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1.  Mutant enzymes challenge all assumptions 
eLife  2014;3:e02171.
Enzymes called Rab GTPases that carry so-called “activating” mutations may never become activated at all.
doi:10.7554/eLife.02171
PMCID: PMC3919269  PMID: 24520166
Membrane traffic; Rab GTPase; nucleotide exchange factor; Human
2.  Hopping rim to rim through the Golgi 
eLife  2013;2:e00903.
A novel approach based on tracking the fate of proteins that become ‘stapled’ to the walls of the Golgi yields insights into the long-sought mechanism of transport through this organelle.
doi:10.7554/eLife.00903
PMCID: PMC3679515  PMID: 23795298
Golgi; Traffic; Membrane; Cell biology; Human
3.  ERK8 is a negative regulator of O-GalNAc glycosylation and cell migration 
eLife  2014;3:e01828.
ER O-glycosylation can be induced through relocalisation GalNAc-Transferases from the Golgi. This process markedly stimulates cell migration and is constitutively activated in more than 60% of breast carcinomas. How this activation is achieved remains unclear. Here, we screened 948 signalling genes using RNAi and imaging. We identified 12 negative regulators of O-glycosylation that all control GalNAc-T sub-cellular localisation. ERK8, an atypical MAPK with high basal kinase activity, is a strong hit and is partially localised at the Golgi. Its inhibition induces the relocation of GalNAc-Ts, but not of KDEL receptors, revealing the existence of two separate COPI-dependent pathways. ERK8 down-regulation, in turn, activates cell motility. In human breast and lung carcinomas, ERK8 expression is reduced while ER O-glycosylation initiation is hyperactivated. In sum, ERK8 appears as a constitutive brake on GalNAc-T relocalisation, and the loss of its expression could drive cancer aggressivity through increased cell motility.
DOI: http://dx.doi.org/10.7554/eLife.01828.001
eLife digest
The likelihood of an individual being able to recover from cancer depends on: where the cancer is within the body, how quickly the disease is detected and how quickly treatment is started. Cancers that have spread from their original location to another part of the body are particular challenging to treat, and cause the vast majority of cancer deaths every year.
Treatments that can recognize and eradicate cancer cells, while leaving nearby healthy cells untouched, are still needed—and so there has been a lot of research into identifying the key differences between healthy cells and cancer cells. For several decades, researchers have been aware that cancer cells have more proteins coated with modified sugars on their cell surfaces than healthy cells. This is caused by the enzymes that add these sugars to the proteins relocating from one location within the cell, the Golgi apparatus, to another, called the endoplasmic reticulum. These specific ‘sugar-coated’ proteins are known to encourage cancer cells to migrate and invade new tissues, but the mechanisms that regulate the addition of these sugar molecules to proteins remains poorly understood.
Now Chia et al. have discovered 12 molecules that regulate this process, including an enzyme called ERK8 that is found at the Golgi apparatus. ERK8 is shown to prevent the relocation of the sugar-adding enzymes from the Golgi to the endoplasmic reticulum, thereby restricting the production of sugar-coated proteins that help the cancer cells to spread within the body. By identifying 12 potential targets for new therapeutics aimed at preventing the spread of cancer, the work of Chia et al. could ultimately help to improve the chances of patients recovering from certain cancers.
DOI: http://dx.doi.org/10.7554/eLife.01828.002
doi:10.7554/eLife.01828
PMCID: PMC3945522  PMID: 24618899
golgi; glycosylation; cell migration; COP-I; retrograde traffic; endoplasmic reticulum; human; mouse
4.  The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module 
Human Molecular Genetics  2012;21(23):5019-5038.
Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P2 modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P2 dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.
doi:10.1093/hmg/dds343
PMCID: PMC3490508  PMID: 22907655
5.  GCC185 plays independent roles in Golgi structure maintenance and AP-1–mediated vesicle tethering 
The Journal of Cell Biology  2011;194(5):779-787.
Two distinct domains of GCC185 function in maintaining Golgi structure or in binding to AP-1 to tether retrograde transport vesicles en route to the Golgi.
GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)–containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.
doi:10.1083/jcb.201104019
PMCID: PMC3171126  PMID: 21875948
6.  Multiple Rab GTPase Binding Sites in GCC185 Suggest a Model for Vesicle Tethering at the Trans-Golgi 
Molecular Biology of the Cell  2009;20(1):209-217.
GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether.
doi:10.1091/mbc.E08-07-0740
PMCID: PMC2613123  PMID: 18946081
7.  A syntaxin 10–SNARE complex distinguishes two distinct transport routes from endosomes to the trans-Golgi in human cells 
The Journal of Cell Biology  2008;180(1):159-172.
Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the Golgi after delivering lysosomal enzymes to the endocytic pathway. This process requires Rab9 guanosine triphosphatase (GTPase) and the putative tether GCC185. We show in human cells that a soluble NSF attachment protein receptor (SNARE) complex comprised of syntaxin 10 (STX10), STX16, Vti1a, and VAMP3 is required for this MPR transport but not for the STX6-dependent transport of TGN46 or cholera toxin from early endosomes to the Golgi. Depletion of STX10 leads to MPR missorting and hypersecretion of hexosaminidase. Mouse and rat cells lack STX10 and, thus, must use a different target membrane SNARE for this process. GCC185 binds directly to STX16 and is competed by Rab6. These data support a model in which the GCC185 tether helps Rab9-bearing transport vesicles deliver their cargo to the trans-Golgi and suggest that Rab GTPases can regulate SNARE–tether interactions. Importantly, our data provide a clear molecular distinction between the transport of MPRs and TGN46 to the trans-Golgi.
doi:10.1083/jcb.200707136
PMCID: PMC2213607  PMID: 18195106
8.  Cholesterol Accumulation Sequesters Rab9 and Disrupts Late Endosome Function in NPC1-deficient Cells* 
The Journal of biological chemistry  2006;281(26):17890-17899.
Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompaniedby stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent man-nose 6-phosphate receptorswere missorted to the lysosome for degradation, a process that was reversed by overexpression of GFP-tagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking.
doi:10.1074/jbc.M601679200
PMCID: PMC3650718  PMID: 16644737
9.  A Functional Role for the GCC185 Golgin in Mannose 6-Phosphate Receptor Recycling 
Molecular Biology of the Cell  2006;17(10):4353-4363.
Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during transport vesicle formation. Rab9-bearing vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing transport vesicles at the TGN.
doi:10.1091/mbc.E06-02-0153
PMCID: PMC1635343  PMID: 16885419
10.  Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells 
The Journal of Cell Biology  2002;156(3):511-518.
Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.
doi:10.1083/jcb.200109030
PMCID: PMC2173336  PMID: 11827983
endosome; Rab9; Golgi complex; Rab7; TIP47
11.  Entry at the trans-Face of the Golgi 
The trans-Golgi network (TGN) receives a select set of proteins from the endocytic pathway—about 5% of total plasma membrane glycoproteins (Duncan and Kornfeld 1988). Proteins that are delivered include mannose 6-phosphate receptors (MPRs), TGN46, sortilin, and various toxins that hitchhike a ride backward through the secretory pathway to intoxicate cells after they exit into the cytoplasm from the endoplasmic reticulum (ER). This article will review work on the molecular players that drive protein transport from the endocytic pathway to the TGN. Distinct requirements have revealed multiple routes for retrograde transport; in addition, the existence of multiple, potential coat proteins and/or cargo adaptors imply that multiple vesicular transfers are likely involved. Several comprehensive reviews have appeared recently and should be sought for additional details (Bonifacino and Rojas 2006; Johannes and Popoff 2008).
Proteins such as mannose 6-phosphate receptors and sortilin move to the trans-Golgi network following endocytosis. The retrograde pathways are more complex than expected, requiring numerous adaptor proteins and multiple vesicle transport steps.
doi:10.1101/cshperspect.a005272
PMCID: PMC3039930  PMID: 21421921
12.  Constructing a Golgi complex 
The Journal of Cell Biology  2001;155(6):873-876.
In this issue, Short et al. report the discovery of a protein named Golgin-45 that is located on the surface of the middle (or medial) cisternae of the Golgi complex. Depletion of this protein disrupts the Golgi complex and leads to the return of a resident, lumenal, medial Golgi enzyme to the endoplasmic reticulum. These findings suggest that Golgin-45 serves as a linchpin for the maintenance of Golgi complex structure, and offer hints as to the mechanisms by which the polarized Golgi complex is constructed.
doi:10.1083/jcb.200109095
PMCID: PMC2150916  PMID: 11739400
13.  Recent advances in understanding Golgi biogenesis 
The Golgi complex is a central processing station for proteins traversing the secretory pathway, yet we are still learning how this compartment is constructed and how cargo moves through it. Recent experiments suggest a key role for Ras-like Rab GTPases and provide important new ideas for how the Golgi may function.
doi:10.3410/B2-32
PMCID: PMC2897732  PMID: 20625450
14.  Multiple routes of protein transport from endosomes to the trans Golgi network 
FEBS letters  2009;583(23):3811-3816.
Proteins use multiple routes for transport from endosomes to the Golgi complex. Shiga and cholera toxins and TGN38/46 are routed from early and recycling endosomes, while mannose 6-phosphate receptors are routed from late endosomes. The identification of distinct molecular requirements for each of these pathways makes it clear that mammalian cells have evolved more complex targeting mechanisms and routes than previously anticipated.
doi:10.1016/j.febslet.2009.10.075
PMCID: PMC2787657  PMID: 19879268
endosome; Golgi; Rab GTPase; mannose 6-phosphate receptors; Shiga and cholera toxins
15.  Unconventional secretion by autophagosome exocytosis 
The Journal of Cell Biology  2010;188(4):451-452.
In this issue, Duran et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911154) and Manjithaya et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911149) use yeast genetics to reveal a role for autophagosome intermediates in the unconventional secretion of an acyl coenzyme A (CoA)–binding protein that lacks an endoplasmic reticulum signal sequence. Medium-chain acyl CoAs are also required and may be important for substrate routing to this pathway.
doi:10.1083/jcb.201001121
PMCID: PMC2828920  PMID: 20156968
16.  Association of β-1,3-N-acetylglucosaminyltransferase 1 and β-1,4-galactosyltransferase 1, trans-Golgi enzymes involved in coupled poly-N-acetyllactosamine synthesis 
Glycobiology  2009;19(6):655-664.
Poly-N-acetyllactosamine (polyLacNAc) is a linear carbohydrate polymer composed of alternating N-acetylglucosamine and galactose residues involved in cellular functions ranging from differentiation to metastasis. PolyLacNAc also serves as a scaffold on which other oligosaccharides such as sialyl Lewis X are displayed. The polymerization of the alternating N-acetylglucosamine and galactose residues is catalyzed by the successive action of UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) and UDP-Gal:βGlcNAc β-1,4-galactosyltransferase, polypeptide 1 (B4GALT1), respectively. The functional association between these two glycosyltransferases led us to investigate whether the enzymes also associate physically. We show that B3GNT1 and B4GALT1 colocalize by immunofluorescence microscopy, interact by coimmunoprecipitation, and affect each other's subcellular localization when one of the two proteins is artificially retained in the endoplasmic reticulum. These results demonstrate that B3GNT1 and B4GALT1 physically associate in vitro and in cultured cells, providing insight into possible mechanisms for regulation of polyLacNAc production.
doi:10.1093/glycob/cwp035
PMCID: PMC2682609  PMID: 19261593
endoplasmic reticulum; enzyme complexes; glycosyltransferase; Golgi complex; poly-N-acetyllactosamine
17.  RhoBTB3: A Rho GTPase-family ATPase required for endosome to Golgi transport 
Cell  2009;137(5):938-948.
Summary
Rho GTPases are key regulators of the actin-based cytoskeleton; Rab GTPases are key regulators of membrane traffic. We report here that the atypical Rho GTPase family member, RhoBTB3, binds directly to Rab9 GTPase, and functions with Rab9 in protein transport from endosomes to the trans Golgi network. Gene replacement experiments show that RhoBTB3 function in cultured cells requires both RhoBTB3’s N-terminal, Rho-related domain, and C-terminal sequences that are important for Rab9 interaction.9 Biochemical analysis reveals that RhoBTB3 binds and hydrolyzes ATP rather than GTP. Rab9 binding opens the auto-inhibited RhoBTB3 protein to permit maximal ATP hydroysis. Because RhoBTB3 interacts with TIP47 on membranes, we propose that it may function to release this cargo selection protein from vesicles to permit their efficient docking and fusion at the Golgi.
doi:10.1016/j.cell.2009.03.043
PMCID: PMC2801561  PMID: 19490898
18.  Journeys through the Golgi—taking stock in a new era 
The Journal of Cell Biology  2009;187(4):449-453.
The Golgi apparatus is essential for protein sorting and transport. Many researchers have long been fascinated with the form and function of this organelle. Yet, despite decades of scrutiny, the mechanisms by which proteins are transported across the Golgi remain controversial. At a recent meeting, many prominent Golgi researchers assembled to critically evaluate the core issues in the field. This report presents the outcome of their discussions and highlights the key open questions that will help guide the field into a new era.
doi:10.1083/jcb.200909011
PMCID: PMC2779233  PMID: 19948493
19.  Recent advances in understanding Golgi biogenesis 
The Golgi complex is a central processing station for proteins traversing the secretory pathway, yet we are still learning how this compartment is constructed and how cargo moves through it. Recent experiments suggest a key role for Ras-like Rab GTPases and provide important new ideas for how the Golgi may function.
doi:10.3410/B2-32
PMCID: PMC2897732  PMID: 20625450
20.  Dual GTPase regulation of the GCC185 Golgin: Communication between adjacent Rab6 and Arl1 binding sites 
Cell  2008;132(2):286-298.
Summary
GCC185 is a large coiled coil protein at the trans Golgi network that is required for receipt of transport vesicles inbound from late endosomes, and for anchoring non-centrosomal microtubules that emanate from the Golgi. Here we demonstrate that recruitment of GCC185 to the Golgi is mediated by two Golgi-localized small GTPases of the Rab and Arl families. GCC185 binds Rab6 and mutation of residues needed for Rab binding abolishes Golgi localization. The crystal structure of Rab6 bound to the GCC185 Rab binding domain reveals that Rab6 recognizes a two-fold symmetric surface on a coiled coil immediately adjacent to a C-terminal GRIP domain. Unexpectedly, Rab6 binding promotes association of Arl1 with the GRIP domain. We present a structure-derived model for dual GTPase membrane attachment that highlights the potential ability of Rab GTPases to reach binding partners at a significant distance from the membrane via their unstructured and membrane-anchored, hypervariable domains.
doi:10.1016/j.cell.2007.11.048
PMCID: PMC2344137  PMID: 18243103
21.  Clues to Neuro-Degeneration in Niemann-Pick Type C Disease from Global Gene Expression Profiling 
PLoS ONE  2006;1(1):e19.
Background
Niemann-Pick Type C (NPC) disease is a neurodegenerative disease that is characterized by the accumulation of cholesterol and glycosphingolipids in the late endocytic pathway. The majority of NPC cases are due to mutations in the NPC1 gene. The precise function of this gene is not yet known.
Methodology/Principal Findings
Using cDNA microarrays, we analyzed the genome-wide expression patterns of human fibroblasts homozygous for the I1061T NPC1 mutation that is characterized by a severe defect in the intracellular processing of low density lipoprotein-derived cholesterol. A distinct gene expression profile was identified in NPC fibroblasts from different individuals when compared with fibroblasts isolated from normal subjects. As expected, NPC1 mutant cells displayed an inappropriate homeostatic response to accumulated intracellular cholesterol. In addition, a number of striking parallels were observed between NPC disease and Alzheimer's disease.
Conclusions/Significance
Many genes involved in the trafficking and processing of amyloid precursor protein and the microtubule binding protein, tau, were more highly expressed. Numerous genes important for membrane traffic and the cellular regulation of calcium, metals and other ions were upregulated. Finally, NPC fibroblasts exhibited a gene expression profile indicative of oxidative stress. These changes are likely contributors to the pathophysiology of Niemann-Pick Type C disease.
doi:10.1371/journal.pone.0000019
PMCID: PMC1762405  PMID: 17183645
22.  Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis 
Molecular Biology of the Cell  2003;14(4):1624-1637.
Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L.
doi:10.1091/mbc.E02-08-0494
PMCID: PMC153127  PMID: 12686614
23.  Role of Nectin in Formation of E-Cadherin–based Adherens Junctions in Keratinocytes: Analysis with the N-Cadherin Dominant Negative Mutant 
Molecular Biology of the Cell  2003;14(4):1597-1609.
E-Cadherin is a Ca2+-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin–based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin–based AJs in keratinocytes.
doi:10.1091/mbc.E02-10-0632
PMCID: PMC153125  PMID: 12686612
24.  Morphology and Dynamics of Clathrin/GGA1-coated Carriers Budding from the Trans-Golgi NetworkV⃞ 
Molecular Biology of the Cell  2003;14(4):1545-1557.
Sorting of transmembrane proteins and their ligands at various compartments of the endocytic and secretory pathways is mediated by selective incorporation into clathrin-coated intermediates. Previous morphological and biochemical studies have shown that these clathrin-coated intermediates consist of spherical vesicles with a diameter of 60–100 nm. Herein, we report the use of fluorescent imaging of live cells to demonstrate the existence of a different type of transport intermediate containing associated clathrin coats. Clathrin and the adaptors GGA1 and adaptor protein-1, labeled with different spectral variants of the green fluorescent protein, are shown to colocalize to the trans-Golgi network and to a population of vesicles and tubules budding from it. These intermediates are highly pleiomorphic and move toward the peripheral cytoplasm for distances of up to 10 μm with average speeds of ∼1 μm/s. The labeled clathrin and GGA1 cycle on and off membranes with half-times of 10–20 s, independently of vesicle budding. Our observations indicate the existence of a novel type of trans-Golgi network-derived carriers containing associated clathrin, GGA1 and adaptor protein-1 that are larger than conventional clathrin-coated vesicles, and that undergo long-range translocation in the cytoplasm before losing their coats.
doi:10.1091/mbc.02-07-0109
PMCID: PMC153121  PMID: 12686608
25.  Procyclin Null Mutants of Trypanosoma brucei Express Free Glycosylphosphatidylinositols on Their Surface 
Molecular Biology of the Cell  2003;14(4):1308-1318.
Procyclins are abundant, glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of procyclic (insect) form trypanosomes. To investigate whether trypanosomes are able to survive without a procyclin coat, all four procyclin genes were deleted sequentially. Bloodstream forms of the null mutant exhibited no detectable phenotype and were able to differentiate to procyclic forms. Initially, differentiated null mutant cells were barely able to grow, but after an adaptation period of 2 mo in culture they proliferated at the same rate as wild-type trypanosomes. Analysis of these culture-adapted null mutants revealed that they were covered by free GPIs. These were closely related to the mature procyclin anchor in structure and were expressed on the surface in numbers comparable with that of procyclin in wild-type cells. However, free GPIs were smaller than the procyclin anchor, indicative of a lower number of poly-N-acetyllactosamine repeats, and a proportion contained diacylphosphatidic acid. Free GPIs are also expressed by wild-type cells, although to a lesser extent. These have been overlooked in the past because they partition in a solvent fraction (chloroform/water/methanol) that is normally discarded when GPI-anchored proteins are purified.
doi:10.1091/mbc.E02-10-0694
PMCID: PMC153102  PMID: 12686589

Results 1-25 (108)