Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking.
AuthorsLiang C, Lee JS, Inn KS, Gack MU, Li Q, Roberts EA, Vergne I, Deretic V, Feng P, Akazawa C, Jung JU,
JournalNat Cell Biol
PubMed ID18552835
'Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the ... More
A cytosolic calcium transient is not necessary for degranulation or oxidative burst in immune complex-stimulated neutrophils.
AuthorsSeetoo KF, Schonhorn JE, Gewirtz AT, Zhou MJ, McMenamin ME, Delva L, Simons ER
JournalJ Leukoc Biol
PubMed ID9307071
'Receptor-mediated activation of neutrophils (PMN) initiates possibly interdependent events, including a rapid transient increase in [Ca2+]i, implicated as a second messenger. To investigate whether this transient is required for eventual degranulation, PMN were incubated with an intracellular Ca2+ chelator (BAPTA), then exposed to chemotactic peptide [N-formyl-methionyl-leucyl-phenylalanine (fMLP)l with or without ... More
Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines.
AuthorsBarratt-Boyes SM, Zimmer MI, Harshyne LA, Meyer EM, Watkins SC, Capuano S, Murphey-Corb M, Falo LD, Donnenberg AD
JournalJ Immunol
PubMed ID10679086
'Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have ... More
A novel methodology for the investigation of intracellular proteolytic processing in intact cells.
AuthorsReis RC, Sorgine MH, Coelho-Sampaio T
JournalEur J Cell Biol
PubMed ID9548376
'Taking advantage of the unique spectral properties of the fluorescent probe FL-Bodipy, we have developed a new methodology to study processing of exogenous proteins in intact cells. FL-Bodipy was conjugated to bovine serum albumin (BSA) at a molar ratio of 29 probe molecules to 1 albumin equivalent. The resulting conjugate ... More
Assays to assess autophagy induction and fusion of autophagic vacuoles with a degradative compartment, using monodansylcadaverine (MDC) and DQ-BSA.
AuthorsVázquez CL, Colombo MI,
JournalMethods Enzymol
PubMed ID19200877
In this chapter we describe the use of monodasylcadaverine (MDC) and DQ-BSA, two practical and convenient tools to study the autophagic pathway. MDC is a lysosomotropic compound useful for the identification of autophagic vesicles by fluorescence microscopy and, in addition, to assess autophagy induction via the accumulation of MDC-labeled vacuoles. ... More
Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation.
AuthorsPhan TG, Green JA, Gray EE, Xu Y, Cyster JG,
JournalNat Immunol
PubMed ID19503106
Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-alpha1beta2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on ... More
Real-time spectrofluorometric assays for the lumenal environment of the maturing phagosome.
AuthorsYates RM, Russell DG,
JournalMethods Mol Biol
PubMed ID18425459
The ultimate goal of phagosomal maturation is the delivery of internalized, particulate cargo to acidic, hydrolytically competent compartments capable of mediating its degradation. Here we outline in detail three fluorometric techniques that allow the study of phagosomal maturation in macrophages by quantifying functionally important features of the lumenal environment of ... More
Extracellular antigen processing and presentation by immature dendritic cells.
In antigen presentation to CD4(+) T cells, proteins are degraded to peptide fragments and loaded onto class II MHC molecules in a process involving the peptide exchange factors H-2M (murine) or HLA-DM (human). In many antigen-presenting cells these processes occur in intracellular endosomal compartments, where peptides are generated and loaded ... More
Oscillatory pericellular proteolysis and oxidant deposition during neutrophil locomotion.
To better understand the mechanism of leukocyte migration in complex environments, model extracellular matrices were prepared using gelatin, Hanks' solution, Bodipy-BSA (fluorescent upon proteolysis), and dihydrotetramethylrosamine or hydroethidine (fluorescent upon oxidation). Using quantitative microfluorometry, neutrophil-mediated extracellular pulses of reactive oxygen metabolites (ROMs) and pericellular proteolysis were periodically observed showing that ... More
Rac1-induced endocytosis is associated with intracellular proteolysis during migration through a three-dimensional matrix.
AuthorsAhram M, Sameni M, Qiu RG, Linebaugh B, Kirn D, Sloane BF
JournalExp Cell Res
PubMed ID11035924
Transfection of Rat1 fibroblasts with an activated form of rac1 (V12rac1) stimulated cell migration in vitro compared to transfection of Rat1 fibroblasts with vector only or with dominant negative rac1 (N17rac1). To investigate the involvement of proteases in this migration, we used a novel confocal assay to evaluate the ability ... More
Imaging proteolysis by living human breast cancer cells.
AuthorsSameni M, Moin K, Sloane BF
JournalNeoplasia
PubMed ID11228542
Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549) through the use of quenched-fluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation ... More
Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator.
Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These ... More
The intraflagellar transport protein IFT20 controls lysosome biogenesis by regulating the post-Golgi transport of acid hydrolases.
Authors
JournalCell Death Differ
PubMed ID31142807
MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B.
Authors
JournalEMBO J
PubMed ID32716134
mTOR Inhibition Restores Amino Acid Balance in Cells Dependent on Catabolism of Extracellular Protein.
Authors
JournalMol Cell
PubMed ID28918901
Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth.
Authors
JournalNat Cell Biol
PubMed ID33686253
Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection.