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1.  Association of CHMP4B and Autophagy with Micronuclei: Implications for Cataract Formation 
BioMed Research International  2014;2014:974393.
Autophagy is a mechanism of cellular self-degradation that is very important for cellular homeostasis and differentiation. Components of the endosomal sorting complex required for transport (ESCRT) machinery are required for endosomal sorting and also for autophagy and the completion of cytokinesis. Here we show that the ESCRT-III subunit CHMP4B not only localizes to normal cytokinetic bridges but also to chromosome bridges and micronuclei, the latter surrounded by lysosomes and autophagosomes. Moreover, CHMP4B can be co-immunoprecipitated with chromatin. Interestingly, a CHMP4B mutation associated with autosomal dominant posterior polar cataract abolishes the ability of CHMP4B to localize to micronuclei. We propose that CHMP4B, through its association with chromatin, may participate in the autophagolysosomal degradation of micronuclei and other extranuclear chromatin. This may have implications for DNA degradation during lens cell differentiation, thus potentially protecting lens cells from cataract development.
doi:10.1155/2014/974393
PMCID: PMC3967805  PMID: 24741567
2.  Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation 
The Journal of Cell Biology  2013;202(2):331-349.
SNX18 promotes autophagosome formation by remodeling membranes and providing membrane to forming autophagosomes.
The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.
doi:10.1083/jcb.201205129
PMCID: PMC3718966  PMID: 23878278
3.  A ZO-1/α5β1-Integrin Complex Regulates Cytokinesis Downstream of PKCε in NCI-H460 Cells Plated on Fibronectin 
PLoS ONE  2013;8(8):e70696.
Recently, we demonstrated that integrin adhesion to the extracellular matrix at the cleavage furrow is essential for cytokinesis of adherent cells. Here, we report that tight junction protein ZO-1 (Zonula Occludens-1) is required for successful cytokinesis in NCI-H460 cells plated on fibronectin. This function of ZO-1 involves interaction with the cytoplasmic domain of α5-integrin to facilitate recruitment of active fibronectin-binding integrins to the base of the cleavage furrow. In the absence of ZO-1, or a functional ZO-1/α5β1-integrin complex, proper actin-dependent constriction between daughter cells is impaired and cells fail cytokinesis. Super-resolution microscopy reveals that in ZO-1 depleted cells the furrow becomes delocalized from the matrix. We also show that PKCε-dependent phosphorylation at Serine168 is required for ZO-1 localization to the furrow and successful cell division. Altogether, our results identify a novel regulatory pathway involving the interplay between ZO-1, α5-integrin and PKCε in the late stages of mammalian cell division.
doi:10.1371/journal.pone.0070696
PMCID: PMC3742740  PMID: 23967087
4.  The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport 
Journal of cell science  2006;119(Pt 19):3944-3957.
The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2]. A shared feature of fab1Δ yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P2, in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.
doi:10.1242/jcs.03153
PMCID: PMC1904490  PMID: 16954148
PIKfyve; Fab1p; Early endosome; Phosphatidylinositol (3,5)-bisphosphate; Endosomal sorting
5.  The Rabs: A family at the root of metazoan evolution 
BMC Biology  2012;10:68.
Eukaryotic cells are distinguished by their compartmentalization into membrane-enclosed organelles that exchange membranes and content in a highly ordered manner. Central in defining membrane identity are the Rabs, a large family of small GTPases that localize to distinct membranes and recruit specific regulators of membrane traffic. Two recent papers, including one by Klöpper et al. in BMC Biology, present phylogenomic evidence that the Rab repertoire was established very early in eukaryotic evolution, and correlates with interspecies variations in organelles.
See research article http://www.biomedcentral.com/1741-7007/10/71
doi:10.1186/1741-7007-10-68
PMCID: PMC3414739  PMID: 22873178
6.  Molecular Mechanisms of Ubiquitin-Dependent Membrane Traffic 
Annual Review of Biophysics  2011;40:119-142.
Over the past fourteen years, ubiquitination has emerged as a centrally important mechanism governing the subcellular trafficking of proteins. Ubiquitination, interaction with sorting factors that contain ubiquitin binding domains, and finally deubiquitination govern the itineraries of cargo proteins that include yeast carboxypeptidase S, the epithelial sodium channel ENaC, and epidermal growth factor receptor. The molecular structures and mechanisms of the paradigmatic HECT and RING domain ubiquitin ligases, JAMM and USP domain deubiquitinating enzymes, and numerous ubiquitin binding domains involved in these pathways, have been worked out in recent years and are described.
doi:10.1146/annurev-biophys-042910-155404
PMCID: PMC3272705  PMID: 21332354
Lysosome; vacuole; yeast genetics; EGF; EGF receptor; growth factor receptor; epithelial sodium channel; ENaC; yeast genetics; carboxypeptidase S; protein structure; crystal structure; ubiquitin; RING domain; HECT domain; JAMM domain; isopeptidase; ubiquitin ligase; deubiquitinating enzyme; ubiquitin binding domain; ESCRT complex
7.  Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1 
Biochemical Journal  2011;441(Pt 1):399-406.
Beclin 1, a subunit of the class III phosphatidylinositol 3-kinase complex, is a tumour suppressor with a central role in endocytic trafficking, cytokinesis and the cross-regulation between autophagy and apoptosis. Interestingly, not only reduced expression but also overexpression of Beclin 1 is correlated with cancer development and metastasis. Thus it seems necessary for the cell to balance the protein levels of Beclin 1. In the present study we describe a regulatory link between Beclin 1 and the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). We establish Nedd4 as a novel binding partner of Beclin 1 and demonstrate that Nedd4 polyubiquitinates Beclin 1 with Lys11- and Lys63-linked chains. Importantly, Nedd4 expression controls the stability of Beclin 1, and depletion of the Beclin 1-interacting protein VPS34 causes Nedd4-mediated proteasomal degradation of Beclin 1 via Lys11-linked polyubiquitin chains. Beclin 1 is thus the first tumour suppressor reported to be controlled by Lys11-linked polyubiquitination.
doi:10.1042/BJ20111424
PMCID: PMC3242507  PMID: 21936852
cancer; homologous with E6-associated protein C-terminus (HECT); neural-precursor-cell-expressed developmentally down-regulated 4 (Nedd4); phosphoinositide; ubiquitination; VPS34; AIP4, atrophin-interacting protein 4; BH3, Bcl-2 homology 3; CCD, coiled-coil domain; CHX, cycloheximide; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; ECD, evolutionarily conserved domain; GST, glutathione transferase; HA, haemagglutinin; HECT, homologous with E6-associated protein C-terminus; HRP, horseradish peroxidase; Nedd4, neural-precursor-cell-expressed developmentally down-regulated 4; PI3K-III, class III PI 3-kinase; siRNA, small interfering RNA; TRAF6, tumour-necrosis-factor-receptor-associated factor 6; Ub, ubiquitin
8.  Autophagy as a trigger for cell death 
Autophagy  2010;6(8):1214-1215.
Autophagy has been reported to contribute to cell death, but the underlying mechanisms remain largely unknown and controversial. We have been studying oogenesis in Drosophila melanogaster as a model system to understand the interplay between autophagy and cell death. Using a novel autophagy reporter we found that autophagy occurs during developmental cell death of nurse cells in late oogenesis. Genetic inhibition of autophagy-related genes atg1, atg13 and vps34 results in late-stage egg chambers containing persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor of apoptosis is a novel mechanism of triggering cell death.
doi:10.4161/auto.6.8.13694
PMCID: PMC3973654  PMID: 20935512
apoptosis; autophagy; Drosophila; IAPs; nurse cells; oogenesis; programmed cell death
9.  The selective macroautophagic degradation of aggregated proteins requires the phosphatidylinositol 3-phosphate binding protein Alfy 
Molecular cell  2010;38(2):265-279.
There is growing evidence that macroautophagic cargo is not limited to bulk cytosol in response to starvation, and can occur selectively for substrates including aggregated proteins. It remains unclear, however, if starvation-induced and selective macroautophagy share identical adapter molecules to capture their cargo. Here we report that Alfy, a phosphatidylinositol 3-phosphate binding protein, is central to the selective elimination of aggregated proteins. We report that the loss of Alfy inhibits the clearance of inclusions, with little to no effect on the starvation response. Alfy is recruited to intracellular inclusions and scaffolds a complex between p62(SQSTM1)-positive proteins and the autophagic effectors Atg5, Atg12, Atg16L and LC3. Alfy overexpression leads to elimination of aggregates in an Atg5-dependent manner, and likewise, to protection in a neuronal and Drosophila model of polyglutamine toxicity. We propose that Alfy plays a key role in selective macroautophagy, by bridging cargo to the molecular machinery that builds autophagosomes.
doi:10.1016/j.molcel.2010.04.007
PMCID: PMC2867245  PMID: 20417604
10.  A Tumor-Associated Mutation of FYVE-CENT Prevents Its Interaction with Beclin 1 and Interferes with Cytokinesis 
PLoS ONE  2011;6(3):e17086.
The tumor suppressor activity of Beclin 1 (BECN1), a subunit of class III phosphatidylinositol 3-kinase complex, has been attributed to its regulation of apoptosis and autophagy. Here, we identify FYVE-CENT (ZFYVE26), a phosphatidylinositol 3-phosphate binding protein important for cytokinesis, as a novel interacting protein of Beclin 1. A mutation in FYVE-CENT (R1945Q) associated with breast cancer abolished the interaction between FYVE-CENT and Beclin 1, and reduced the localization of these proteins at the intercellular bridge during cytokinesis. Breast cancer cells containing the FYVE-CENT R1945Q mutation displayed a significant increase in cytokinetic profiles and bi - multinuclear phenotype. Both Beclin 1 and FYVE-CENT were found to be downregulated in advanced breast cancers. These findings suggest a positive feedback loop for recruitment of FYVE-CENT and Beclin 1 to the intercellular bridge during cytokinesis, and reveal a novel potential tumor suppressor mechanism for Beclin 1.
doi:10.1371/journal.pone.0017086
PMCID: PMC3063775  PMID: 21455500
11.  Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis 
The Journal of Cell Biology  2010;190(4):523-531.
Blocking autophagy protects the apoptosis inhibitor dBruce from destruction and promotes nurse cell survival in developing egg chambers.
Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.
doi:10.1083/jcb.201002035
PMCID: PMC2928014  PMID: 20713604
12.  Structure and functions of stable intercellular bridges formed by incomplete cytokinesis during development 
Cytokinesis, the final step of cell division, normally proceeds to completion in living organisms, so that daughter cells physically separate by abscission. In certain tissues and developmental stages, on the other hand, the cytokinesis process is incomplete, giving rise to cells interconnected in syncytia by stable intercellular bridges. This evolutionarily conserved physiological process occurs in the female and male germline in species ranging from insects to humans, and has also been observed in some somatic tissues in invertebrates. Stable intercellular bridges have fascinated cell biologists ever since they were first described more than 50 years ago, and even though substantial progress has been made concerning their ultrastructure and molecular composition, much remains to be understood about their biological functions. Another major question is by which mechanisms complete versus incomplete cytokinesis is determined. In this mini-review we will try to give an overview of the current knowledge about the structure, composition and functions of stable intercellular bridges, and discuss recent insights into the molecular control of the incomplete cytokinesis process.
doi:10.4161/cib.4.1.13550
PMCID: PMC3073259  PMID: 21509167
incomplete cytokinesis; stable intercellular bridge; cytokinesis; intercellular communication; ring canal; oogenesis; spermatogenesis; anillin; MKLP1; Pav-Klp; cindr
13.  Ubiquitination of α-integrin cytoplasmic tails 
Recent findings have shown that ubiquitination is involved in regulating several proteins required for cell adhesion and migration. We showed that α5 integrin is ubiquitinated at its cytoplasmic lysines in response to fibronectin binding, and that this is required for its sorting to lysosomes together with fibronectin. Here we speculate whether other α integrin tails may also be ubiquitinated, and discuss the significance of ubiquitin linkages in the regulation of cell adhesion and migration.
doi:10.4161/cib.3.6.13176
PMCID: PMC3038070  PMID: 21331246
integrin; ubiquitin; cell migration; endocytosis; lysosome
14.  Self-eating from an ER-associated cup 
The Journal of Cell Biology  2008;182(4):621-622.
Since the first morphological description of autophagosomes in the early 1960s, two critical questions have been a matter of intense investigation and debate: what is the origin of the autophagosomal membrane and how is it formed? A study by Axe et al. (E.L. Axe, S.A. Walker, M. Manifava, P. Chandra, H.L. Roderick, A. Habermann, G. Griffiths, and N.T. Ktistakis. 2008. J. Cell Biol. 182:685–701) provides evidence that cup-shaped protrusions from the endoplasmic reticulum, named omegasomes, serve as platforms for autophagosome biogenesis in mammalian cells.
doi:10.1083/jcb.200807061
PMCID: PMC2518700  PMID: 18725534
15.  Disruption of Vps4 and JNK Function in Drosophila Causes Tumour Growth 
PLoS ONE  2009;4(2):e4354.
Several regulators of endocytic trafficking have recently been identified as tumour suppressors in Drosophila. These include components of the endosomal sorting complex required for transport (ESCRT) machinery. Disruption of subunits of ESCRT-I and –II leads to cell-autonomous endosomal accumulation of ubiquitinated receptors, loss of apicobasal polarity and epithelial integrity, and increased cell death. Here we report that disruption of the ATPase dVps4, the most downstream component of the ESCRT machinery, causes the same array of cellular phenotypes. We find that loss of epithelial integrity and increased apoptosis, but not loss of cell polarity, require the activation of JNK signalling. Abrogation of JNK signalling prevents apoptosis in dVps4 deficient cells. Indeed double deficiency in dVps4 and JNK signalling leads to the formation of neoplastic tumours. We conclude that dvps4 is a tumour suppressor in Drosophila and that JNK is central to the cell-autonomous phenotypes of ESCRT-deficient cells.
doi:10.1371/journal.pone.0004354
PMCID: PMC2632753  PMID: 19194501
16.  Regulation of Early Endosomal Entry by the Drosophila Tumor Suppressors Rabenosyn and Vps45 
Molecular Biology of the Cell  2008;19(10):4167-4176.
The small GTPase Rab5 has emerged as an important regulator of animal development, and it is essential for endocytic trafficking. However, the mechanisms that link Rab5 activation to cargo entry into early endosomes remain unclear. We show here that Drosophila Rabenosyn (Rbsn) is a Rab5 effector that bridges an interaction between Rab5 and the Sec1/Munc18-family protein Vps45, and we further identify the syntaxin Avalanche (Avl) as a target for Vps45 activity. Rbsn and Vps45, like Avl and Rab5, are specifically localized to early endosomes and are required for endocytosis. Ultrastructural analysis of rbsn, Vps45, avl, and Rab5 null mutant cells, which show identical defects, demonstrates that all four proteins are required for vesicle fusion to form early endosomes. These defects lead to loss of epithelial polarity in mutant tissues, which overproliferate to form neoplastic tumors. This work represents the first characterization of a Rab5 effector as a tumor suppressor, and it provides in vivo evidence for a Rbsn–Vps45 complex on early endosomes that links Rab5 to the SNARE fusion machinery.
doi:10.1091/mbc.E08-07-0716
PMCID: PMC2555928  PMID: 18685079
17.  Ubc4/5 and c-Cbl Continue to Ubiquitinate EGF Receptor after Internalization to Facilitate Polyubiquitination and Degradation 
Molecular Biology of the Cell  2008;19(8):3454-3462.
c-Cbl is the E3 ubiquitin ligase that ubiquitinates the epidermal growth factor (EGF) receptor (EGFR). On the basis of localization, knockdown, and in vitro activity analyses, we have identified the E2 ubiquitin-conjugating enzyme that cooperates with c-Cbl as Ubc4/5. Upon EGF stimulation, both Ubc4/5 and c-Cbl were relocated to the plasma membrane and then to Hrs-positive endosomes, strongly suggesting that EGFR continues to be ubiquitinated after internalization. Our time-course experiment showed that EGFR undergoes polyubiquitination, which seemed to be facilitated during the transport to Hrs-positive endosomes. Use of a conjugation-defective ubiquitin mutant suggested that receptor polyubiquitination is required for efficient interaction with Hrs and subsequent sorting to lysosomes. Abrupt inhibition of the EGFR kinase activity resulted in dissociation of c-Cbl from EGFR. Concomitantly, EGFR was rapidly deubiquitinated and its degradation was delayed. We propose that sustained tyrosine phosphorylation of EGFR facilitates its polyubiquitination in endosomes and counteracts rapid deubiquitination, thereby ensuring Hrs-dependent lysosomal sorting.
doi:10.1091/mbc.E07-10-0988
PMCID: PMC2488299  PMID: 18508924
18.  Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain 
The Journal of Cell Biology  2008;180(6):1065-1071.
p62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.
doi:10.1083/jcb.200711108
PMCID: PMC2290837  PMID: 18347073
19.  An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor 
The Journal of Cell Biology  2008;180(6):1205-1218.
Down-regulation of activated and ubiquitinated growth factor (GF) receptors by endocytosis and subsequent lysosomal degradation ensures attenuation of GF signaling. The ubiquitin-binding adaptor protein Eps15 (epidermal growth factor receptor [EGFR] pathway substrate 15) functions in endocytosis of such receptors. Here, we identify an Eps15 isoform, Eps15b, and demonstrate its expression in human cells and conservation across vertebrate species. Although both Eps15 and Eps15b interact with the endosomal sorting protein Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) in vitro, we find that Hrs specifically binds Eps15b in vivo (whereas adaptor protein 2 preferentially interacts with Eps15). Although Eps15 mainly localizes to clathrin-coated pits at the plasma membrane, Eps15b localizes to Hrs-positive microdomains on endosomes. Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect. Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR. These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.
doi:10.1083/jcb.200708115
PMCID: PMC2373575  PMID: 18362181
20.  Mechanisms and functions of endocytosis 
The Journal of Cell Biology  2008;180(1):7-11.
A recent EMBO-FEBS workshop entitled Endocytic Systems: Mechanism and Function, organized by Howard Riezman in Villars-sur-Ollon (Switzerland), showcased the multifaceted approaches and model systems used to study endocytosis. The meeting revealed how endocytosis controls multiple aspects of biology, ranging from development to immunity and neurotransmission.
doi:10.1083/jcb.200711073
PMCID: PMC2213624  PMID: 18195098
21.  Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease 
The Journal of Cell Biology  2007;179(3):485-500.
The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations.
doi:10.1083/jcb.200702115
PMCID: PMC2064794  PMID: 17984323
22.  The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport 
Journal of cell science  2006;119(Pt 19):3944-3957.
The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2]. A shared feature of fab1Δ yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P2, in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.
doi:10.1242/jcs.03153
PMCID: PMC1904490  PMID: 16954148
PIKfyve; Fab1p; Early endosome; Phosphatidylinositol (3,5)-bisphosphate; Endosomal sorting
23.  Fab1 Phosphatidylinositol 3-Phosphate 5-Kinase Controls Trafficking but Not Silencing of Endocytosed Receptors 
Molecular Biology of the Cell  2006;17(9):3989-4001.
The trafficking of endocytosed receptors through phosphatidylinositol 3-phosphate [PtdIns(3)P]-containing endosomes is thought to attenuate their signaling. Here, we show that the PtdIns(3)P 5-kinase Fab1/PIKfyve controls trafficking but not silencing of endocytosed receptors. Drosophila fab1 mutants contain undetectable phosphatidylinositol 3,5-bisphosphate levels, show profound increases in cell and organ size, and die at the pupal stage. Mutant larvae contain highly enlarged multivesicular bodies and late endosomes that are inefficiently acidified. Clones of fab1 mutant cells accumulate Wingless and Notch, similarly to cells lacking Hrs, Vps25, and Tsg101, components of the endosomal sorting machinery for ubiquitinated membrane proteins. However, whereas hrs, vps25, and tsg101 mutant cell clones accumulate ubiquitinated cargo, this is not the case with fab1 mutants. Even though endocytic receptor trafficking is impaired in fab1 mutants, Notch, Wingless, and Dpp signaling is unaffected. We conclude that Fab1, despite its importance for endosomal functions, is not required for receptor silencing. This is consistent with the possibility that Fab1 functions at a late stage in endocytic receptor trafficking, at a point when signal termination has occurred.
doi:10.1091/mbc.E06-03-0239
PMCID: PMC1556381  PMID: 16837550
24.  The ESCRT-III Subunit hVps24 Is Required for Degradation but Not Silencing of the Epidermal Growth Factor Receptor 
Molecular Biology of the Cell  2006;17(6):2513-2523.
The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.
doi:10.1091/mbc.E05-10-0915
PMCID: PMC1474783  PMID: 16554368
25.  p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death 
The Journal of Cell Biology  2005;171(4):603-614.
Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.
doi:10.1083/jcb.200507002
PMCID: PMC2171557  PMID: 16286508

Results 1-25 (35)