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1.  EGF receptor trafficking: consequences for signaling and cancer 
Trends in Cell Biology  2014;24(1):26-34.
Highlights
•EGF receptor endocytic traffic can regulate signaling and cell survival.•Signaling from activated EGFR occurs at the endosome as well as the cell surface.•Endocytosis can have positive and negative effects on signaling and tumorigenesis.•EGFR traffic promoted by antineoplastic therapy is important in tumor resistance.
The ligand-stimulated epidermal growth factor receptor (EGFR) has been extensively studied in the analysis of molecular mechanisms regulating endocytic traffic and the role of that traffic in signal transduction. Although such studies have largely focused on mitogenic signaling and dysregulated traffic in tumorigenesis, there is growing interest in the potential role of EGFR traffic in cell survival and the consequent response to cancer therapy. Here we review recent advances in our understanding of molecular mechanisms regulating ligand-stimulated EGFR activation, internalization, and post-endocytic sorting. The role of EGFR overexpression/mutation and new modulators of EGFR traffic in cancer and the response to cancer therapeutics are also discussed. Finally, we speculate on the relationship between EGFR traffic and cell survival.
doi:10.1016/j.tcb.2013.11.002
PMCID: PMC3884125  PMID: 24295852
epidermal growth factor receptor (EGFR); endocytosis; trafficking; ubiquitination; oncogenes; antineoplastic therapy
2.  Expression of OA1 limits the fusion of a subset of MVBs with lysosomes – a mechanism potentially involved in the initial biogenesis of melanosomes 
Journal of Cell Science  2013;126(22):5143-5152.
Summary
Multivesicular endosomes/bodies (MVBs) deliver proteins, such as activated EGF receptor (EGFR), to the lysosome for degradation, and, in pigmented cells, MVBs containing PMEL are an initial stage in melanosome biogenesis. The mechanisms regulating numbers and fate of different populations of MVB are unclear. Here, we focus on the role of the G-protein-coupled receptor OA1 (also known as GPR143), which is expressed exclusively in pigmented cells and mutations in which cause the most common type of ocular albinism. When exogenously expressing PMEL, HeLa cells have been shown to form MVBs resembling early stage melanosomes. To focus on the role of OA1 in the initial stages of melanosome biogenesis we take advantage of the absence of the later stages of melanosome maturation in HeLa cells to determine whether OA1 activity can regulate MVB number and fate. Expression of wild-type but not OA1 mutants carrying inactivating mutations or deletions causes MVB numbers to increase. Whereas OA1 expression has no effect on delivery of EGFR-containing MVBs to the lysosome, it inhibits the lysosomal delivery of PMEL and PMEL-containing MVBs accumulate. We propose that OA1 activity delays delivery of PMEL-containing MVBs to the lysosome to allow time for melanin synthesis and commitment to melanosome biogenesis.
doi:10.1242/jcs.128561
PMCID: PMC3828590  PMID: 24006264
Lysosomes; Multivesicular bodies; OA1
3.  Melanosome Maturation Defect in Rab38-deficient Retinal Pigment Epithelium Results in Instability of Immature Melanosomes during Transient Melanogenesis 
Molecular Biology of the Cell  2007;18(10):3914-3927.
Pathways of melanosome biogenesis in retinal pigment epithelial (RPE) cells have received less attention than those of skin melanocytes. Although the bulk of melanin synthesis in RPE cells occurs embryonically, it is not clear whether adult RPE cells continue to produce melanosomes. Here, we show that progression from pmel17-positive premelanosomes to tyrosinase-positive mature melanosomes in the RPE is largely complete before birth. Loss of functional Rab38 in the “chocolate” (cht) mouse causes dramatically reduced numbers of melanosomes in adult RPE, in contrast to the mild phenotype previously shown in skin melanocytes. Choroidal melanocytes in cht mice also have reduced melanosome numbers, but a continuing low level of melanosome biogenesis gradually overcomes the defect, unlike in the RPE. Partial compensation by Rab32 that occurs in skin melanocytes is less effective in the RPE, presumably because of the short time window for melanosome biogenesis. In cht RPE, premelanosomes form but delivery of tyrosinase is impaired. Premelanosomes that fail to deposit melanin are unstable in both cht and tyrosinase-deficient RPE. Together with the high levels of cathepsin D in immature melanosomes of the RPE, our results suggest that melanin deposition may protect the maturing melanosome from the activity of lumenal acid hydrolases.
doi:10.1091/mbc.E07-03-0268
PMCID: PMC1995718  PMID: 17671165
4.  Correction: Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium 
PLoS ONE  2013;8(5):10.1371/annotation/83a88285-e6a0-41fb-ae67-4315c21e5090.
doi:10.1371/annotation/83a88285-e6a0-41fb-ae67-4315c21e5090
PMCID: PMC3654017
5.  Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium 
PLoS ONE  2013;8(2):e57769.
The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (ChmFlox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the ChmFlox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.
doi:10.1371/journal.pone.0057769
PMCID: PMC3584022  PMID: 23460904
6.  The role of EGF receptor ubiquitination in regulating its intracellular traffic 
Traffic (Copenhagen, Denmark)  2011;13(2):329-337.
Progression of activated EGF receptor (EGFR) through the endocytic pathway regulates EGFR signaling. Here we show that a non-ubiquitinated EGFR mutant, unable to bind the endosomal sorting complex required for transport (ESCRT) component, Hrs, is not efficiently targeted onto intraluminal vesicles (ILVs) of multivesicular endosomes/bodies (MVBs). Moreover, ubiquitination and ESCRT engagement of activated EGFR is required for EGF-stimulated ILV formation. Non-ubiquitinated EGFRs enter clathrin-coated tubules emanating from MVBs and show enhanced recycling to the plasma membrane, compared to wild type EGFR.
doi:10.1111/j.1600-0854.2011.01305.x
PMCID: PMC3261333  PMID: 22017370
EGF receptor; multivesicular bodies; intralumenal vesicles; recycling; ubiquitination
7.  Methamphetamine-induced nitric oxide promotes vesicular transport in blood–brain barrier endothelial cells 
Neuropharmacology  2013;65(C):74-82.
Methamphetamine's (METH) neurotoxicity is thought to be in part due to its ability to induce blood–brain barrier (BBB) dysfunction. Here, we investigated the effect of METH on barrier properties of cultured rat primary brain microvascular endothelial cells (BMVECs). Transendothelial flux doubled in response to METH, irrespective of the size of tracer used. At the same time, transendothelial electrical resistance was unchanged as was the ultrastructural appearance of inter-endothelial junctions and the distribution of key junction proteins, suggesting that METH promoted vesicular but not junctional transport. Indeed, METH significantly increased uptake of horseradish peroxidase into vesicular structures. METH also enhanced transendothelial migration of lymphocytes indicating that the endothelial barrier against both molecules and cells was compromised. Barrier breakdown was only observed in response to METH at low micromolar concentrations, with enhanced vesicular uptake peaking at 1 μM METH. The BMVEC response to METH also involved rapid activation of endothelial nitric oxide synthase and its inhibition abrogated METH-induced permeability and lymphocyte migration, indicating that nitric oxide was a key mediator of BBB disruption in response to METH. This study underlines the key role of nitric oxide in BBB function and describes a novel mechanism of drug-induced fluid-phase transcytosis at the BBB.
Highlights
► A mechanism for methamphetamine-induced blood–brain barrier disruption is proposed. ► Effect of methamphetamine in brain microvascular endothelial cells was studied. ► Methamphetamine induces fluid-phase endocytosis (pinocytosis). ► Methamphetamine also enhances inflammatory lymphocyte transmigration. ► Both effects are mediated by endothelial nitric oxide synthase.
doi:10.1016/j.neuropharm.2012.08.021
PMCID: PMC3546162  PMID: 22960442
Methamphetamine; Fluid-phase transcytosis; Blood–brain barrier; Transendothelial leukocyte migration; Nitric oxide
8.  Trans-endocytosis of CD80 and CD86: a molecular basis for the cell extrinsic function of CTLA-4 ** 
Science (New York, N.Y.)  2011;332(6029):600-603.
CTLA-4 is an essential negative regulator of T cell immune responses whose mechanism of action is the subject of debate. CTLA-4 also shares two ligands (CD80 and CD86) with a stimulatory receptor, CD28. Here we show that CTLA-4 can capture its ligands from opposing cells by a process of trans-endocytosis. Following removal, these costimulatory ligands are degraded inside CTLA-4-expressing cells resulting in impaired costimulation via CD28. Acquisition of CD86 from antigen presenting cells is stimulated by TCR engagement and observed in vitro and in vivo. These data reveal a mechanism of immune regulation whereby CTLA-4 acts as an effector molecule to inhibit CD28 costimulation by the cell-extrinsic depletion of ligands, accounting for many of the known features of the CD28-CTLA-4 system.
doi:10.1126/science.1202947
PMCID: PMC3198051  PMID: 21474713
CTLA-4; CD86; T cell; dendritic cell; suppression
9.  Retinal Pigment Epithelium Defects Accelerate Photoreceptor Degeneration in Cell Type–Specific Knockout Mouse Models of Choroideremia 
In this study, the authors provide insight into the pathogenesis of choroideremia, which is caused by the disruption of intracellular vesicular transport. They also touch on other issues, such as the photoreceptor-RPE relationship and aging of the RPE.
Purpose.
Choroideremia (CHM) is a progressive X-linked degeneration of three ocular layers (photoreceptors, retinal pigment epithelium, and choroid), with a complex and still largely unclear pathogenesis. To investigate the pathophysiology of CHM, the authors engineered mice with a cell type–specific Chm/Rep1 knockout (KO).
Methods.
A mouse line carrying a conditional allele ChmFlox was crossed with the transgenic line IRBP-Cre to achieve Chm KO, specifically in the photoreceptor layer, and Tyr-Cre to produce Chm KO, specifically in the retinal pigment epithelial and other pigmented cells. ChmFlox, Tyr-Cre+ and ChmFlox, IRBP-Cre+ mice were mated to produce mice with Chm KO in both layers. All mouse lines were studied by histology, electron microscopy, electroretinography (ERG), scanning laser ophthalmoscopy (SLO), and biochemical methods.
Results.
In ChmFlox, IRBP-Cre+ mice the authors observed the progressive degeneration of photoreceptors in the presence of normal retinal pigment epithelium (RPE). ChmFlox, Tyr-Cre+ mice exhibited coat color dilution and pigment abnormalities of the RPE in the presence of an intact outer nuclear layer. In 6- to 8-month-old ChmFlox, Tyr-Cre+, IRBP-Cre+ mice, the degeneration of photoreceptors was accelerated compared with ChmFlox, IRBP-Cre+ mice but became leveled with age, such that it was comparable at 12 to 14 months. Detailed ERG and SLO analysis supported the histopathologic findings.
Conclusions.
Defects in photoreceptors and RPE can arise because of intrinsic defects caused cell autonomously by the Chm KO. However, when both photoreceptors and RPE are diseased, the dynamics of the degenerative process are altered. Photoreceptor functional deficit and cell death manifest much earlier, suggesting that the diseased RPE accelerates photoreceptor degeneration.
doi:10.1167/iovs.09-4892
PMCID: PMC3066613  PMID: 20445111
10.  Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina 
Molecular Biology of the Cell  2009;20(17):3896-3904.
The daily phagocytosis of shed photoreceptor outer segments by pigment epithelial cells is critical for the maintenance of the retina. In a subtractive polymerase chain reaction analysis, we found that functional differentiation of human ARPE19 retinal pigment epithelial (RPE) cells is accompanied by up-regulation of annexin (anx) A2, a major Src substrate and regulator of membrane–cytoskeleton dynamics. Here, we show that anx A2 is recruited to the nascent phagocytic cup in vitro and in vivo and that it fully dissociates once the phagosome is internalized. In ARPE19 cells depleted of anx A2 by using small interfering RNA and in ANX A2−/− mice the phagocytosis of outer segments was impaired, and in ANX A2−/− mice there was an accumulation of phagocytosed outer segments in the RPE apical processes, indicative of retarded phagosome transport. We show that anx A2 is tyrosine phosphorylated at the onset of phagocytosis and that the synchronized activation of focal adhesion kinase and c-Src is abnormal in ANX A2−/− mice. These findings reveal that anx A2 is involved in the circadian regulation of outer segment phagocytosis, and they provide new insight into the protein machinery that regulates phagocytic function in RPE cells.
doi:10.1091/mbc.E08-12-1204
PMCID: PMC2735488  PMID: 19587120
11.  Multivesicular bodies: co-ordinated progression to maturity 
Current Opinion in Cell Biology  2008;20(4-24):408-414.
Multivesicular endosomes/bodies (MVBs) sort endocytosed proteins to different destinations. Many lysosomally directed membrane proteins are sorted onto intralumenal vesicles, whilst recycling proteins remain on the perimeter membrane from where they are removed via tubular extensions. MVBs move to the cell centre during this maturation process and, when all recycling proteins have been removed, fuse with lysosomes. Recent advances have identified endosomal-sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways in intralumenal vesicle formation and mechanisms for sorting recycling cargo into tubules. Cytoskeletal motors, through interactions with these machineries and by regulating MVB movement, help to co-ordinate events leading to a mature, fusion-competent MVB.
doi:10.1016/j.ceb.2008.04.001
PMCID: PMC2577128  PMID: 18502633
12.  The Ternary Rab27a–Myrip–Myosin VIIa Complex Regulates Melanosome Motility in the Retinal Pigment Epithelium 
Traffic (Copenhagen, Denmark)  2007;8(5):486-499.
The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a–Myrip–MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery.
doi:10.1111/j.1600-0854.2007.00548.x
PMCID: PMC1920545  PMID: 17451552
melanosome motility; Myrip; Rab27a; RPE
13.  Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia 
Journal of Clinical Investigation  2006;116(2):386-394.
Choroideremia (CHM) is an X-linked degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid, caused by loss of function of the CHM/REP1 gene. REP1 is involved in lipid modification (prenylation) of Rab GTPases, key regulators of intracellular vesicular transport and organelle dynamics. To study the pathogenesis of CHM and to develop a model for assessing gene therapy, we have created a conditional mouse knockout of the Chm gene. Heterozygous-null females exhibit characteristic hallmarks of CHM: progressive degeneration of the photoreceptors, patchy depigmentation of the RPE, and Rab prenylation defects. Using tamoxifen-inducible and tissue-specific Cre expression in combination with floxed Chm alleles, we show that CHM pathogenesis involves independently triggered degeneration of photoreceptors and the RPE, associated with different subsets of defective Rabs.
doi:10.1172/JCI26617
PMCID: PMC1326146  PMID: 16410831
14.  The Role of Rab27a in the Regulation of Melanosome Distribution within Retinal Pigment Epithelial Cells 
Molecular Biology of the Cell  2004;15(5):2264-2275.
Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.
doi:10.1091/mbc.E03-10-0772
PMCID: PMC404021  PMID: 14978221
15.  The Kinetics of Mannose 6-Phosphate Receptor Trafficking in the Endocytic Pathway in HEp-2 Cells: The Receptor Enters and Rapidly Leaves Multivesicular Endosomes without Accumulating in a Prelysosomal Compartment 
Molecular Biology of the Cell  1998;9(4):809-816.
We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor–epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome–lysosome pathway in these cells.
PMCID: PMC25308  PMID: 9529379

Results 1-15 (15)