scholarly journals Single cell optical transfection

2010 ◽  
Vol 7 (47) ◽  
pp. 863-871 ◽  
Author(s):  
David J. Stevenson ◽  
Frank J. Gunn-Moore ◽  
Paul Campbell ◽  
Kishan Dholakia
Keyword(s):  
Plasma Membrane ◽  
Single Cell ◽  
Laser Field ◽  
Small Interfering Rna ◽  
Eukaryotic Cell ◽  
Optical Injection ◽  
Cell Transfection ◽  
Gene Gun ◽  
Transfection Method ◽  
Interfering Rna

The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist—the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology.

scholarly journals Synaptotagmin VII Is Targeted to Dense-core Vesicles and Regulates Their Ca2+-dependent Exocytosis in PC12 Cells

2004 ◽  
Vol 279 (50) ◽  
pp. 52677-52684 ◽  
Author(s):  
Mitsunori Fukuda ◽  
Eiko Kanno ◽  
Megumi Satoh ◽  
Chika Saegusa ◽  
Akitsugu Yamamoto
Keyword(s):  
Plasma Membrane ◽  
Neuropeptide Y ◽  
Cell Line ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Small Interfering Rna ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Pc12 Cell Line ◽  
Interfering Rna

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membraneversussecretory granules). In this study we established a PC12 cell line “stably expressing” the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as thetrans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+-dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (∼60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (∼85–90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expressionversusstable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+sensor for exocytosis in endocrine cells.

scholarly journals Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase IIIα

2008 ◽  
Vol 19 (2) ◽  
pp. 711-721 ◽  
Author(s):  
Andras Balla ◽  
Yeun Ju Kim ◽  
Peter Varnai ◽  
Zsofia Szentpetery ◽  
Zachary Knight ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Small Interfering Rna ◽  
Type Ii ◽  
Measurable Effect ◽  
Down Regulation ◽  
Type Iii ◽  
Hormone Sensitive ◽  
Phosphatidylinositol 4 ◽  
Interfering Rna

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca2+ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca2+ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and Ca2+ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P2 levels in 32P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P2 resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca2+ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.

scholarly journals The Molecular Mechanism of Hepcidin-mediated Ferroportin Down-Regulation

2007 ◽  
Vol 18 (7) ◽  
pp. 2569-2578 ◽  
Author(s):  
Ivana De Domenico ◽  
Diane McVey Ward ◽  
Charles Langelier ◽  
Michael B. Vaughn ◽  
Elizabeta Nemeth ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanism ◽  
Small Interfering Rna ◽  
Multivesicular Body ◽  
Transport Proteins ◽  
Late Endosome ◽  
Phosphorylation Sites ◽  
Down Regulation ◽  
Green Fluorescent ◽  
Interfering Rna

Ferroportin (Fpn) is the only known iron exporter in vertebrates. Hepcidin, a peptide secreted by the liver in response to iron or inflammation, binds to Fpn, inducing its internalization and degradation. We show that after binding of hepcidin, Fpn is tyrosine phosphorylated at the plasma membrane. Mutants of human Fpn that do not get internalized or that are internalized slowly show either absent or impaired phosphorylation. We identify adjacent tyrosines as the phosphorylation sites and show that mutation of both tyrosines prevents hepcidin-mediated Fpn internalization. Once internalized, Fpn is dephosphorylated and subsequently ubiquitinated. An inability to ubiquitinate Fpn does not prevent hepcidin-induced internalization, but it inhibits the degradation of Fpn. Ubiquitinated Fpn is trafficked through the multivesicular body pathway en route to degradation in the late endosome/lysosome. Depletion of proteins involved in multivesicular body trafficking (Endosome Sorting Complex Required for Transport proteins), by small-interfering RNA, reduces the trafficking of Fpn-green fluorescent to the lysosome.

scholarly journals Facilitative Glucose Transporter 9 Expression Affects Glucose Sensing in Pancreatic β-Cells

Endocrinology ◽  
2009 ◽  
Vol 150 (12) ◽  
pp. 5302-5310 ◽  
Author(s):  
Sarah A. Evans ◽  
Manuel Doblado ◽  
Maggie M. Chi ◽  
John A. Corbett ◽  
Kelle H. Moley
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Small Interfering Rna ◽  
Glucose Sensing ◽  
Low Density ◽  
Β Cells ◽  
Microsome Fraction ◽  
Glucose Stimulated Insulin Secretion ◽  
Rna Knockdown ◽  
Interfering Rna

Abstract Facilitative glucose transporters (GLUTs) including GLUT9, accelerate the facilitative diffusion of glucose across the plasma membrane. Studies in GLUT2-deficient mice suggested the existence of another GLUT in the mammalian β-cell responsible for glucose sensing. The objective of this study was to determine the expression and function of GLUT9 in murine and human β-cells. mRNA and protein expression levels were determined for both isoforms of GLUT9 in murine and human isolated islets as well as insulinoma cell lines (MIN6). Immunohistochemistry and subcellular localization were performed to localize the protein within the cell. Small interfering RNA knockdown of GLUT9 was used to determine the effect of this transporter, in the presence of GLUT2, on cell metabolism and insulin secretion in MIN6 and INS cells. In this report we demonstrate that GLUT9a and GLUT9b are expressed in pancreatic islets and that this expression localizes to insulin-containing β-cells. Subcellular localization studies indicate that mGLUT9b is found associated with the plasma membrane as well as in the high-density microsome fraction and low-density microsome fraction, whereas mGLUT9a appears to be located only in the high-density microsome and low-density microsome under basal conditions. Functionally GLUT9 appears to participate in the regulation of glucose-stimulated insulin secretion in addition to GLUT2. small interfering RNA knockdown of GLUT9 results in reduced cellular ATP levels that correlate with reductions in glucose-stimulated insulin secretion in MIN6 and INS cells. These studies confirm the expression of GLUT9a and GLUT9b in murine and human β-cells and suggest that GLUT9 may participate in glucose-sensing in β-cells.

scholarly journals Requirement for Chloride Channel Function during the Hepatitis C Virus Life Cycle

2015 ◽  
Vol 89 (7) ◽  
pp. 4023-4029 ◽  
Author(s):  
Zsofia Igloi ◽  
Bjorn-Patrick Mohl ◽  
Jonathan D. Lippiat ◽  
Mark Harris ◽  
Jamel Mankouri
Keyword(s):  
Plasma Membrane ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Chloride Channel ◽  
Small Interfering Rna ◽  
Channel Blockers ◽  
Virus Life Cycle ◽  
Channel Inhibition ◽  
Interfering Rna

Hepatocytes express an array of plasma membrane and intracellular ion channels, yet their role during the hepatitis C virus (HCV) life cycle remains largely undefined. Here, we show that HCV increases intracellular hepatic chloride (Cl−) influx that can be inhibited by selective Cl−channel blockers. Through pharmacological and small interfering RNA (siRNA)-mediated silencing, we demonstrate that Cl−channel inhibition is detrimental to HCV replication. This represents the first observation of the involvement of Cl−channels during the HCV life cycle.

Phospholipase C-dependent Ca2+-sensing pathways leading to endocytosis and inhibition of the plasma membrane vacuolar H+-ATPase in osteoclasts

2010 ◽  
Vol 299 (3) ◽  
pp. C570-C578 ◽  
Author(s):  
Hiromu Sakai ◽  
Yoshie Moriura ◽  
Takuya Notomi ◽  
Junko Kawawaki ◽  
Keiko Ohnishi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Bone Resorption ◽  
Cell Surface ◽  
Phospholipase C ◽  
Small Interfering Rna ◽  
Proton Extrusion ◽  
Inhibitory Peptide ◽  
Low Levels ◽  
Interfering Rna ◽  
Cell Capacitance

In osteoclasts, elevation of extracellular Ca2+ is an endogenous signal that inhibits bone resorption. We recently found that an elevation of extracellular Ca2+ decreased proton extrusion through the plasma membrane vacuolar H+-ATPase (V-ATPase) rapidly. In this study we investigated mechanisms underlying this early Ca2+-sensing response, particularly in reference to the activity of the plasma membrane V-ATPase and to membrane retrieval. Whole cell clamp recordings allowed us to measure the V-ATPase currents and the cell capacitance ( Cm) simultaneously. Cm is a measure of cell surface. Extracellular Ca2+ (2.5–40 mM) decreased Cm and the V-ATPase current simultaneously. The decreased Cm, together with the enhanced uptake of a lipophilic dye (FM1–43), indicated that Ca2+ facilitated endocytosis. The endocytosis was blocked by dynamin inhibitors (dynasore and dynamin-inhibitory peptide), by small interfering RNA (siRNA) targeting for dynanmin-2 and also by bafilomycin A1, a blocker of V-ATPases. The extracellular Ca2+-induced endocytosis and inhibition of the V-ATPase current were diminished by a phospholipase C inhibitor (U73122) and siRNA targeting for phospholipase C γ2 subunit. Holding the cytosolic Ca2+ at either high (0.5–5 μM) or low levels or inhibiting calmodulin by an inhibitor (W7) or an antibody (anti-CaM) decreased the stimulated endocytosis and the inhibition of the V-ATPase current. These data suggest that extracellular Ca2+ facilitated dynamin- and V-ATPase-dependent endocytosis in association with an inhibition of the plasma membrane V-ATPase. Phospholipase C, cytosolic Ca2+, and calmodulin were involved in the signaling pathways. Membrane retrieval and the plasma membrane V-ATPase activity may cooperate during the early phase of Ca2+-sensing response in osteoclasts.

scholarly journals Mapping of R-SNARE function at distinct intracellular GLUT4 trafficking steps in adipocytes

2008 ◽  
Vol 180 (2) ◽  
pp. 375-387 ◽  
Author(s):  
Dumaine Williams ◽  
Jeffrey E. Pessin
Keyword(s):  
Plasma Membrane ◽  
Small Interfering Rna ◽  
Osmotic Shock ◽  
Snare Proteins ◽  
Fusion Reactions ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Site Of Action ◽  
Interfering Rna

The functional trafficking steps used by soluble NSF attachment protein receptor (SNARE) proteins have been difficult to establish because of substantial overlap in subcellular localization and because in vitro SNARE-dependent binding and fusion reactions can be promiscuous. Therefore, to functionally identify the site of action of the vesicle-associated membrane protein (VAMP) family of R-SNAREs, we have taken advantage of the temporal requirements of adipocyte biosynthetic sorting of a dual-tagged GLUT4 reporter (myc-GLUT4-GFP) coupled with small interfering RNA gene silencing. Using this approach, we confirm the requirement of VAMP2 and VAMP7 for insulin and osmotic shock trafficking from the vesicle storage sites, respectively, and fusion with the plasma membrane. Moreover, we identify a requirement for VAMP4 for the initial biosynthetic entry of GLUT4 from the Golgi apparatus into the insulin-responsive vesicle compartment, VAMP8, for plasma membrane endocytosis and VAMP2 for sorting to the specialized insulin-responsive compartment after plasma membrane endocytosis.

scholarly journals AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture

2003 ◽  
Vol 164 (1) ◽  
pp. 133-144 ◽  
Author(s):  
Christelle Benaud ◽  
Benoît J. Gentil ◽  
Nicole Assard ◽  
Magalie Court ◽  
Jerome Garin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Small Interfering Rna ◽  
Kidney Cells ◽  
Cortical Actin ◽  
Madin Darby Canine Kidney ◽  
Annexin 2 ◽  
Interfering Rna ◽  
Darby Canine Kidney ◽  
Canine Kidney

Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell–cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca2+-dependent cell–cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2–specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

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