to prepare Shrub for the next round of polymerisation on the endosomal membrane (Troost, 2012).

The presented genetic studies suggest an antagonistic relationship between Lgd and several components of the ESCRT complexes with respect to Notch activation. This implies that activation of Notch in lgd cells depends on the function of the ESCRT complexes and therefore indicates that it must occur in a different manner in lgd cells to that in ESCRT-mutant cells. The results suggest that loss of lgd function somehow affects the activity of Shrub, which in turn results in the activation of Notch. It is important to point out that the antagonism between lgd and ESCRT is observed only with respect to activation of Notch signalling. With respect to endosome morphology, they appear to act synergistically because a reduction of shrub function by half results in a dramatic enlargement of endosomes of lgd hypomorphic cells, which normally do not exhibit such a defect. The results therefore reveal a complex relationship between Lgd and the ESCRT function and further work is required to resolve this relationship in detail (Troost, 2012).

Because activation of Notch is not possible without release of the Notch intracellular domain (NICD) into the cytosol, it is assumed that a fraction or all of Notch must somehow remain at the limiting membrane of the endosome and is not incorporated into ILVs in lgd cells. There are three possibilities for how this might be achieved: no ILVs form; Notch might not be efficiently incorporated into the ILVs; or ILVs might back-fuse with the limiting membrane of the maturing endosome. Back-fusion has been documented to occur in vertebrate cells. The current results suggest that loss of lgd function results in a reduction in the activity of Shrub. Therefore, the possibility is favored that the loss of lgd function results in a less efficient incorporation of Notch into the ILVs due to the reduced activity of Shrub (Troost, 2012).

Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome

The tumour suppressor Lethal (2) giant discs (Lgd) is a regulator of endosomal trafficking of the Notch signalling receptor as well as other transmembrane proteins in Drosophila. The loss of its function results in an uncontrolled ligand-independent activation of the Notch signalling receptor. This study investigated the consequences of loss of lgd function and the requirements for the activation of Notch. The activation of Notch in lgd cells was shown to be independent of Kuz and dependent on γ-secretase. The lgd cells were found to have a defect that delays degradation of transmembrane proteins, which are residents of the plasma membrane. Furthermore, the results show that the activation of Notch in lgd cells occurs in the lysosome. By contrast, the pathway is activated at an earlier phase in mutants of the gene that encodes the ESCRT-III component Shrub, which is an interaction partner of Lgd. It was further shown that activation of Notch appears to be a general consequence of loss of lgd function. In addition, electron microscopy of lgd cells revealed that they contain enlarged multi-vesicular bodies. The presented results further elucidate the mechanism of uncontrolled Notch activation upon derailed endocytosis (Schneider, 2013).

Notch signalling is involved in many homeostatic and developmental processes in all metazoans and uncontrolled activation is a cause of disease in humans. Hence, it is important to unravel the mechanisms of its normal as well as its uncontrolled activation. Previous work has shown that loss of lgd function results in the ligand-independent activation of the Notch signalling pathway in imaginal disc cells. This activation was still dependent on the activity of the function of Psn, which encodes a component of the γ-secretase complex. This work extends the characterisation of lgd and defines the condition under which Notch is activated in lgd cells more precisely. Moreover, activation of Notch was shown to be a consequence of loss of lgd function also in another tissue, the follicle epithelium. This suggests that it is a general consequence of loss of lgd function in cells in Drosophila. It was confirmed that the γ-secretase complex is necessary for the activation of Notch in lgd cells. In addition, the first EM analysis is presented of lgd mutant cells, which indicates that a fraction of the endosomes of lgd cells is indeed enlarged and contain ILVs (Schneider, 2013).

Previously a model was suggested of how Notch is activated in lgd cells (Troost, 2012). Several of the uncertainties of this model are resolved by the results of the current work. This study showed that activation of Notch requires the fusion of the endosome with the lysosome. Thus, activation probably occurs in the lysosome and not the ME. This finding has several implications. First, it indicates that the defect occurs during endosome maturation in lgd cells, since fusion of the ME with the lysosome does not result in activation of Notch in wild-type cells. Secondly, it indicates that although lgd cells are defective in degradation of trans-membrane proteins, the MEs eventually fuse with lysosomes. Thus, degradation is delayed rather than prevented. This delay in fusion can explain the observation that lgd cells contain a fraction of moderately enlarged MVBs, because it allows the MEs to undergo more homotypic fusions and, thus, to grow to a larger size than normal over time. Thirdly, it indicates that the observed accumulation of Notch in MEs to high levels is not per se the cause of activation of the pathway. This notion is also supported by the observation that although all lgd cells of the follicular epithelium activate the Notch-pathway, not all show strong accumulation of Notch in MEs (Schneider, 2013).

A prerequisite for all Notch activation is that the NICD must face the cytosol so that it can access the nucleus after its release. During normal degradation, Notch is incorporated into ILVs and the NICD is separated from the cytosol. Thus, it cannot access the nucleus even if cleavage would occur. This consideration implies that in lgd cells the formation of ILVs must either fail or Notch is inefficiently incorporated in them. EM analysis revealed that the enlarged MEs contained ILVs. Thus, it appears that ILV formation is at least not strongly affected by loss of lgd. It has been recently shown that Lgd physically interacts with Shrub and is required for its full function (Troost, 2012). A similar interaction has been reported between the mammalian orthologues of Lgd interact and the orthologues of Shrub, CHMP4a, b, c (Martinelli, 2012; Usami, 2012). Moreover, in vitro experiments suggest that Lgd1 and Lgd2 can influence the polymerisation of CHMP proteins (Martinelli, 2012). The possibility is therefore favored that upon loss of lgd function, Notch is inefficiently incorporated into the ILVs due to a reduction, but not abolishment in the activity of shrub. Consequently, a fraction or all of Notch is not incorporated into ILVs and remains in the limiting membrane of the ME (NLM fraction). This NLM fraction is then activated in a ligand-independent manner. However, generating a NLM fraction is a prerequisite for activation of Notch in lgd cells, but it is not sufficient. Loss of hrs function prevents activation of Notch in lgd cells. In hrs mutants, the formation of ILVs is suppressed and, as a result, cargo (Notch) remains in the LM. (Schneider, 2013).

The current results provide more information about the further requirements of Notch activation in lgd cells. Activation of Notch is not only independent of the ligands, but also of kuz. Kuz is required for ecto-domain shedding, which is a prerequisite for RIP by the γ-secretase complex. Since the activity of the γ-secretase complex is necessary for activation of Notch in lgd cells, ecto-domain shedding of Notch must occur by an alternative manner in lgd cells. Alternative ecto-domain shedding of the NLM fraction in the lysosome can be easily explained by the degradation of the NECD, which extends into the lumen by the activated acidic hydrolases. In this way a NEXT-like fragment could be generated that serves as a substrate for the γ-secretase complex. It has been shown that the γ-secretase complex is active in the lysosome and it is likely that it can perform RIP on the NEXT-like fragment to release NICD into the cytosol. In agreement with this scenario is the finding that NδEGF is activated in lgd, but not in wild-type cells. This suggests that the remaining lin12 repeats of NδEGF that are protruding into the lumen change their conformation or become degraded in lgd cells. Activation of Notch in lgd cells requires the function of the activity of vATPase. This proton pump is required for the acidification of the lumen of MEs, which in turn is a prerequisite for the activation of the acidic hydrolases in the lysosome. The function of vATPase during activation of the Notch pathway in lgd cells could be twofold: Its activity is the prerequisite for the activation of hydrolases. Additionally, the resulting acidification of the lumen might also denature NECD or even resolve the Ca2+ salt bridges between NICD and NECD. It is worth pointing out that the function of vATPase during Notch activation in lgd cells is different from that during ligand-dependent activation. This activation appears to occur in the EE, whose luminal pH is significantly higher. However, the data suggest that the S1 cleavage is required in addition. Thus, it is probably an interplay of several factors that causes the activation of Notch (Schneider, 2013).

The results refine a previously suggested model of Notch activation in lgd cells . It suggests that the loss of lgd function results in a reduction in the activity of Shrub. As a consequence a fraction or all of Notch remains at the limiting membrane of the ME. Upon fusion with the lysosome the activated hydrolases, possibly in combination of the acidic environment causes alternative ecto-domain shedding. This creates a NEXT-like substrate for the γ-secretase complex, which releases NICD into the cytosol (Schneider, 2013).

The model is very similar to that suggested for the regulation of activation of Notch by the E3-ligase Deltex. Recent work reports an intimate functional relationship between the arrestin-like protein Kurtz (Krz), Deltex (Dx) and Shrub upon regulation of Notch (Hori, 2011). The authors suggested a model in which Shrub downregulates the activity of Notch by incorporating the poly-ubiquitinated receptor into ILVs, while Dx antagonises this incorporation and promotes activation. Dx exerts its function by regulating the ubiquitination status of NICD. Its overexpression shifts the equilibrium from poly- to mono-ubiquitylation. It will be interesting to determine how Lgd fits into this scheme. Qualitatively, the phenotype of loss of lgd function is similar to that of Dx overexpression, although the level of Notch activation is stronger. This would suggest that Lgd antagonises the function of Dx. The model proposed by Hori (2011) suggests that Dx prevents the incorporation of Notch into ILVs by Shrub. Lgd appears to influence the activity of Shrub by direct physical interaction (Troost, 2012). Thus, the possibility is favored that the relationship between Dx and Lgd is indirect and mediated through Shrub (Schneider, 2013).

The experiments indicate that a process is required for activation of Notch in lgd cells that has limited capacity. A possibility is that the affected process is the cleavage of Notch the γ-secretase complex, as it was observed that the ectopic activation of Notch in lgd cells is suppressed through reduction of the activity of the complex. If this is true it has to be explained how other transmembrane proteins that can compete with Notch are transformed into substrates for the γ-secretase complex. A possibility is that they also undergo alternative ecto-domain shedding (Schneider, 2013).

This study has confirmed that loss of shrub function results in the activation of the Notch pathway also during oogenesis. Further support is provided for the previously drawn conclusion that the activation of Notch in shrub cells occurs through another mechanism than in the case of lgd, because it was found that the activation of Notch is suppressed if Rab7 is depleted in lgd but not shrub cells (Troost, 2012). This finding indicates that the activation in shrub cells occurs in the ME rather than in the lysosome. Thus, activation of Notch can occur in several endosomal compartments (Schneider, 2013).

It is possible that different levels of inactivation of shrub trigger different modes of Notch activation. The loss of shrub function results in a strong activation of Notch and loss of epithelial integrity (Hori, 2011; Troost, 2012). However, slight reduction of shrub function only weakly activates Notch and appears to have little effect on the epithelial integrity (Hori, 2011). This raises the possibility that activation of Notch through reduction of shrub activity might occur through a different mechanism than upon abolishment of shrub function. It would be interesting to investigate whether the slight activation of Notch occurs in the lysosome or endosome (Schneider, 2013).

Synergy between the ESCRT-III complex and Deltex defines a ligand-independent Notch signal

The Notch signaling pathway defines a conserved mechanism that regulates cell fate decisions in metazoans. Signaling is modulated by a broad and multifaceted genetic circuitry, including members of the endocytic machinery. Several individual steps in the endocytic pathway have been linked to the positive or negative regulation of the Notch receptor. In seeking genetic elements involved in regulating the endosomal/lysosomal degradation of Notch, mediated by the molecular synergy between the ubiquitin ligase Deltex and Kurtz, the nonvisual beta-arrestin in Drosophila, this study identified Shrub, a core component of the ESCRT-III complex as a key modulator of this synergy. Shrub promotes the lysosomal degradation of the receptor by mediating its delivery into multivesicular bodies (MVBs). However, the interplay between Deltex, Kurtz, and Shrub can bypass this path, leading to the activation of the receptor. This analysis shows that Shrub plays a pivotal rate-limiting step in late endosomal ligand-independent Notch activation, depending on the Deltex-dependent ubiquitinylation state of the receptor. This activation mode of the receptor emphasizes the complexity of Notch signal modulation in a cell and has significant implications for both development and disease (Hori, 2011).

The extraordinary sensitivity of normal development to the dosage of the Notch receptor is manifested through the haploinsufficient and triplomutant behavior of the Notch locus. Dosage sensitivity is consistent with the fact that the Notch signaling mechanism relies on stoichiometric interactions rather than enzymatic amplification steps to bring the signal from the surface to the nucleus. This also provides a rationale for the observation that cellular events involved in trafficking/turnover are emerging as major Notch signal-controlling mechanisms. The canonical pathway relies on the activation of the receptor triggered by its interaction with membrane-bound ligands on an apposing cell but the possibility that the receptor can also be activated intracellularly, in a ligand-independent fashion, as several studies, including the present one, suggest, has important implications for the biology and pathobiology of Notch. The rules governing how and where a receptor, trafficking through the endocytic compartments, can be activated, in the presence or absence of the ligand, are still not completely defined. Moreover, it is not understood how such events are integrated into the genetic circuitry that affects the regulation of endosomal compartment assembly and function (Hori, 2011).

This study provides insight into these questions by showing that the interplay between the Notch signal modulator Dx, the nonvisual β-arrestin orthologue Krz, and a critical component of the ESCRT-III complex, Shrub, directs Notch either into a degradation or into a ligand-independent activation path, which is paralleled by distinct ubiquitinylation states of Notch. The ESCRT pathway, recently described as a 'cargo-recognition and membrane-sculpting machine,' defines a complex, multipurpose cellular machinery with cellular roles and molecular mechanisms that are not fully elucidated (Henne, 2011). ESCRT is crucial in mediating the various steps leading to the sorting of membrane proteins into MVBs on their way to lysosomal degradation. The implication of Shrub in Notch signaling-related processes was revealed through an unbiased genetic screen for isogenic modifiers of the dx-krz-dependent phenotype, which is based on the endosomal/lysosomal degradation of the Notch receptor. This study is not the first to provide a general link between Notch signaling with the ESCRT machinery, but both the genetic screen as well as the subsequent analysis points to the differential and major role of the ESCRT-III complex in the dx-krz-dependent, ligand-independent mode of Notch signaling described in this study (Hori, 2011).

Several studies established that as the Notch receptor enters an endocytic path, it can be activated inside the cell in both a ligand-dependent as well as ligand-independent fashion. Consequently, several elements of the endosomal machinery including elements of the ESCRT complexes were shown to influence the intracellular accumulation and activation of the Notch receptor. The current data are compatible with these studies and indeed extend and complement them. These studies are not directly comparable, not only because of differing genetic backgrounds, a crucial element in evaluating genetic interactions, but also because this study is analyzing the impact of ESCRT function on the modulation of Notch signaling via the synergistic action of Dx and Krz, which may well define a different but specific path. Considering the entire body of work related to various aspects of Notch receptor trafficking it seems that there may be several, distinct ways the receptor can be activated after entering the endocytic path. Some studies link early endosomes with the activation of the receptor, whereas others implicate late endosomal compartments with ligand-independent activation of Notch. Particularly relevant to the activation mode documented in this study are the genetic studies of Wilkin (2008) that associated Notch activation with the HOPS (homotypic fusion and vacuole protein sorting) and AP-3 (adaptor protein-3) complexes, demonstrating the existence of a Notch activation path that is dependent on late endosomal compartments (Hori, 2011).

This study found that the expression of Shrub triggers a dramatic subcellular shift of the Notch receptor to MVBs, consistent with the fact that ESCRT-III mediates the cargo de-ubiquitination, budding, and scission of intraluminal vesicles, which control the delivery of the cargo to the lysosomes. The down-regulation of Notch signals by Shrub is apparently associated with the recruitment of Notch in intraluminal vesicles and its eventual degradation. On the other hand, disrupting the cellular equilibrium between Dx and Shrub by down-regulating Shrub and/or up-regulating Dx activates the receptor in a ligand-independent manner. It is also clear that the mode of Notch activation document in this study is independent of the ligands and is linked to the ubiquitinylation status of the Notch receptor, which in turn is modulated by Dx and Krz. Krz was shown to modulate Notch activity through its ability to regulate the levels of the Notch protein. It is noted with interest that although β-arrestins have been implicated as adaptors during clathrin-dependent endocytosis, Ram8, an arrestin homologous protein in yeast, has also been associated with the recruitment of the ESCRT machinery to MVBs loaded with a G-coupled receptor cargo. If Krz has a similar relationship with the ESCRT machinery, it may be involved in sorting Notch on MVBs and hence the eventual recruitment of Notch in intraluminal vesicles for degradation, a notion compatible with the observation that Krz enhances the Shrub-dependent down-regulation of Notch (Hori, 2011).

In order for the receptor to enter intraluminal vesicles, a de-ubiquitinylation of the cargo must take place. It is noteworthy that Snf7 (the Shrub orthologue in yeast) recruits the de-ubiquitinating enzyme Doa4 necessary for such cargo de-ubiquitination. In cell culture studies, where the relative subcellular localization of Notch, Shrub, and Dx could clearly be followed, localization of Notch, Shrub, and Dx with MVB membranes was observed. Because this subcellular phenotype is paralleled by a dramatic ligand-independent activation of the receptor and a shift from poly- to a mono-ubiquitination status of the receptor, this leads to a suggestion that Dx, which physically interacts with Notch, interferes with processes that are essential for loading the receptor on intraluminal vesicles (Hori, 2011).

It is clear that a more detailed analysis of subcellular dynamics in vivo is necessary to address many of the questions raised by the present study. On the basis of the data presented in this study a model is presented for the activation mode that was uncovered. It is suggested that the ESCRT-III component Shrub can regulate receptor cycling, diverting it to a signaling path, a fate modulated by Dx and Krz. Thus, Notch signaling can be attenuated inside the cell in a ligand-independent fashion. It remains to be determined how such intracellular signaling serves the developmental logic of Notch which couples the fate of one cell to that of the next door cellular neighbor. It is possible for such mode of Notch action to be useful to modulate the fate of a cell that, for example, circulates and is thus not necessarily in contact with a ligand-expressing neighbor. Irrespective of the potential role ligand-independent activation may play in normal development, activating the receptor can have profound pathological consequences. Therefore, understanding pathways capable of modulating Notch activity in an intracellular, ligand-independent manner is of great importance (Hori, 2011).

Lgd regulates the activity of the BMP/Dpp signalling pathway during Drosophila oogenesis

The tumour suppressor gene lethal (2) giant discs (lgd) is involved in endosomal trafficking of transmembrane proteins in Drosophila. Loss of function results in the ligand-independent activation of the Notch pathway in all imaginal disc cells and follicle cells. Analysis of lgd loss of function has largely been restricted to imaginal discs and suggests that no other signalling pathway is affected. The devotion of Lgd to the Notch pathway was puzzling given that lgd loss of function also affects trafficking of components of other signalling pathways, such as the Dpp (a Drosophila BMP) pathway. Moreover, Lgd physically interacts with Shrub, a fundamental component of the ESCRT trafficking machinery, whose loss of function results in the activation of several signalling pathways. This study shows that during oogenesis lgd loss of function causes ectopic activation of the Drosophila BMP signalling pathway. This activation occurs in somatic follicle cells as well as in germline cells. The activation in germline cells causes an extra round of division, producing egg chambers with 32 instead of 16 cells. Moreover, more germline stem cells were formed. The lgd mutant cells are defective in endosomal trafficking, causing an accumulation of the type I Dpp receptor Thickveins in maturing endosomes, which probably causes activation of the pathway. Taken together, these results show that lgd loss of function causes various effects among tissues and can lead to the activation of signalling pathways other than Notch. They further show that there is a role for the endosomal pathway during oogenesis (Morawa, 2015).

In recent years, it has been established that the tumour suppressor gene lethal (2) giant discs (lgd) [l(2)gd1 - FlyBase] has a function in the endosomal pathway and in the regulation of the activity of the Notch pathway in Drosophila melanogaster. Loss of its function leads to a defect in endosomal trafficking of transmembrane proteins, which accumulate in maturing endosomes (MEs). This defect causes the constitutive activation of the Notch pathway in a ligand-independent manner. lgd encodes a member of an evolutionary conserved family whose members contain four repeats of the novel DM14 domain followed by one C2 domain. A mutation in one of the two human orthologs, LGD2 (also named Aki, Freud-1, TAPE and Cc2d1a), causes mental retardation, and this protein might be a tumour suppressor in liver cells (Morawa, 2015).

In Drosophila the Notch receptor is activated by two ligands, Delta (Dl) and Serrate (Ser). Their binding initiates the S2-cleavage of the extracellular domain of Notch (NECD). The cleaved NECD is then trans-endocytosed together with the ligand into the signal-sending cell. The cleavage is performed by a metalloproteinase encoded by kuzbanian (kuz) in Drosophila. The resulting intermediate Notch extracellular truncation (NEXT) fragment is cleaved by the γ-secretase complex, which contains Aph-1 and Presenilin (Psn). This S3-cleavage releases the Notch intracellular domain (NICD) into the cytosol from where it migrates into the nucleus to activate the target genes together with Suppressor of Hairless [Su(H)]. Previous work has shown that the constitutive activation of Notch observed in lgd mutant cells requires the activity of the γ-secretase complex (Morawa, 2015).

Notch traffics constitutively through the endosomal pathway to be degraded in the lysosom. Trafficking is initiated by endocytosis, which results in the formation of early endosomal vesicles. These vesicles fuse to form the early endosome (EE). Receptors destined for degradation remain in the ME, which eventually fuses with the lysosome where the cargo is degraded. During maturation of the endosome, receptors are concentrated in domains of its limiting membrane (LM) and are translocated into the lumen of the ME through pinching off this part as intraluminal vesicles (ILVs). The formation of ILVs achieves the separation of the NICDs of receptors from the cytosol. This step is important for the complete degradation of receptors as well as the termination of signalling through activated receptors (Morawa, 2015).

The events in the endosomal pathway are controlled by small GTPases, chiefly Rab5 and Rab7 (Huotari, 2011). Rab5 orchestrates events in the EEs, such as the initiation of ILV formation through recruitment of endosomal sorting complexes required for transport (ESCRT)-0, the first of five sequentially acting complexes, termed ESCRT-0-ESCRT-III and Vps4 (reviewed by Hurley, 2010). ESCRT-0 initiates the sequential recruitment of the complexes and concentrates ubiquitylated receptors in regions of ILV formation. ESCRT-0 consists of Hrs and Stam. The central component of the last acting ESCRT-III complex in Drosophila is encoded by shrub. It encodes the Drosophila ortholog of mammalian CHMP4 family proteins and yeast Snf7. Shrub is recruited to the LM of the MEs where it polymerises into filaments to perform the abscission of ILVs in concert with the Vps4 complex. As a result of the action of the ESCRT complexes, the ME contains an increasing number of ILVs and is called a multi-vesicular body (MVB). A recent paper provides evidence that suggests that LGD2 also interacts with CHMP4 during budding of HIV. Although the loss of the function of the ESCRT-I, -II and -III results in activation of the Notch and other signalling pathways in Drosophila, that of ESCRT-0 does not. An explanation for this puzzling fact is that ESCRT-0 performs an additional function that is required for activation in ESCRT-I, -II and -III mutants. This might consist of clustering of cargo at sites of ILV formation. The additional function of ESCRT-0 is also required for the activation of the Notch pathway in lgd mutant cells because concomitant loss of ESCRT-0 and Lgd activity suppresses Notch activation (Morawa, 2015).

It has been shown that Lgd physically interacts with the ESCRT-III core component Shrub (Troost, 2012). This interaction is important for the full activity of Shrub in vivo. Genetic experiments have revealed that the activation of Notch in lgd mutant cells requires the Rab7-mediated fusion of the ME with the lysosome (Schneider, 2013). Furthermore, every other manipulation that prevents fusion of the ME with the lysosome, such as loss of Rab7 function, suppresses activation of Notch in lgd mutant cells (Schneider, 2013). This requirement for fusion explains a paradox in the relationship between Lgd and Shrub: the activation of Notch in lgd mutant discs is suppressed if the activity of shrub is reduced to 50%, although their individual loss results in Notch activation (Troost, 2012). The paradox could be explained by the observation that the MEs of these cells lose association with Rab7 and fail to fuse with the lysosome (Schneider, 2013). Thus, Notch pathway activation fails because the endosomes are more dysfunctional than in lgd mutant cells (Morawa, 2015).

In Drosophila, loss of function of ESCRT-I, -II and -III in imaginal discs results in the ectopic activation of the Notch pathway. In addition, the activity of the Drosophila BMP pathway, called the Decapentaplegic (Dpp) pathway, is enhanced. Moreover, loss of ESCRT function causes loss of epithelial integrity. However, previous analysis in imaginal discs indicates that neither the activity of other major signalling pathways, such as the Dpp pathway, nor the epithelial integrity is affected by the loss of lgd function (Schneider, 2013). Hence, the phenotype of ESCRT mutants is more severe than that of loss-of-function lgd mutants despite their intimate relationship (Morawa, 2015).

The analysis of signalling pathways in lgd mutants described above was restricted to imaginal discs and indicated that only the Notch pathway is affected. It is not known whether these pathways are affected in other mutant tissues. The development of the oocyte of Drosophila is an excellent system to study cellular processes, such as cell signalling and, thus, to answer this question. During oogenesis the progenies of a germline stem cell (GSC) develops through distinct and recognisable stages into a mature egg in the ovariole. The ovariole is subdivided into an anterior germarium, which contains two kinds of stem cells and a posterior part that consists of a string of increasingly older egg chambers (ECs) with 16 germline cells (GCs) surrounded by a somatic follicle epithelium. The ECs bud off the germarium and mature upon their posterior migration. The GSCs are located at the anterior tip of the germarium and are surrounded by somatic cap cells, which form the niche required for their survival. The niche can accommodate 2-3 GSCs. These divide asymmetrically to give rise to another GSC and a cystoblast that begins to differentiate. The cystoblast undergoes four rounds of mitotic divisions to give rise to a cyst with 16 GCs. The cyst is encapsulated by somatic follicle cells (FCs) to generate the EC. The FCs are generated by two somatic stem cells located at the exit of the germarium. One GC in each EC will differentiate into the oocyte, whereas the other 15 differentiate into polyploid nurse cells (Morawa, 2015).

Several signals are required to maintain the GSC population. These are emitted at the tip of germarium by cap cells. The most important one is Dpp. Although anterior cap cells are probably responsible for the majority of the Dpp signal, the extent of its expression domain remains unclear. Dpp activates the co-receptor Thickveins (Tkv; type 1) and type 2 receptors on GSC. The result of Dpp signalling is the suppression of the expression of bag of marbles (bam) in the GSC. When a GSC divides, the daughter attached to the cap cell receives the Dpp signal, which suppresses bam expression and maintains the GSC fate. The non-contacting daughter cell does not receive sufficient signal and produces Bam, which causes its differentiation as a cystoblast. Thus, Bam silencing is the hallmark of asymmetry in the female germline of Drosophila. The short range of the Dpp signal is achieved through degradation of the activated Tkv receptor in the cystoblast by a specific mechanism that involves the kinase Fused (Fu) and the E3 ligase Smurf. If the mechanism is disturbed, for example, by inactivation of fu, the number of GSCs increases and an extra round of division of the cyst cells occurs, frequently giving rise to ECs with 32 instead of 16 GCs (Morawa, 2015).

One well-characterised event during oogenesis that is mediated through the Notch signalling pathway is the switch from the mitotic cycle to the endocycle in FCs of ECs, which occurs at between stage 6 and 7. This switch is triggered by a Dl signal from the GCs and initiates the differentiation of FCs through activation of Hindsight (Hnt) expression. It was previously shown that the loss of function of lgd in FCs results in precocious activation of the Notch pathway and expression of Hnt (Schneider, 2013). As in wing discs, the activation was independent of Kuz, and depended on Hrs and fusion of the ME with the lysosome (Schneider, 2013). Hence, the activation is caused by the same mechanism as in the wing disc (Morawa, 2015).

The consequence of loss of lgd function in the germline was investigated previously using the allele lgdd3. ECs were observed with more than 40 GCs, a feature characteristic of tumour suppressor mutants. However, the analysis was hampered by the uncertain nature of lgdd3 and the lack of appropriate markers (Morawa, 2015).

This study further investigated the function of lgd during oogenesis. Its loss causes activation of the Dpp pathway in addition to the Notch pathway in FCs. In the germline, loss of function of lgd causes an extra mitotic division that produces ECs with 32 GCs. In addition, it causes formation of more GSCs. Both phenotypes rely on ectopic activation of the Dpp. These results indicate that these phenotypes are caused by a failure to degrade Tkv. Moreover, the loss of function of shrub was found to result in a similar phenotype (Morawa, 2015).

The previously conducted analysis of the function of lgd was largely restricted to imaginal discs. It suggested that loss of lgd function specifically activates the Notch pathway. Moreover, the loss of lgd function in FCs results in activation of the Notch pathway in the same manner as in imaginal disc cells. The devotion of Lgd to the Notch pathway was puzzling, given that it also controls trafficking of components of several signalling pathways, such as Tkv. Lgd functionally interacts with Shrub, whose loss of function affects several signalling pathways in imaginal disc cells. Thus, it would be conceivable that these pathways are also activated in lgd mutant cells. This study shows that the Dpp pathway is ectopically activated upon loss of function of lgd in the ovary. This activation occurs in FCs as well as GCs. Only the Dpp, and not the Notch, pathway is activated in lgd mutant GCs. This observation is different from what has been reported for mutants of the ESCRT-II component Vps25, where Dpp signalling was enhanced, but the pathway not ectopically activated (Morawa, 2015).

This study failed to detect any phenotype that could be attributed to the observed ectopic activation of the Dpp pathway in lgd mutant FCs, but its ectopic activation in the mutant germline causes an extra round of cell division. Moreover, more GSCs were observed and expression of Bam-GFP was suppressed in a fraction of germaria. These findings suggest that the range of the Dpp signal is extended and, thus, maintains the GSC fate in more distantly located cells (Morawa, 2015).

It has been recently shown that the Dpp-activated form of the Tkv receptor is specifically degraded in cyst cells to suppress the ectopic activation of the pathway. This degradation through the endosomal pathway is mediated by a complex consisting of the E3 ligase Smurf and Fu, and restricts the activity of the pathway to GSCs in the cap cell niche. The loss of fu function causes a loss of degradation of the activated receptor in progenies of the GSCs and, thus, increases the range of the pathway in GCs. Hence, ectopic activation occurs cell autonomously, but is induced by the Dpp ligand. The ectopic activity induced by loss of fu function causes a variety of phenotypes, including egg chambers that contain 32 GCs, as was observed upon loss of lgd function. Loss of function of lgd has been shown to result in a defect in the degradation of Tkv and other cargo proteins, such as Notch and Dl, in FCs and GCs. This study also found that the range of the Dpp pathway is increased in the absence of lgd function. It is therefore likely that the activation of the pathway is caused by a general failure of degradation that also affects the degradation of the activated form of Tkv. In further support of this notion, genetic interactions were found between fu, lgd and shrub (Morawa, 2015).

It has been proposed that in lgd cells, transmembrane proteins destined to become degraded in the lysosome are not completely incorporated in ILVs of MEs (Schneider, 2013). This hypothesis has been strongly supported by recent work of the Schweisguth laboratory (Couturier, 2014). Consequently, a fraction of Notch and Tkv remains at the LM and their intracellular domains stay in contact with the cytosol. Activated Tkv in this fraction continues to signal as long as the ME exists. It is therefore proposed that it is the defect in incorporation of activated Tkv into ILVs that causes the ectopic activation of the Dpp pathway. In this scenario, defects that increase the lifetime of the lgd mutant ME, such as loss of Dmon1 function, enhances and prolongs the activity of the Dpp pathway. This is what was observed. The incorporation of transmembrane proteins into ILVs requires their previous ubiquitylation by E3 ligases. Hence, the loss of the function of the E3 ligase Fu, which normally ubiquitylates activated Tkv, also prevents incorporation of activated Tkv in ILVs (Morawa, 2015).

Loss of shrub function causes a similar defect to that of lgd. Shrub is the central element of the ESCRT-III complex, which is required for ILV scission and interacts physically with Lgd (Troost, 2012). Loss of shrub function is expected to result in all Tkv remaining on the LM. Thus, it was no surprise to see that loss of its function in the FCs also causes the activation of the Dpp pathway. More surprising was the finding that reduction of Shrub activity by only 50% is sufficient to initiate the activation of the Dpp pathway in the germline. Given that that phenotypes of shrub/+ cells are stronger than those of lgd mutant cells, it is likely that reduction of the activity of shrub by 50% causes a larger fraction of Tkv to remain on the LM than in lgd mutant cells. The phenotypes of shrub/+ cells are strongly enhanced by heterozygousity of fu, indicating that there is an intimate functional relationship and that, as in the case of lgd, a defect in Tkv degradation causes the activation of the Dpp pathway (Morawa, 2015).

Because of the similarity in the phenotype and their intimate relationship, it is puzzling to find an antagonistic relationship between shrub and lgd. However, this antagonism was observed only in respect to the formation of supernumerary GCs, but not in respect to formation of supernumerary dad-lacZ-positive GSCs. A similar complex situation was found for the relationship of shrub and lgd in imaginal discs, where the reduction of shrub activity suppressed the activation of the Notch pathway, but enhanced the morphological defect of endosomes in lgd cells (Troost, 2012). Finding explanation for this complex relationship will be key to understanding the function of Lgd. One possibility is that the function of the ME is disturbed more severely in the double mutants than in each single mutant. In agreement with this is the observation in the imaginal disc, where the MEs lost their association with Rab7 in shrub lgd/lgd+ cells (Morawa, 2015).

A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila

The apical extracellular matrix plays a central role in epithelial tube morphogenesis. In the Drosophila tracheal system, Serpentine (Serp), a secreted chitin deacetylase expressed by the tracheal cells plays a key role in regulating tube length. This study shows that the fly fat body, which is functionally equivalent to the mammalian liver, also contributes to tracheal morphogenesis. Serp is expressed by the fat body, and the secreted Serp is taken up by the tracheal cells and translocated to the lumen to functionally support normal tracheal development. This process is defective in rab9 and shrub/vps32 mutants and in wild-type embryos treated with a secretory pathway inhibitor, leading to an abundant accumulation of Serp in the fat body. Fat body-derived Serp reaches the tracheal lumen after establishment of epithelial barrier function and is retained in the lumen in a chitin synthase-dependent manner. These results thus reveal that the fat body, a mesodermal organ, actively contributes to tracheal development (Dong, 2014).

The coiled-coil protein Shrub controls neuronal morphogenesis in Drosophila

The diversity of neuronal cells, especially in the size and shape of their dendritic and axonal arborizations, is a striking feature of the mature nervous system. Dendritic branching is a complex process, and the underlying signaling mechanisms remain to be further defined at the mechanistic level. This paper reports the identification of shrub mutations that increased dendritic branching. Single-cell clones of shrub mutant dendritic arborization (DA) sensory neurons in Drosophila larvae showed ectopic dendritic and axonal branching, indicating a cell-autonomous function for shrub in neuronal morphogenesis. shrub encodes an evolutionarily conserved coiled-coil protein homologous to the yeast protein Snf7, a key component in the ESCRT-III (endosomal sorting complex required for transport) complex that is involved in the formation of endosomal compartments known as multivesicular bodies (MVBs). Mouse orthologs can substitute for Shrub in mutant Drosophila embryos and loss of Shrub function caused abnormal distribution of several early or late endosomal markers in DA sensory neurons. These findings demonstrate that the novel coiled-coil protein Shrub functions in the endosomal pathway and plays an essential role in neuronal morphogenesis (Sweeney, 2006).

The ESCRT complexes have been associated with sev- eral neurodegenerative diseases. The hereditary spastic paraplegia protein Spastin interacts with CHMP1B, an ESCRT-III-complex-related endosomal protein (Reid, 2005). A specific mutation within the CHMP2B gene, which encodes the ortholog of Vps2, another component of the ESCRT-III complex, was found in a Danish frontotemporal dementia family (Skibinski, 2007). Further understanding of the molecular and physiological consequences of defects in ESCRT complexes may offer new insights into age-dependent neurodegenerative disorders (Sweeney, 2006)


Search PubMed for articles about Drosophila Shrub

Babatz, F., Naffin, E. and Klambt, C. (2018). The Drosophila blood-brain barrier adapts to cell growth by unfolding of pre-existing septate junctions. Dev Cell 47(6): 697-710 e693. PubMed ID: 30482667

Couturier, L., Trylinski, M., Mazouni, K., Darnet, L. and Schweisguth, F. (2014). A fluorescent tagging approach in Drosophila reveals late endosomal trafficking of Notch and Sanpodo. J Cell Biol 207: 351-363. PubMed ID: 25365996

Daniel, E., Daude, M., Kolotuev, I., Charish, K., Auld, V. and Le Borgne, R. (2018). Coordination of septate junctions assembly and completion of cytokinesis in proliferative epithelial tissues. Curr Biol 28(9): 1380-1391 e1384. PubMed ID: 29706514

Dong, B., Kakihara, K., Otani, T., Wada, H. and Hayashi, S. (2013). Rab9 and retromer regulate retrograde trafficking of luminal protein required for epithelial tube length control. Nat Commun 4: 1358. PubMed ID: 23322046

Eikenes, A. H., Malerod, L., Christensen, A. L., Steen, C. B., Mathieu, J., Nezis, I. P., Liestol, K., Huynh, J. R., Stenmark, H. and Haglund, K. (2015). ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo. PLoS Genet 11: e1004904. PubMed ID: 25635693

Gallagher, C. M. and Knoblich, J. A. (2006). The conserved c2 domain protein lethal (2) giant discs regulates protein trafficking in Drosophila. Dev Cell 11: 641-653. PubMed ID: 17084357

Hadjighassem, M. R., Austin, M. C., Szewczyk, B., Daigle, M., Stockmeier, C. A. and Albert, P. R. (2009). Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression. Biol Psychiatry 66: 214-222. PubMed ID: 19423080

Henne, W. M., Buchkovich, N. J. and Emr, S. D. (2011). The ESCRT pathway. Dev Cell 21: 77-91. PubMed ID: 21763610

Hori, K., Sen, A., Kirchhausen, T. and Artavanis-Tsakonas, S. (2011). Synergy between the ESCRT-III complex and Deltex defines a ligand-independent Notch signal. J Cell Biol 195: 1005-1015. PubMed ID: 22162134

Huotari, J. and Helenius, A. (2011). Endosome maturation. EMBO J 30: 3481-3500. PubMed ID: 21878991

Hurley, J. H. (2010). The ESCRT complexes. Crit Rev Biochem Mol Biol 45: 463-487. PubMed ID: 20653365

Martinelli, N., Hartlieb, B., Usami, Y., Sabin, C., Dordor, A., Miguet, N., Avilov, S. V., Ribeiro, E. A., Jr., Gottlinger, H. and Weissenhorn, W. (2012). CC2D1A is a regulator of ESCRT-III CHMP4B. J Mol Biol 419: 75-88. PubMed ID: 22406677

Morawa, K. S., Schneider, M. and Klein, T. (2015). Lgd regulates the activity of the BMP/Dpp signalling pathway during Drosophila oogenesis. Development 142: 1325-1335. PubMed ID: 25804739

Nakamura, A., Naito, M., Tsuruo, T. and Fujita, N. (2008). Freud-1/Aki1, a novel PDK1-interacting protein, functions as a scaffold to activate the PDK1/Akt pathway in epidermal growth factor signaling. Mol Cell Biol 28: 5996-6009. PubMed ID: 18662999

Ou, X. M., Lemonde, S., Jafar-Nejad, H., Bown, C. D., Goto, A., Rogaeva, A. and Albert, P. R. (2003). Freud-1: A neuronal calcium-regulated repressor of the 5-HT1A receptor gene. J Neurosci 23: 7415-7425. PubMed ID: 12917378

Pannen, H., Rapp, T. and Klein, T. (2020). The ESCRT machinery regulates retromer dependent transcytosis of septate junction components in Drosophila. Elife 9. PubMed ID: 33377869

Reid, E., Connell, J., Edwards, T. L., Duley, S., Brown, S. E. and Sanderson, C. M. (2005). The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B. Hum Mol Genet 14: 19-38. PubMed ID: 15537668

Saksena, S., Wahlman, J., Teis, D., Johnson, A. E. and Emr, S. D. (2009). Functional reconstitution of ESCRT-III assembly and disassembly. Cell 136: 97-109. PubMed ID: 19135892

Sears, J. C. and Broadie, K. (2017). Fragile X Mental Retardation Protein regulates activity-dependent membrane trafficking and trans-synaptic signaling mediating synaptic remodeling. Front Mol Neurosci 10: 440. PubMed ID: 29375303

Skibinski, G., Parkinson, N. J., Brown, J. M., Chakrabarti, L., Lloyd, S. L., Hummerich, H., Nielsen, J. E., Hodges, J. R., Spillantini, M. G., Thusgaard, T., Brandner, S., Brun, A., Rossor, M. N., Gade, A., Johannsen, P., Sorensen, S. A., Gydesen, S., Fisher, E. M. and Collinge, J. (2005). Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nat Genet 37: 806-808. PubMed ID: 16041373

Sweeney, N. T., Brenman, J. E., Jan, Y. N. and Gao, F. B. (2006). The coiled-coil protein Shrub controls neuronal morphogenesis in Drosophila. Curr Biol 16: 1006-1011. PubMed ID: 16713958

Schneider, M., Troost, T., Grawe, F., Martinez-Arias, A. and Klein, T. (2013). Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome. J Cell Sci 126: 645-656. PubMed ID: 23178945

Steigemann, P., Wurzenberger, C., Schmitz, M. H., Held, M., Guizetti, J., Maar, S. and Gerlich, D. W. (2009). Aurora B-mediated abscission checkpoint protects against tetraploidization. Cell 136: 473-484. PubMed ID: 19203582

Troost, T., Jaeckel, S., Ohlenhard, N. and Klein, T. (2012). The tumour suppressor Lethal (2) giant discs is required for the function of the ESCRT-III component Shrub/CHMP4. J. Cell Sci. 125: 763-776. PubMed Citation: 22389409

Usami, Y., Popov, S., Weiss, E. R., Vriesema-Magnuson, C., Calistri, A. and Gottlinger, H. G. (2012). Regulation of CHMP4/ESCRT-III function in human immunodeficiency virus type 1 budding by CC2D1A. J Virol 86: 3746-3756. PubMed ID: 22258254

Wilkin, M., Tongngok, P., Gensch, N., Clemence, S., Motoki, M., Yamada, K., Hori, K., Taniguchi-Kanai, M., Franklin, E., Matsuno, K. and Baron, M. (2008). Drosophila HOPS and AP-3 complex genes are required for a Deltex-regulated activation of notch in the endosomal trafficking pathway. Dev Cell 15: 762-772. PubMed ID: 19000840

Yamazaki, Y., Palmer, L., Alexandre, C., Kakugawa, S., Beckett, K., Gaugue, I., Palmer, R. H. and Vincent, J. P. (2016). Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs. Nat Cell Biol 18(4): 451-457. PubMed ID: 26974662

Zhao, M., Li, X. D. and Chen, Z. (2010). CC2D1A, a DM14 and C2 domain protein, activates NF-kappaB through the canonical pathway. J Biol Chem 285: 24372-24380. PubMed ID: 20529849

Biological Overview

date revised: 12 September 2022

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