Autophagy Monitoring Assay II: Imaging Autophagy Induction in LLC-PK1 Cells Using GFP-LC3 Protein Fusion Construct

2017 ◽  
pp. 211-219 ◽  
Author(s):  
Pavan P. Adiseshaiah ◽  
Sarah L. Skoczen ◽  
Jamie C. Rodriguez ◽  
Timothy M. Potter ◽  
Krishna Kota ◽  
...  
Keyword(s):  
Protein Fusion ◽  
Fusion Construct ◽  
Autophagy Induction

scholarly journals Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools

1998 ◽  
Vol 143 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Péter Várnai ◽  
Tamás Balla
Keyword(s):  
Fluorescent Probe ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Protein Fusion ◽  
Fusion Construct ◽  
Cellular Expression ◽  
Ph Domains ◽  
Green Fluorescent

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol– labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.

Molecular characterization of volume-sensitive SKCachannels in human liver cell lines

2002 ◽  
Vol 282 (1) ◽  
pp. G116-G122 ◽  
Author(s):  
Richard Roman ◽  
Andrew P. Feranchak ◽  
Marlyn Troetsch ◽  
Jeffrey C. Dunkelberg ◽  
Gordon Kilic ◽  
...  
Keyword(s):  
Cell Volume ◽  
Human Liver ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Chinese Hamster Ovary Cells ◽  
Cell Volume Regulation ◽  
Chinese Hamster ◽  
Protein Fusion ◽  
Fusion Construct ◽  
Human Liver Cell

In human liver, Ca2+-dependent changes in membrane K+permeability play a central role in coordinating functional interactions between membrane transport, metabolism, and cell volume. On the basis of the observation that K+conductance is partially sensitive to the bee venom toxin apamin, we aimed to assess whether small-conductance Ca2+-sensitive K+(SKCa) channels are expressed endogenously and contribute to volume-sensitive K+efflux and cell volume regulation. We isolated a full-length 2,140-bp cDNA (hSK2) highly homologous to rat brain rSK2 cDNA, including the putative apamin-sensitive pore domain, from a human liver cDNA library. Identical cDNAs were isolated from primary human hepatocytes, human HuH-7 hepatoma cells, and human Mz-ChA-1 cholangiocarcinoma cells. Transduction of Chinese hamster ovary cells with a recombinant adenovirus encoding the hSK2-green fluorescent protein fusion construct resulted in expression of functional apamin-sensitive K+channels. In Mz-ChA-1 cells, hypotonic (15% less sodium glutamate) exposure increased K+current density (1.9 ± 0.2 to 37.5 ± 7.1 pA/pF; P < 0.001). Apamin (10–100 nM) inhibited K+current activation and cell volume recovery from swelling. Apamin-sensitive SKCachannels are functionally expressed in liver and biliary epithelia and likely contribute to volume-sensitive changes in membrane K+permeability. Accordingly, the hSK2 protein is a potential target for pharmacological modulation of liver transport and metabolism through effects on membrane K+permeability.

scholarly journals Dynamic Distribution of Chemoattractant Receptors in Living Cells During Chemotaxis and Persistent Stimulation

1997 ◽  
Vol 139 (2) ◽  
pp. 365-374 ◽  
Author(s):  
Zhan Xiao ◽  
Ning Zhang ◽  
Douglas B. Murphy ◽  
Peter N. Devreotes
Keyword(s):  
Single Cells ◽  
Chimeric Protein ◽  
Green Fluorescence Protein ◽  
Living Cells ◽  
Cell Polarization ◽  
Asymmetric Distribution ◽  
Protein Fusion ◽  
Surface Binding ◽  
Distribution Profile ◽  
Fusion Construct

While the localization of chemoattractant receptors on randomly oriented cells has been previously studied by immunohistochemistry, the instantaneous distribution of receptors on living cells undergoing directed migration has not been determined. To do this, we replaced cAR1, the primary cAMP receptor of Dictyostelium, with a cAR1-green fluorescence protein fusion construct. We found that this chimeric protein is functionally indistinguishable from wild-type cAR1. By time-lapse imaging of single cells, we observed that the receptors remained evenly distributed on the cell surface and all of its projections during chemotaxis involving turns and reversals of polarity directed by repositioning of a chemoattractant-filled micropipet. Thus, cell polarization cannot result from a gradient-induced asymmetric distribution of chemoattractant receptors. Some newly extended pseudopods at migration fronts showed a transient drop in fluorescence signals, suggesting that the flow of receptors into these zones may slightly lag behind the protrusion process. Challenge with a uniform increase in chemoattractant, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile. Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization. The chimeric receptors were able to “cap” rapidly during treatment with Con A, suggesting that they are mobile in the plane of the cell membrane. This capping was not influenced by pretreatment with chemoattractant.

scholarly journals A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0125010 ◽  
Author(s):  
Matthew C. Clifton ◽  
David M. Dranow ◽  
Alison Leed ◽  
Ben Fulroth ◽  
James W. Fairman ◽  
...  
Keyword(s):  
Binding Protein ◽  
Maltose Binding Protein ◽  
Protein Fusion ◽  
Fusion Construct ◽  
Maltose Binding Protein Fusion

Early signalling events of autophagy

2013 ◽  
Vol 55 ◽  
pp. 1-15 ◽  
Author(s):  
Laura E. Gallagher ◽  
Edmond Y.W. Chan
Keyword(s):  
Protein Kinase ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Mammalian Cells ◽  
Mammalian Target Of Rapamycin ◽  
Interacting Protein ◽  
The Core ◽  
Autophagy Gene ◽  
Autophagy Induction ◽  
Cellular Pathways

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1–ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1–ULK1 signalling module.

Autophagy and ageing: implications for age-related neurodegenerative diseases

2013 ◽  
Vol 55 ◽  
pp. 119-131 ◽  
Author(s):  
Bernadette Carroll ◽  
Graeme Hewitt ◽  
Viktor I. Korolchuk
Keyword(s):  
Neurodegenerative Diseases ◽  
Energy Availability ◽  
Future Studies ◽  
Intracellular Degradation ◽  
Age Related ◽  
Autophagy Induction ◽  
Social Importance ◽  
Cellular Dysfunction ◽  
Autophagy Activity ◽  
Intense Research

Autophagy is a process of lysosome-dependent intracellular degradation that participates in the liberation of resources including amino acids and energy to maintain homoeostasis. Autophagy is particularly important in stress conditions such as nutrient starvation and any perturbation in the ability of the cell to activate or regulate autophagy can lead to cellular dysfunction and disease. An area of intense research interest is the role and indeed the fate of autophagy during cellular and organismal ageing. Age-related disorders are associated with increased cellular stress and assault including DNA damage, reduced energy availability, protein aggregation and accumulation of damaged organelles. A reduction in autophagy activity has been observed in a number of ageing models and its up-regulation via pharmacological and genetic methods can alleviate age-related pathologies. In particular, autophagy induction can enhance clearance of toxic intracellular waste associated with neurodegenerative diseases and has been comprehensively demonstrated to improve lifespan in yeast, worms, flies, rodents and primates. The situation, however, has been complicated by the identification that autophagy up-regulation can also occur during ageing. Indeed, in certain situations, reduced autophagosome induction may actually provide benefits to ageing cells. Future studies will undoubtedly improve our understanding of exactly how the multiple signals that are integrated to control appropriate autophagy activity change during ageing, what affect this has on autophagy and to what extent autophagy contributes to age-associated pathologies. Identification of mechanisms that influence a healthy lifespan is of economic, medical and social importance in our ‘ageing’ world.

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