Time-lapse Imaging of Individual BKCa Channels in Live Cells Using Site-specific Labeling of Quantum Dots

The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP–Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

Download Full-text

Time-Lapse Imaging of Fluorescently Labeled Live Cells in the Embryonic Mammalian Forebrain

Cold Spring Harbor Protocols ◽

10.1101/pdb.err068353 ◽

2011 ◽

Vol 2012 (1) ◽

pp. pdb.err068353-pdb.err068353

Author(s):

S. C. Noctor

Keyword(s):

Time Lapse ◽

Live Cells ◽

Time Lapse Imaging

Download Full-text

A nanobody-based toolset to monitor and modify the mitochondrial GTPase Miro1

10.1101/2021.12.10.472061 ◽

2021 ◽

Author(s):

Funmilayo O Fagbadebo ◽

Philipp D Kaiser ◽

Katharina Zittlau ◽

Natascha Bartlick ◽

Teresa R Wagner ◽

...

Keyword(s):

Time Lapse ◽

Model Systems ◽

Live Cells ◽

Mitochondrial Outer Membrane ◽

Potential Biomarker ◽

Time Lapse Imaging ◽

Lateral Sclerosis ◽

Neuronal Disorders ◽

Research Tools

The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time-lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.

Download Full-text

Identification of multiple kinetic populations of DNA-binding proteins in live cells

10.1101/509620 ◽

2019 ◽

Cited By ~ 2

Author(s):

Han N. Ho ◽

Daniel Zalami ◽

Jürgen Köhler ◽

Antoine M. van Oijen ◽

Harshad Ghodke

Keyword(s):

Dna Binding ◽

Fluorescence Imaging ◽

Single Molecule ◽

Binding Proteins ◽

Dynamic Range ◽

Time Lapse ◽

Dna Binding Proteins ◽

Live Cells ◽

Single Molecule Fluorescence ◽

Time Lapse Imaging

ABSTRACTUnderstanding how multi-protein complexes function in cells requires detailed quantitative understanding of their association and dissociation kinetics. Analysis of the heterogeneity of binding lifetimes enables interrogation of the various intermediate states formed during the reaction. Single-molecule fluorescence imaging permits the measurement of reaction kinetics inside living organisms with minimal perturbation. However, poor photo-physical properties of fluorescent probes limit the dynamic range and accuracy of measurements of off rates in live cells. Time-lapse single-molecule fluorescence imaging can partially overcome the limits of photobleaching, however, limitations of this technique remain uncharacterized. Here, we present a structured analysis of which timescales are most accessible using the time-lapse imaging approach and explore uncertainties in determining kinetic sub-populations. We demonstrate the effect of shot noise on the precision of the measurements, as well as the resolution and dynamic range limits that are inherent to the method. Our work provides a convenient implementation to determine theoretical errors from measurements and to support interpretation of experimental data.STATEMENT OF SIGNIFICANCEMeasuring lifetimes of interactions between DNA-binding proteins and their substrates is important for understanding how they function in cells. In principle, time-lapse imaging of fluorescently-tagged proteins using single-molecule methods can be used to identify multiple sub-populations of DNA-binding proteins and determine binding lifetimes lasting for several tens of minutes. Despite this potential, currently available guidelines for the selection of binding models are unreliable, and the practical implementation of this approach is limited. Here, using experimental and simulated data we identify the minimum size of the dataset required to resolve multiple populations reliably and measure binding lifetimes with desired accuracy. This work serves to provide a guide to data collection, and measurement of DNA-binding lifetimes from single-molecule time-lapse imaging data.

Download Full-text

Nuclear Aggresomes Form by Fusion of PML-associated Aggregates

Molecular Biology of the Cell ◽

10.1091/mbc.e05-01-0019 ◽

2005 ◽

Vol 16 (10) ◽

pp. 4905-4917 ◽

Cited By ~ 55

Author(s):

Lianwu Fu ◽

Ya-sheng Gao ◽

Albert Tousson ◽

Anish Shah ◽

Tung-Ling L. Chen ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Fluorescence Recovery After Photobleaching ◽

Nuclear Architecture ◽

Time Lapse ◽

Promyelocytic Leukemia ◽

Live Cells ◽

Aggregate Formation ◽

Pml Bodies ◽

Nuclear Domains ◽

Time Lapse Imaging

Nuclear aggregates formed by proteins containing expanded poly-glutamine (poly-Q) tracts have been linked to the pathogenesis of poly-Q neurodegenerative diseases. Here, we show that a protein (GFP170*) lacking poly-Q tracts forms nuclear aggregates that share characteristics of poly-Q aggregates. GFP170*aggregates recruit cellular chaperones and proteasomes, and alter the organization of nuclear domains containing the promyelocytic leukemia (PML) protein. These results suggest that the formation of nuclear aggregates and their effects on nuclear architecture are not specific to poly-Q proteins. Using GFP170*as a model substrate, we explored the mechanistic details of nuclear aggregate formation. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses show that GFP170*molecules exchange rapidly between aggregates and a soluble pool of GFP170*, indicating that the aggregates are dynamic accumulations of GFP170*. The formation of cytoplasmic and nuclear GFP170*aggregates is microtubule-dependent. We show that within the nucleus, GFP170*initially deposits in small aggregates at or adjacent to PML bodies. Time-lapse imaging of live cells shows that small aggregates move toward each other and fuse to form larger aggregates. The coalescence of the aggregates is accompanied by spatial rearrangements of the PML bodies. Significantly, we find that the larger nuclear aggregates have complex internal substructures that reposition extensively during fusion of the aggregates. These studies suggest that nuclear aggregates may be viewed as dynamic multidomain inclusions that continuously remodel their components.

Download Full-text

Time-Lapse Imaging of Fluorescently Labeled Live Cells in the Embryonic Mammalian Forebrain

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot066605 ◽

2011 ◽

Vol 2011 (11) ◽

pp. pdb.prot066605-pdb.prot066605 ◽

Cited By ~ 4

Author(s):

S. C. Noctor

Keyword(s):

Time Lapse ◽

Live Cells ◽

Time Lapse Imaging

Download Full-text

Ni–nitrilotriacetic acid-modified quantum dots as a site-specific labeling agent of histidine-tagged proteins in live cells

Chemical Communications ◽

10.1039/b719434j ◽

2008 ◽

pp. 1910 ◽

Cited By ~ 41

Author(s):

Junwon Kim ◽

Hye-Young Park ◽

Jaeseung Kim ◽

Jiyoung Ryu ◽

Do Yoon Kwon ◽

...

Keyword(s):

Quantum Dots ◽

Nitrilotriacetic Acid ◽

Live Cells ◽

Site Specific ◽

A Site

Download Full-text

Direct Visualization of Amlodipine Intervention into Living Cells by Means of Fluorescence Microscopy

Molecules ◽

10.3390/molecules26102997 ◽

2021 ◽

Vol 26 (10) ◽

pp. 2997

Author(s):

Christine Quentin ◽

Rūta Gerasimaitė ◽

Alexandra Freidzon ◽

Levon S. Atabekyan ◽

Gražvydas Lukinavičius ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Aqueous Solutions ◽

Time Lapse ◽

Drug Uptake ◽

Live Cells ◽

Direct Visualization ◽

Cellular Environment ◽

Time Lapse Imaging ◽

The Impact

Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring.

Download Full-text