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1.
J Cell Biol ; 222(2)2023 02 06.
Article in English | MEDLINE | ID: mdl-36416725

ABSTRACT

Fibronectin (FN) is an essential structural and regulatory component of the extracellular matrix (ECM), and its binding to integrin receptors supports cell adhesion, migration, and signaling. Here, using live-cell microscopy of fibroblasts expressing FN tagged with a pH-sensitive fluorophore, we show that FN is secreted predominantly at the ventral surface of cells in an integrin-independent manner. Locally secreted FN then undergoes ß1 integrin-dependent fibrillogenesis. We find that the site of FN secretion is regulated by cell polarization, which occurs in bursts under stabilized lamellipodia at the leading edge. Moreover, analysis of FN secretion and focal adhesion dynamics suggest that focal adhesion formation precedes FN deposition and that deposition continues during focal adhesion disassembly. Lastly, we show that the polarized FN deposition in spreading and migrating cells requires both intact microtubules and myosin II-mediated contractility. Thus, while FN secretion does not require integrin binding, the site of exocytosis is regulated by membrane and cytoskeletal dynamics with secretion occurring after new adhesion formation.


Subject(s)
Fibronectins , Microtubules , Myosin Type II , Pseudopodia , Cytoskeletal Proteins/metabolism , Fibroblasts/metabolism , Fibronectins/genetics , Fibronectins/metabolism , Integrins/metabolism , Microtubules/genetics , Microtubules/metabolism , Myosin Type II/genetics , Myosin Type II/metabolism , Pseudopodia/genetics , Pseudopodia/metabolism , Extracellular Matrix/metabolism , Exocytosis
2.
Nat Commun ; 11(1): 4271, 2020 08 26.
Article in English | MEDLINE | ID: mdl-32848153

ABSTRACT

Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore (Yale-595) and a bio-orthogonal labeling strategy that enables two-color super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize dual organelle dynamics in hard-to-transfect cell lines by super-resolution for over an order of magnitude longer than with tagged proteins. The extended time domain possible using these tools reveals dynamic nanoscale targeting between different organelles.


Subject(s)
Fluorescent Dyes , Microscopy, Fluorescence/methods , Nanotechnology/methods , Organelles/metabolism , Cell Line , Fluorescent Dyes/chemistry , HeLa Cells , Human Umbilical Vein Endothelial Cells , Humans , Imaging, Three-Dimensional , Microscopy, Confocal , Photobleaching , Time-Lapse Imaging
3.
Nat Commun ; 8(1): 432, 2017 09 05.
Article in English | MEDLINE | ID: mdl-28874656

ABSTRACT

The Golgi is composed of a stack of cis, medial, trans cisternae that are biochemically distinct. The stable compartments model postulates that permanent cisternae communicate through bi-directional vesicles, while the cisternal maturation model postulates that transient cisternae biochemically mature to ensure anterograde transport. Testing either model has been constrained by the diffraction limit of light microscopy, as the cisternae are only 10-20 nm thick and closely stacked in mammalian cells. We previously described the unstacking of Golgi by the ectopic adhesion of Golgi cisternae to mitochondria. Here, we show that cargo processing and transport continue-even when individual Golgi cisternae are separated and "land-locked" between mitochondria. With the increased spatial separation of cisternae, we show using three-dimensional live imaging that cis-Golgi and trans-Golgi remain stable in their composition and size. Hence, we provide new evidence in support of the stable compartments model in mammalian cells.The different composition of Golgi cisternae gave rise to two different models for intra-Golgi traffic: one where stable cisternae communicate via vesicles and another one where cisternae biochemically mature to ensure anterograde transport. Here, the authors provide evidence in support of the stable compartments model.


Subject(s)
Golgi Apparatus/metabolism , Mammals/metabolism , Animals , Biological Transport , Coated Vesicles/metabolism , Fluorescence Recovery After Photobleaching , Golgi Apparatus/ultrastructure , Golgi Matrix Proteins , HeLa Cells , Humans , Membrane Fusion , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondria/ultrastructure
4.
Angew Chem Int Ed Engl ; 56(35): 10408-10412, 2017 08 21.
Article in English | MEDLINE | ID: mdl-28679029

ABSTRACT

Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes of the high-density plasma membrane probe DiI-TCO and the photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI-SiR. Using DiI-SiR, we visualized filopodia dynamics in HeLa cells over 25 min at 0.5 s temporal resolution, and visualized dynamic contact-mediated repulsion events in primary mouse hippocampal neurons over 9 min at 2 s temporal resolution. HIDE probes such as DiI-SiR are non-toxic and do not require transfection, and their apparent photostability significantly improves the ability to monitor dynamic processes in live cells at super-resolution over biologically relevant timescales.


Subject(s)
Cell Membrane/chemistry , Fluorescent Dyes/chemistry , Nanotechnology , Optical Imaging , HeLa Cells , Humans , Microscopy, Fluorescence , Molecular Structure , Tumor Cells, Cultured
5.
J Cell Mol Med ; 21(11): 2950-2962, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28544529

ABSTRACT

Type 2 diabetes is caused by defects in both insulin sensitivity and insulin secretion. Glucose triggers insulin secretion by causing exocytosis of insulin granules from pancreatic ß-cells. High circulating cholesterol levels and a diminished capacity of serum to remove cholesterol from ß-cells are observed in diabetic individuals. Both of these effects can lead to cholesterol accumulation in ß-cells and contribute to ß-cell dysfunction. However, the molecular mechanisms by which cholesterol accumulation impairs ß-cell function remain largely unknown. Here, we used total internal reflection fluorescence microscopy to address, at the single-granule level, the role of cholesterol in regulating fusion pore dynamics during insulin exocytosis. We focused particularly on the effects of cholesterol overload, which is relevant to type 2 diabetes. We show that excess cholesterol reduced the number of glucose-stimulated fusion events, and modulated the proportion of full fusion and kiss-and-run fusion events. Analysis of single exocytic events revealed distinct fusion kinetics, with more clustered and compound exocytosis observed in cholesterol-overloaded ß-cells. We provide evidence for the involvement of the GTPase dynamin, which is regulated in part by cholesterol-induced phosphatidylinositol 4,5-bisphosphate enrichment in the plasma membrane, in the switch between full fusion and kiss-and-run fusion. Characterization of insulin exocytosis offers insights into the role that elevated cholesterol may play in the development of type 2 diabetes.


Subject(s)
Cholesterol/pharmacology , Glucose/metabolism , Insulin-Secreting Cells/drug effects , Insulin/metabolism , Membrane Fusion/drug effects , Secretory Vesicles/drug effects , Animals , Cell Line, Tumor , Cell Membrane/drug effects , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Dynamins/genetics , Dynamins/metabolism , Exocytosis , Gene Expression Regulation , Glucose/pharmacology , Humans , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Mice , Microscopy, Fluorescence/methods , Models, Biological , Phosphatidylinositol 4,5-Diphosphate/metabolism , Secretory Vesicles/metabolism , Signal Transduction
6.
Ann Surg ; 266(6): 1075-1083, 2017 12.
Article in English | MEDLINE | ID: mdl-27611607

ABSTRACT

OBJECTIVE: We asked if leptin and its cognate receptor were present in normal and diseased parathyroid glands, and if so, whether they had any functional effects on parathyroid hormone (PTH) secretion in parathyroid neoplasms. BACKGROUND: The parathyroid glands acting through PTH play a critical role in the regulation of serum calcium. Based on leptin's recently discovered role in bone metabolism, we hypothesized these glands were the sites of a functional interaction between these 2 hormones. METHODS: From July 2010 to July 2011, 96 patients were enrolled in a prospective study of leptin and hyperparathyroidism, all of whom were enrolled based on their diagnosis of hyperparathyroidism, and their candidacy for surgical intervention provided informed consent. Immediately after parathyroidectomy, 100 to 300 mg of adenomatous or hyperplastic diseased parathyroid tissue was prepared and processed according to requirements of the following: in situ hybridization, immunohistochemistry, immunofluorescence by conventional and spinning disc confocal microscopy, electron microscopy, parathyroid culture, whole organ explant, and animal model assays. RESULTS: Leptin, leptin receptor (long isoform), and PTH mRNA transcripts and protein were detected in an overlapping fashion in parathyroid chief cells in adenoma and hyperplastic glands, and also in normal parathyroid by in situ hybridization, qRT-PCR, and immunohistochemistry. Confocal microscopy confirmed active exogenous leptin uptake in cultured parathyroid cells. PTH secretion in explants increased in response to leptin and decreased with leptin receptor signaling inhibition by AG490, a JAK2/STAT3 inhibitor. Ob/ob mice injected with mouse leptin exhibited increased PTH levels from baseline. CONCLUSIONS: Taken together, these data suggest that leptin is a functionally active product of the parathyroid glands and stimulates PTH release.


Subject(s)
Leptin/metabolism , Parathyroid Glands/metabolism , Parathyroid Hormone/metabolism , Adenoma/metabolism , Animals , Cells, Cultured , Humans , Hyperparathyroidism/metabolism , Hyperplasia/metabolism , Immunohistochemistry , Mice, Knockout , Microscopy, Confocal , Microscopy, Fluorescence , Microscopy, Immunoelectron , Parathyroid Glands/pathology , Parathyroid Neoplasms/metabolism , Prospective Studies , RNA, Messenger/metabolism , Receptors, Leptin/antagonists & inhibitors , Receptors, Leptin/metabolism
7.
Proc Natl Acad Sci U S A ; 113(24): 6677-82, 2016 06 14.
Article in English | MEDLINE | ID: mdl-27247384

ABSTRACT

One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers.


Subject(s)
CD8 Antigens/metabolism , Cell Membrane/metabolism , Secretory Vesicles/metabolism , Sphingomyelins/metabolism , trans-Golgi Network/metabolism , Biological Transport, Active/drug effects , Biological Transport, Active/physiology , CD8 Antigens/genetics , Cell Membrane/genetics , Cnidarian Venoms/pharmacology , HeLa Cells , Humans , Secretory Vesicles/genetics , Sphingomyelins/genetics , trans-Golgi Network/genetics
8.
Nat Commun ; 7: 10778, 2016 Mar 04.
Article in English | MEDLINE | ID: mdl-26940217

ABSTRACT

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution.


Subject(s)
Heterocyclic Compounds, 4 or More Rings/chemistry , Luminescent Proteins , Microscopy, Fluorescence/methods , Nanotechnology/methods , Rhodamines/chemistry , Animals , COS Cells , Chlorocebus aethiops , HeLa Cells , Humans
9.
Proc Natl Acad Sci U S A ; 109(35): E2316-23, 2012 Aug 28.
Article in English | MEDLINE | ID: mdl-22847441

ABSTRACT

Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase(OCRL)), which acts on PI(4,5)P(2) and PI(3,4,5)P(3), was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase(OCRL) to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P(2) and PI(3,4,5)P(3) biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase(OCRL) recruitment and PI(4,5)P(2) dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P(2) biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P(3) synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.


Subject(s)
Endocytosis/physiology , Endocytosis/radiation effects , Light , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositols/metabolism , Actins/metabolism , Amino Acid Motifs/physiology , Animals , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Binding Sites/physiology , COS Cells , Cell Membrane/metabolism , Cell Membrane/radiation effects , Chlorocebus aethiops , Cryptochromes/genetics , Cryptochromes/metabolism , Humans , KCNQ2 Potassium Channel/physiology , KCNQ3 Potassium Channel/physiology , Membrane Potentials/physiology , Membrane Potentials/radiation effects , PC12 Cells , Phosphatidylinositol 3-Kinases/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation/physiology , Phosphorylation/radiation effects , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/physiology , Signal Transduction/radiation effects
10.
PLoS One ; 7(3): e32655, 2012.
Article in English | MEDLINE | ID: mdl-22403691

ABSTRACT

PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at the plasma membrane of endothelial cells (ECs), on structures such as caveolae, fenestrae and transendothelial channels. Knockout of PV1 in mice results in in utero and perinatal mortality. To be able to interpret the complex PV1 knockout phenotype, it is critical to determine whether the formation of diaphragms is the only cellular role of PV1. We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level. Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys. The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs. The absence of caveolae in the lung ECs did not affect the transcription or translation of PV1, but it caused a sharp increase in PV1 protein internalization rate via a clathrin- and dynamin-independent pathway followed by degradation in lysosomes. Thus, PV1 is retained on the cell surface of ECs by structures capable of forming diaphragms, but undergoes rapid internalization and degradation in the absence of these structures, suggesting that formation of diaphragms is the only role of PV1.


Subject(s)
Carrier Proteins/metabolism , Caveolae/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Membrane Proteins/metabolism , Animals , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Diaphragm/cytology , Lung/cytology , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Protein Transport , Transcription, Genetic
11.
Cell Host Microbe ; 11(1): 46-57, 2012 Jan 19.
Article in English | MEDLINE | ID: mdl-22264512

ABSTRACT

The intracellular bacterial pathogen Legionella pneumophila subverts host membrane transport pathways to promote fusion of vesicles exiting the endoplasmic reticulum (ER) with the pathogen-containing vacuole. During infection there is noncanonical pairing of the SNARE protein Sec22b on ER-derived vesicles with plasma membrane (PM)-localized syntaxin proteins on the vacuole. We show that the L. pneumophila Rab1-targeting effector DrrA is sufficient to stimulate this noncanonical SNARE association and promote membrane fusion. DrrA activation of the Rab1 GTPase on PM-derived organelles stimulated the tethering of ER-derived vesicles with the PM-derived organelle, resulting in vesicle fusion through the pairing of Sec22b with the PM syntaxin proteins. Thus, the effector protein DrrA stimulates a host membrane transport pathway that enables ER-derived vesicles to remodel a PM-derived organelle, suggesting that Rab1 activation at the PM is sufficient to promote the recruitment and fusion of ER-derived vesicles.


Subject(s)
Bacterial Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Legionella pneumophila/pathogenicity , Membrane Fusion , Qa-SNARE Proteins/metabolism , R-SNARE Proteins/metabolism , Cell Line , Endoplasmic Reticulum/metabolism , Humans , Protein Binding , Vacuoles/metabolism , Vacuoles/microbiology , rab1 GTP-Binding Proteins/metabolism
12.
Curr Opin Chem Biol ; 15(6): 822-30, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22079055

ABSTRACT

Cellular compartmentalization into discrete organelles is maintained by membrane trafficking including vesiculation and tubulation. Recent advances in superresolution imaging have begun to bring these small and dynamic events into focus. Most nanoscopes exploit, and are limited by, switching dyes ON and OFF. Using ground state depletion to switch dyes into long-lived dark states can exploit specific photophysical properties of dyes, such as redox potential or pK(a), and expand the repertoire of nanoscopy probes for multicolor imaging. Seeing is not enough, and new technologies based on homodimerization, heterodimerization and selective release can manipulate membrane trafficking in pulse-chase and light-controlled ways. Herein we highlight the utility and promise of these strategies and discuss their current limitations.


Subject(s)
Cell Membrane/metabolism , Fluorescent Dyes/analysis , Microscopy, Fluorescence/methods , Molecular Imaging/methods , Molecular Probes/analysis , Proteins/analysis , Animals , Biological Transport , Cell Membrane/chemistry , Dimerization , Fluorescent Dyes/chemical synthesis , Gene Knockdown Techniques , Humans , Molecular Probes/chemical synthesis , Oxidation-Reduction , Photochemical Processes , Proteins/chemistry , Proteins/metabolism , RNA Interference , Transport Vesicles/chemistry , Transport Vesicles/metabolism
13.
Med Image Comput Comput Assist Interv ; 14(Pt 1): 629-36, 2011.
Article in English | MEDLINE | ID: mdl-22003671

ABSTRACT

Multi-angle total internal reflection fluorescence microscopy (MA-TIRFM) is a new generation of TIRF microscopy to study cellular processes near dorsal cell membrane in 4 dimensions (3D+t). To perform quantitative analysis using MA-TIRFM, it is necessary to track subcellular particles in these processes. In this paper, we propose a method based on a MAP framework for automatic particle tracking and apply it to track clathrin coated pits (CCPs). The expectation maximization (EM) algorithm is employed to solve the MAP problem. To provide the initial estimations for the EM algorithm, we develop a forward filter based on the most probable trajectory (MPT) filter. Multiple linear models are used to model particle dynamics. For CCP tracking, we use two linear models to describe constrained Brownian motion and fluorophore variation according to CCP properties. The tracking method is evaluated on synthetic data and results show that it has high accuracy. The result on real data confirmed by human expert cell biologists is also presented.


Subject(s)
Image Processing, Computer-Assisted/methods , Microscopy, Electron/methods , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Microscopy/methods , Algorithms , Automation , Cell Biology , Clathrin/metabolism , Coated Pits, Cell-Membrane/metabolism , Computer Simulation , Fluorescent Dyes/pharmacology , Humans , Models, Statistical , Reproducibility of Results
14.
J Cell Biol ; 193(4): 643-53, 2011 May 16.
Article in English | MEDLINE | ID: mdl-21555461

ABSTRACT

Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from approximately 60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.


Subject(s)
Adipocytes/metabolism , Exocytosis , Glucose Transporter Type 4/metabolism , Insulin/metabolism , Transport Vesicles/metabolism , 3T3-L1 Cells , Animals , Biosensing Techniques , Carrier Proteins/genetics , Carrier Proteins/metabolism , Glucose Transporter Type 4/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Intracellular Signaling Peptides and Proteins , Kinetics , Membrane Fusion , Mice , Microscopy, Fluorescence , Microscopy, Video , Phospholipase D/metabolism , RNA Interference , Recombinant Fusion Proteins/metabolism , Transfection , Vesicle-Associated Membrane Protein 2/genetics , Vesicle-Associated Membrane Protein 2/metabolism
15.
J Cell Biol ; 181(7): 1047-54, 2008 Jun 30.
Article in English | MEDLINE | ID: mdl-18573914

ABSTRACT

Membrane trafficking during cytokinesis is not well understood. We used advanced live cell imaging techniques to track exocytosis of single vesicles to determine whether constitutively exocytosed membrane is focally delivered to the cleavage furrow. Ultrasensitive three-dimensional confocal time-lapse imaging of the temperature-sensitive membrane cargo protein vesicular stomatitis virus protein-yellow fluorescent protein revealed that vesicles from both daughter cells traffic out of the Golgi and into the furrow, following curvilinear paths. Immunolocalization and photobleaching experiments indicate that individual vesicles accumulate at the midbody and generate a reserve vesicle pool that is distinct from endosomal and lysosomal compartments. Total internal reflection fluorescence microscopy imaging provided direct evidence that Golgi-derived vesicles from both daughter cells not only traffic to the furrow region but dock and fuse there, supporting a symmetrically polarized exocytic delivery model. In contrast, quantitative analysis of midbody abscission showed inheritance of the midbody remnant by one daughter cell, indicating that cytokinesis is composed of both symmetrical and asymmetrical stages.


Subject(s)
Cell Membrane/metabolism , Cytokinesis , Exocytosis , Animals , Biological Transport , Fluorescence Recovery After Photobleaching , Golgi Apparatus/metabolism , HeLa Cells , Humans , Intracellular Membranes/metabolism , Mammals , Microscopy, Confocal , Microscopy, Fluorescence , Recombinant Fusion Proteins/metabolism , Transport Vesicles/metabolism
16.
Neoplasia ; 9(12): 1099-110, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18084617

ABSTRACT

Overexpression of the epidermal growth factor receptor (EGFR) in epithelial tumors is associated with poor prognosis and is the target for a number of cancer therapeutics. Monoclonal antibody (mAb) 806 is a novel anti-EGFR antibody with significant therapeutic efficacy in tumor models when used as a single agent, and displays synergistic antitumor activity in combination with other EGFR therapeutics. Unlike other EGFR antibodies, mAb 806 is selective for tumor cells and does not bind to normal tissue, making it an ideal candidate for generation of radioisotope or toxin conjugates. Ideally, antibodies suited to these therapeutic applications must bind to and actively internalize their cognate receptor. We investigated the intracellular trafficking of fluorescently tagged mAb 806 in live cells and analyzed its biodistribution in a tumor xenografted nude mouse model. Following binding to EGFR, mAb 806 was internalized through dynamin-dependent, clathrin-mediated endocytosis. Internalized mAb 806 localized to early endosomes and subsequently trafficked to and accumulation in lysosomal compartments. Furthermore, biodistribution analysis in nude mice showed specific uptake and retention of radiolabeled mAb 806 to human tumor xenografts. These results highlight the potential use of mAb 806 for generation of conjugates suitable for diagnostic and therapeutic use in patients with EGFR-positive malignancies.


Subject(s)
Antibodies, Monoclonal/pharmacokinetics , ErbB Receptors/immunology , Neoplasm Proteins/immunology , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Biological Transport , Cell Line, Tumor , Clathrin-Coated Vesicles/metabolism , Endocytosis , Endosomes/metabolism , Epitopes/immunology , Humans , Immunoconjugates/pharmacokinetics , Lysosomes/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Tissue Distribution , Transfection
17.
Proc Natl Acad Sci U S A ; 103(52): 19777-82, 2006 Dec 26.
Article in English | MEDLINE | ID: mdl-17170139

ABSTRACT

Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-nitrosylation. Although intracellular S-nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein S-nitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimide-sensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-nitrosylation reactions that can regulate intracellular transport processes.


Subject(s)
Nitric Oxide Synthase Type III/metabolism , Nitric Oxide/metabolism , S-Nitrosothiols/metabolism , Animals , Cattle , Cell Line , Chlorocebus aethiops , Golgi Apparatus/metabolism , Kinetics , Nitric Oxide Synthase Type III/genetics , Protein Transport
18.
Nat Cell Biol ; 8(9): 971-7, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16906144

ABSTRACT

The intracellular pathogen Legionella pneumophila avoids fusion with lysosomes and subverts membrane transport from the endoplasmic reticulum to create an organelle that supports bacterial replication. Transport of endoplasmic reticulum-derived vesicles to the Legionella-containing vacuole (LCV) requires bacterial proteins that are translocated into host cells by a type IV secretion apparatus called Dot/Icm. Recent observations have revealed recruitment of the host GTPase Rab1 to the LCV by a process requiring the Dot/Icm system. Here, a visual screen was used to identify L. pneumophila mutants with defects in Rab1 recruitment. One of the factors identified in this screen was DrrA, a new Dot/Icm substrate protein translocated into host cells. We show that DrrA is a potent and highly specific Rab1 guanine nucleotide-exchange factor (GEF). DrrA can disrupt Rab1-mediated secretory transport to the Golgi apparatus by competing with endogenous exchange factors to recruit and activate Rab1 on plasma membrane-derived organelles. These data establish that intracellular pathogens have the capacity to directly modulate the activation state of a specific member of the Rab family of GTPases and thus further our understanding of the mechanisms used by bacterial pathogens to manipulate host vesicular transport.


Subject(s)
Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Legionella pneumophila/physiology , rab1 GTP-Binding Proteins/metabolism , Animals , Bacterial Proteins/genetics , Biological Transport, Active , Cell Membrane/metabolism , Cells, Cultured , DNA-Binding Proteins/genetics , Female , Golgi Apparatus/metabolism , Guanine Nucleotide Exchange Factors/genetics , Legionella pneumophila/genetics , Legionella pneumophila/metabolism , Mice , Mutation , Protein Transport , Vacuoles/metabolism , Vacuoles/microbiology , rab1 GTP-Binding Proteins/genetics
19.
J Cell Biol ; 161(5): 853-9, 2003 Jun 09.
Article in English | MEDLINE | ID: mdl-12782685

ABSTRACT

Although cell movement is driven by actin, polarization and directional locomotion require an intact microtubule cytoskeleton that influences polarization by modulating substrate adhesion via specific targeting interactions with adhesion complexes. The fidelity of adhesion site targeting is precise; using total internal reflection fluorescence microscopy (TIRFM), we now show microtubule ends (visualized by incorporation of GFP tubulin) are within 50 nm of the substrate when polymerizing toward the cell periphery, but not when shrinking from it. Multiple microtubules sometimes followed similar tracks, suggesting guidance along a common cytoskeletal element. Use of TIRFM with GFP- or DsRed-zyxin in combination with either GFP-tubulin or GFP-CLIP-170 further revealed that the polymerizing microtubule plus ends that tracked close to the dorsal surface consistently targeted substrate adhesion complexes. This supports a central role for the microtubule tip complex in the guidance of microtubules into adhesion foci, and provides evidence for an intimate cross-talk between microtubule tips and substrate adhesions in the range of molecular dimensions.


Subject(s)
Cell Adhesion/physiology , Cell Membrane/metabolism , Eukaryotic Cells/metabolism , Focal Adhesions/metabolism , Microtubules/metabolism , Animals , Eukaryotic Cells/cytology , Goldfish , Green Fluorescent Proteins , Luminescent Proteins , Microscopy, Fluorescence , Microtubule-Associated Proteins/metabolism , Molecular Structure , Neoplasm Proteins , Polymers/metabolism , Recombinant Fusion Proteins , Tubulin/metabolism
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