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

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

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Beaded of Goldschmidt, an Antimorphic Allele of Serrate, Encodes a Protein Lacking Transmembrane and Intracellular Domains

Genetics ◽

10.1093/genetics/145.2.359 ◽

1997 ◽

Vol 145 (2) ◽

pp. 359-374 ◽

Cited By ~ 2

Author(s):

Neil A Hukriede ◽

Robert J Fleming

Keyword(s):

Essential Gene ◽

Protein Product ◽

Homologous Region ◽

Wing Margin ◽

Loss Of Function ◽

Wild Type ◽

Rich Region ◽

Coding Regions ◽

Dna Lesion ◽

Intracellular Domains

Serrate (Ser) is an essential gene in Drosophila melanogaster best known for the Ser dominant (SerD) allele and its effects on wing development. Animals heterozygous or homozygous for (SerD) are viable and exhibit loss of wing margin tissue and associated bristles and hairs. The Beaded of Goldschmidt (BdG) allele of Ser, when heterozygous to wild type, will also produce animals exhibiting loss of wing margin material. However, animals homozygous for BdG exhibit a larval lethal phenotype comparable to animals homozygous for loss-of-function Ser alleles. BdG is a partial duplication of the Ser locus with a single 5′ Ser-homologous region and two distinct 3′ regions. Meiotic recombination between BdG and a wild-type Ser chromosome demonstrated that only one DNA lesion, caused by the insertion of a transposable roo element into the coding regions of the Ser transcript, appears capable of generating BdG phenotypes. Due to the roo insertion, the protein product is predicted to be prematurely truncated and lack an extracellular cysteine-rich region along with the transmembrane and intracellular domains found within the normal SERRATE (SER) protein. The loss of these protein domains apparently contributes to the antimorphic nature of this mutation.

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Efficient Cargo Sorting by ESCRT-I and the Subsequent Release of ESCRT-I from Multivesicular Bodies Requires the Subunit Mvb12

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0588 ◽

2007 ◽

Vol 18 (2) ◽

pp. 636-645 ◽

Cited By ~ 45

Author(s):

Matt Curtiss ◽

Charles Jones ◽

Markus Babst

Keyword(s):

Protein Complex ◽

Transmembrane Proteins ◽

Multivesicular Body ◽

Multivesicular Bodies ◽

Rapid Degradation ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Complex Functions ◽

Vacuolar Protein ◽

Cargo Sorting

The endosomal sorting complex required for transport (ESCRT)-I protein complex functions in recognition and sorting of ubiquitinated transmembrane proteins into multivesicular body (MVB) vesicles. It has been shown that ESCRT-I contains the vacuolar protein sorting (Vps) proteins Vps23, Vps28, and Vps37. We identified an additional subunit of yeast ESCRT-I called Mvb12, which seems to associate with ESCRT-I by binding to Vps37. Transient recruitment of ESCRT-I to MVBs results in the rapid degradation of Mvb12. In contrast to mutations in other ESCRT-I subunits, which result in strong defects in MVB cargo sorting, deletion of MVB12 resulted in only a partial sorting phenotype. This trafficking defect was fully suppressed by overexpression of the ESCRT-II complex. Mutations in MVB12 did not affect recruitment of ESCRT-I to MVBs, but they did result in delivery of ESCRT-I to the vacuolar lumen via the MVB pathway. Together, these observations suggest that Mvb12 may function in regulating the interactions of ESCRT-I with cargo and other proteins of the ESCRT machinery to efficiently coordinate cargo sorting and release of ESCRT-I from the MVB.

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Abrogating ALIX Interactions Results in Stuttering of the ESCRT Machinery

Viruses ◽

10.3390/v12091032 ◽

2020 ◽

Vol 12 (9) ◽

pp. 1032 ◽

Cited By ~ 1

Author(s):

Shilpa Gupta ◽

Mourad Bendjennat ◽

Saveez Saffarian

Keyword(s):

Binding Site ◽

Linear Model ◽

Residence Time ◽

Live Imaging ◽

Assembly Model ◽

Wild Type ◽

Cellular Membranes ◽

Virion Assembly ◽

Endosomal Sorting ◽

Escrt Machinery

Endosomal sorting complexes required for transport (ESCRT) proteins assemble on budding cellular membranes and catalyze their fission. Using live imaging of HIV virions budding from cells, we followed recruitment of ESCRT proteins ALIX, CHMP4B and VPS4. We report that the ESCRT proteins transiently co-localize with virions after completion of virion assembly for durations of 45 ± 30 s. We show that mutagenizing the YP domain of Gag which is the primary ALIX binding site or depleting ALIX from cells results in multiple recruitments of the full ESCRT machinery on the same virion (referred to as stuttering where the number of recruitments to the same virion >3). The stuttering recruitments are approximately 4 ± 3 min apart and have the same stoichiometry of ESCRTs and same residence time (45 ± 30 s) as the single recruitments in wild type interactions. Our observations suggest a role for ALIX during fission and question the linear model of ESCRT recruitment, suggesting instead a more complex co-assembly model.

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Effect of waxy (Low Amylose) on Fungal Infection of Sorghum Grain

Phytopathology ◽

10.1094/phyto-09-14-0255-r ◽

2015 ◽

Vol 105 (6) ◽

pp. 786-796 ◽

Cited By ~ 5

Author(s):

Deanna L. Funnell-Harris ◽

Scott E. Sattler ◽

Patrick M. O’Neill ◽

Kent M. Eskridge ◽

Jeffrey F. Pedersen

Keyword(s):

Fungal Infection ◽

Plant Introduction ◽

Starch Granules ◽

Loss Of Function ◽

Wild Type ◽

Starch Properties ◽

Alternaria Sp ◽

Fusarium Thapsinum ◽

Sorghum Grain ◽

Reduced Activity

Loss of function mutations in waxy, encoding granule bound starch synthase (GBSS) that synthesizes amylose, results in starch granules containing mostly amylopectin. Low amylose grain with altered starch properties has increased usability for feed, food, and grain-based ethanol. In sorghum, two classes of waxy (wx) alleles had been characterized for absence or presence of GBSS: wxa (GBSS−) and wxb (GBSS+, with reduced activity). Field-grown grain of wild-type; waxy, GBSS−; and waxy, GBSS+ plant introduction accessions were screened for fungal infection. Overall, results showed that waxy grains were not more susceptible than wild-type. GBSS− and wild-type grain had similar infection levels. However, height was a factor with waxy, GBSS+ lines: short accessions (wxb allele) were more susceptible than tall accessions (undescribed allele). In greenhouse experiments, grain from accessions and near-isogenic wxa, wxb, and wild-type lines were inoculated with Alternaria sp., Fusarium thapsinum, and Curvularia sorghina to analyze germination and seedling fitness. As a group, waxy lines were not more susceptible to these pathogens than wild-type, supporting field evaluations. After C. sorghina and F. thapsinum inoculations most waxy and wild-type lines had reduced emergence, survival, and seedling weights. These results are valuable for developing waxy hybrids with resistance to grain-infecting fungi.

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A non-canonical role for p27Kip1 in restricting proliferation of corneal endothelial cells during development

10.1101/721621 ◽

2019 ◽

Author(s):

Dennis M. Defoe ◽

Huiying Rao ◽

David J. Harris ◽

Preston D. Moore ◽

Jan Brocher ◽

...

Keyword(s):

Protein Function ◽

Corneal Endothelium ◽

Clustering Algorithm ◽

Critical Factor ◽

Cell Number ◽

Loss Of Function ◽

Wild Type ◽

Tissue Organization ◽

Density Based Clustering ◽

Mutant Cells

AbstractThe cell cycle regulator p27Kip1 is a critical factor controlling cell number in many lineages. While its anti-proliferative effects are well-established, the extent to which this is a result of its function as a cyclin-dependent kinase (CDK) inhibitor or through other known molecular interactions is not clear. To genetically dissect its role in the developing corneal endothelium, we examined mice harboring two loss-of-function alleles, a null allele (p27−) that abrogates all protein function and a knockin allele (p27CK−) that targets only its interaction with cyclins and CDKs. Whole-animal mutants, in which all cells are either homozygous knockout or knockin, exhibit identical proliferative increases (∼0.6-fold) compared with wild-type tissues. On the other hand, use of mosaic analysis with double markers (MADM) to produce infrequently-occurring clones of wild-type and mutant cells within the same tissue environment uncovers a roughly three- and six-fold expansion of individual p27CK−/CK− and p27−/− cells, respectively. Mosaicism also reveals distinct migration phenotypes, with p27−/− cells being highly restricted to their site of production and p27CK−/CK− cells more widely scattered within the endothelium. Using a density-based clustering algorithm to quantify dispersal of MADM-generated clones, a four-fold difference in aggregation is seen between the two types of mutant cells. Overall, our analysis reveals that, in developing mouse corneal endothelium, p27 regulates cell number by acting cell autonomously, both through its interactions with cyclins and CDKs and through a cyclin-CDK-independent mechanism(s). Combined with its parallel influence on cell motility, it constitutes a potent multi-functional effector mechanism with major impact on tissue organization.

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Role of SpoVG in Asymmetric Septation inBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.181.11.3392-3401.1999 ◽

1999 ◽

Vol 181 (11) ◽

pp. 3392-3401 ◽

Cited By ~ 41

Author(s):

Kiyoshi Matsuno ◽

Abraham L. Sonenshein

Keyword(s):

Cell Division ◽

Signal Transduction Pathway ◽

Negative Regulator ◽

Stage I ◽

Loss Of Function ◽

Wild Type ◽

Gene Coding ◽

Additional Defect ◽

Cell Division Inhibitor ◽

Mutant Cells

ABSTRACT Deletion of the citC gene, coding for isocitrate dehydrogenase, arrests sporulation of Bacillus subtilis at stage I after bipolar localization of the cell division protein FtsZ but before formation of the asymmetric septum. A spontaneous extragenic suppressor mutation that overcame the stage I block was found to map within the spoVG gene. The suppressing mutation and otherspoVG loss-of-function mutations enabled citCmutant cells to form asymmetric septa and to activate the forespore-specific sigma factor ςF. However, little induction of mother cell-specific, ςE-dependent sporulation genes was observed in a citC spoVG double mutant, indicating that there is an additional defect(s) in compartmentalized gene expression in the citC mutant. These other defects could be partially overcome by reducing the synthesis of citrate, by buffering the medium, or by adding excess MnCl2. Overexpression of the spoVG gene in wild-type cells significantly delayed ςF activation. Increased expression and stability of SpoVG in citC mutant cells may contribute to the citC mutant phenotype. Inactivation of the spoVG gene caused a population of otherwise wild-type cells to produce a small number of minicells during growth and caused sporulating cells to complete asymmetric septation more rapidly than normal. Unlike the case for inactivation of the cell division inhibitor gene minD, many of these minicells contained DNA and appeared only when the primary sporulation signal transduction pathway, the Spo0A phosphorelay, was active. These results suggest that SpoVG interferes with or is a negative regulator of the pathway leading to asymmetric septation.

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A unique mutation in the Enhancer of split gene complex affects the fates of the mystery cells in the developing Drosophila eye

Development ◽

10.1242/dev.115.1.89 ◽

1992 ◽

Vol 115 (1) ◽

pp. 89-101 ◽

Cited By ~ 10

Author(s):

J.A. Fischer-Vize ◽

P.D. Vize ◽

G.M. Rubin

Keyword(s):

Compound Eye ◽

Photoreceptor Cells ◽

Neural Cells ◽

Loss Of Function ◽

Wild Type ◽

Partial Loss ◽

Eye Disc ◽

Mutant Cells ◽

Enhancer Of Split

An unusual recessive allele of the Drosophila groucho gene, which encodes a transducin-like protein, affects the fates of specific cells in the eye disc. groucho is one of several transcription units in the Enhancer of split complex. Most groucho mutations are zygotic lethal due to the proliferation of embryonic neural cells at the expense of epidermal cells. In contrast, flies homozygous for the mutant allele described here, groBFP2, are viable but have abnormal eyes. The Drosophila compound eye is composed of several hundred identical facets, or ommatidia, each of which contains eight photoreceptor cells, R1-R8. In groBFP2 mutant retinas, most of the facets contain eight normally determined photoreceptor cells and one or two additional R-cells of the R3/4 subtype. The extra photoreceptors appear to arise from the mystery cells, which are part of the precluster that initiates the ommatidium, but do not normally become neurons. groBFP2 behaves as a partial loss-of-function mutant. Analysis of ommatidia mosaic for wild-type and groBFP2 mutant cells suggests that the focus of action of the groBFP2 mutation is outside of the photoreceptor cells. These results imply that one function of groucho is in a pathway whereby neuralization of the mystery cells is inhibited by other non-neural cells in the eye disc. In addition, determination of R3/4 photoreceptors usually requires contact with R2 and R5. Specification of the mystery cells as ectopic R3/4 subtype photoreceptors in groBFP2 mutant eye discs implies that induction by R2 or R5 is not absolutely necessary for R3/4 cell determination.

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The roles of the homeobox genes aristaless and Distal-less in patterning the legs and wings of Drosophila

Development ◽

10.1242/dev.125.22.4483 ◽

1998 ◽

Vol 125 (22) ◽

pp. 4483-4493 ◽

Cited By ~ 24

Author(s):

G. Campbell ◽

A. Tomlinson

Keyword(s):

Null Allele ◽

Normal Development ◽

Distal Tibia ◽

Homeobox Genes ◽

Clonal Analysis ◽

Loss Of Function ◽

Wild Type ◽

Adhesive Properties ◽

Wing Imaginal Discs ◽

Mutant Cells

In the leg and wing imaginal discs of Drosophila, the expression domains of the homeobox genes aristaless (al) and Distal-less (Dll) are defined by the secreted signaling molecules Wingless (Wg) and Decapentaplegic (Dpp). Here, the roles played by al and Dll in patterning the legs and wings have been investigated through loss of function studies. In the developing leg, al is expressed at the presumptive tip and a molecularly defined null allele of al reveals that its only function in patterning the leg appears to be to direct the growth and differentiation of the structures at the tip. In contrast, Dll has previously been shown to be required for the development of all of the leg more distal than the coxa. Dll protein can be detected in a central domain in leg discs throughout most of larval development, and in mature discs this domain corresponds to the distal-most region of the leg, the tarsus and the distal tibia. Clonal analysis reveals that late in development these are the only regions in which Dll function is required. However, earlier in development Dll is required in more proximal regions of the leg suggesting it is expressed at high levels in these cells early in development but not later. This reveals a correlation between a temporal requirement for Dll and position along the proximodistal axis; how this may relate to the generation of the P/D axis is discussed. Dll is required in the distal regions of the leg for the expression of tarsal-specific genes including al and bric-a-brac. Dll mutant cells in the leg sort out from wild-type cells suggesting one function of Dll here is to control adhesive properties of cells. Dll is also required for the normal development of the wing, primarily for the differentiation of the wing margin.

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The intracellular deletions of Delta and Serrate define dominant negative forms of the Drosophila Notch ligands

Development ◽

10.1242/dev.122.8.2465 ◽

1996 ◽

Vol 122 (8) ◽

pp. 2465-2474 ◽

Cited By ~ 11

Author(s):

X. Sun ◽

S. Artavanis-Tsakonas

Keyword(s):

Notch Pathway ◽

Notch Signalling ◽

Dominant Negative ◽

Negative Regulator ◽

Loss Of Function ◽

Wild Type ◽

Mutant Forms ◽

Intracellular Domains ◽

Notch Ligands ◽

Enhancer Of Split

We examined the function of the intracellular domains of the two known Drosophila Notch ligands, Delta and Serrate, by expressing wild-type and mutant forms in the developing Drosophila eye under the sevenless promoter. The expression of intracellularly truncated forms of either Delta (sev-DlTM) or Serrate (sev-SerTM) leads to extra photoreceptor phenotypes, similar to the eye phenotypes associated with loss-of-function mutations of either Notch or Delta. Consistent with the notion that the truncated ligands reduce. Notch signalling activity, the eye phenotypes of sev-DlTM and sev-SerTM are enhanced by loss-of-function mutations in the Notch pathway elements, Notch, Delta, mastermind, deltex and groucho, but are suppressed by a duplication of Delta or mutations in Hairless, a negative regulator of the pathway. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split m delta, a target of Notch signalling, is down-regulated by the truncated ligands highly expressed in neighbouring cells. We conclude that the truncated ligands act as antagonists of Notch signalling.

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Membrane manipulations by the ESCRT machinery

F1000Research ◽

10.12688/f1000research.6319.1 ◽

2015 ◽

Vol 4 ◽

pp. 516 ◽

Cited By ~ 6

Author(s):

Greg Odorizzi

Keyword(s):

Plasma Membrane ◽

Transmembrane Proteins ◽

Membrane Dynamics ◽

Endocytic Pathway ◽

Current Understanding ◽

Eukaryotic Cells ◽

Major Research ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Research Findings

The endosomal sorting complexes required for transport (ESCRTs) collectively comprise a machinery that was first known for its function in the degradation of transmembrane proteins in the endocytic pathway of eukaryotic cells. Since their discovery, however, ESCRTs have been recognized as playing important roles at the plasma membrane, which appears to be the original site of function for the ESCRT machinery. This article reviews some of the major research findings that have shaped our current understanding of how the ESCRT machinery controls membrane dynamics and considers new roles for the ESCRT machinery that might be driven by these mechanisms.

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