Delivery of nanoparticles inside the cytosol of live cells for the monitoring of cellular processes (Conference Presentation)

2018 ◽  
Author(s):  
Xizhen Lian ◽  
Alfredo Erazo-Oliveras ◽  
Hong-Cai Zhou ◽  
Jean-Philippe Pellois
Keyword(s):  
Live Cells ◽  
Cellular Processes

scholarly journals Intracellular Ionic Strength Sensing Using NanoLuc

2021 ◽  
Vol 22 (2) ◽  
pp. 677
Author(s):  
Tausif Altamash ◽  
Wesam Ahmed ◽  
Saad Rasool ◽  
Kabir H. Biswas
Keyword(s):  
Thermal Stability ◽  
Phase Separation ◽  
Drug Discovery ◽  
Ionic Strength ◽  
Cellular Signaling ◽  
Live Cells ◽  
Protein Activity ◽  
Cellular Survival ◽  
Cellular Processes ◽  
Wide Range

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

scholarly journals Assay Systems for Profiling Deubiquitinating Activity

2020 ◽  
Vol 21 (16) ◽  
pp. 5638
Author(s):  
Jinhong Cho ◽  
Jinyoung Park ◽  
Eunice EunKyeong Kim ◽  
Eun Joo Song
Keyword(s):  
Protein Degradation ◽  
Protein Interactions ◽  
Live Cells ◽  
Deubiquitinating Enzyme ◽  
Protein Protein Interactions ◽  
Deubiquitinating Enzymes ◽  
Cell Lysates ◽  
Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

scholarly journals Multiplexed dynamic imaging of genomic loci in single cells by combined CRISPR imaging and DNA sequential FISH

2017 ◽  
Author(s):  
Yodai Takei ◽  
Sheel Shah ◽  
Sho Harvey ◽  
Lei S. Qi ◽  
Long Cai
Keyword(s):  
Single Cells ◽  
Embryonic Stem ◽  
Dynamic Imaging ◽  
Live Cells ◽  
Chromosome Dynamics ◽  
Cellular Processes ◽  
Sequential Fish ◽  
Telomere Dynamics ◽  
Subtelomeric Regions

ABSTRACTVisualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labelled and uniquely identified. Here we describe an approach we call “track first and identify later” for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR labeling and DNA sequential fluorescence in situ hybridization (DNA seqFISH). Our approach first labels and tracks chromosomal loci in live cells with the CRISPR system, then barcodes those loci by DNA seqFISH in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.

scholarly journals Fabrication, Characterization and Application of Biomolecule Micropatterns on Cyclic Olefin Polymer (COP) Surfaces with Adjustable Contrast

Biosensors ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 3
Author(s):  
Roland Hager ◽  
Thomas Haselgrübler ◽  
Sandra Haas ◽  
Anna-Maria Lipp ◽  
Julian Weghuber
Keyword(s):  
Protein Interactions ◽  
Total Internal Reflection ◽  
Microcontact Printing ◽  
Activation Parameters ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Promising Alternative ◽  
Cyclic Olefin ◽  
Glass Substrates ◽  
Cellular Processes

Peptide and protein micropatterns are powerful tools for the investigation of various cellular processes, including protein–protein interactions (PPIs). Within recent years, various approaches for the production of functional surfaces have been developed. Most of these systems use glass as a substrate, which has several drawbacks, including high fragility and costs, especially if implemented for fluorescence microscopy. In addition, conventional fabrication technologies such as microcontact printing (µCP) are frequently used for the transfer of biomolecules to the glass surface. In this case, it is challenging to adjust the biomolecule density. Here, we show that cyclic olefin polymer (COP) foils, with their encouraging properties, including the ease of manufacturing, chemical resistance, biocompatibility, low water absorption, and optical clarity, are a promising alternative to glass substrates for the fabrication of micropatterns. Using a photolithography-based approach, we generated streptavidin/biotinylated antibody patterns on COPs with the possibility of adjusting the pattern contrast by varying plasma activation parameters. Our experimental setup was finally successfully implemented for the analysis of PPIs in the membranes of live cells via total internal reflection fluorescence (TIRF) microscopy.

Visualizing membrane trafficking using total internal reflection fluorescence microscopy

2003 ◽  
Vol 31 (4) ◽  
pp. 819-823 ◽  
Author(s):  
V. Beaumont
Keyword(s):  
Fluorescence Microscopy ◽  
Membrane Trafficking ◽  
Cell Biology ◽  
Total Internal Reflection ◽  
Optical Resolution ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Live Cells ◽  
Excitable Cells ◽  
Cellular Processes

There is a dizzying array of fluorescent probes now commercially available to monitor cellular processes, and advances in molecular biology have highlighted the ease with which proteins can now be labelled with fluorophores without loss of functionality. This has led to an explosion in the popularity of fluorescence microscopy techniques. One such specialized technique, total internal reflection fluorescence microscopy (TIR-FM), is ideally suited to gaining insight into events occurring at, or close to, the plasma membrane of live cells with excellent optical resolution. In the last few years, the application of TIR-FM to membrane trafficking events in both non-excitable and excitable cells has been an area of notable expansion and fruition. This review gives a brief overview of that literature, with emphasis on the study of the regulation of exocytosis and endocytosis in excitable cells using TIR-FM. Finally, recent applications of TIR-FM to the study of cellular processes at the molecular level are discussed briefly, providing promise that the future of TIR-FM in cell biology will only get brighter.

scholarly journals Contact-ID, a tool for profiling organelle contact sites, reveals regulatory proteins of mitochondrial-associated membrane formation

2020 ◽  
Vol 117 (22) ◽  
pp. 12109-12120 ◽  
Author(s):  
Chulhwan Kwak ◽  
Sanghee Shin ◽  
Jong-Seok Park ◽  
Minkyo Jung ◽  
Truong Thi My Nhung ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Regulatory Proteins ◽  
Live Cells ◽  
Strong Activity ◽  
Cellular Processes ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Specific Proteins ◽  
Molecular Components ◽  
Organelle Membrane

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle–membrane contact sites in live cells.

scholarly journals CHIMs are versatile cholesterol analogs mimicking and visualizing cholesterol behavior in lipid bilayers and cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Anna L. L. Matos ◽  
Fabian Keller ◽  
Tristan Wegner ◽  
Carla Elizabeth Cadena del Castillo ◽  
David Grill ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Structural Integrity ◽  
Phospholipid Bilayers ◽  
Live Cells ◽  
Computational Studies ◽  
Biologically Relevant ◽  
Cellular Processes ◽  
Limited Applicability ◽  
And Function

AbstractCholesterol is an essential component of cellular membranes regulating the structural integrity and fluidity of biological bilayers and cellular processes such as signal transduction and membrane trafficking. However, tools to investigate the role and dynamics of cholesterol in live cells are still scarce and often show limited applicability. To address this, we previously developed a class of imidazolium-based cholesterol analogs, CHIMs. Here we confirm that CHIM membrane integration characteristics largely mimic those of cholesterol. Computational studies in simulated phospholipid bilayers and biophysical analyses of model membranes reveal that in biologically relevant systems CHIMs behave similarly to natural cholesterol. Importantly, the analogs can functionally replace cholesterol in membranes, can be readily labeled by click chemistry and follow trafficking pathways of cholesterol in live cells. Thus, CHIMs represent chemically versatile cholesterol analogs that can serve as a flexible toolbox to study cholesterol behavior and function in live cells and organisms.

scholarly journals Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

2019 ◽  
Author(s):  
Adam D. Cawte ◽  
Peter J. Unrau ◽  
David S. Rueda
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Live Cell ◽  
Rna Aptamers ◽  
Live Cells ◽  
Structured Illumination ◽  
Rna Molecules ◽  
Cellular Processes ◽  
Rna Imaging

AbstractRNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to fluorophore exchange. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

scholarly journals Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

2020 ◽  
Author(s):  
Bo Meng ◽  
Pedro P. Vallejo Ramirez ◽  
Katharina M. Scherer ◽  
Ezra Bruggeman ◽  
Julia C. Kenyon ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Biological Process ◽  
Full Length ◽  
Live Cells ◽  
Fluorescent Labelling ◽  
Endosomal Membrane ◽  
Cellular Processes ◽  
N Terminus ◽  
Main Component

The scission of lipid membranes is a common biological process, often mediated by ESCRT complexes in concert with VPS4 assembling around the separation point. The functions of the ESCRT-I and ESCRT-III complexes are well established in certain of these cellular processes; however, the role of ESCRT-II remains contentious. Here, we devised a SNAP-tag fluorescent labelling strategy to understand the domain requirements of EAP45, the main component of ESCRT-II, in HIV egress, late endosome recruitment, and cytokinesis. We used TIRF microscopy to measure the spatial co-occurrence of the HIV structural polyprotein Gag with full length EAP45 in both fixed and live cells. Gag colocalises with the full length EAP45 comparably to ALIX, but this is lost on deletion of the EAP45 N terminus. Our findings reveal the H0 domain of the EAP45 protein is essential for linking to ESCRT-I during HIV budding and in anchoring at the late endosomal membrane, however in cytokinesis it is the Glue domain that is critical.

Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications

Lab on a Chip ◽  
2016 ◽  
Vol 16 (11) ◽  
pp. 2014-2024 ◽  
Author(s):  
Lip Ket Chin ◽  
Chau-Hwang Lee ◽  
Bi-Chang Chen
Keyword(s):  
Imaging Techniques ◽  
Super Resolution ◽  
Lab On A Chip ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
Cellular Processes ◽  
High Spatiotemporal Resolution ◽  
Microfluidic Technology ◽  
Resolution Imaging

The synergy of novel super-resolution imaging techniques and microfluidic technology provides new biological and biomedical insights into sub-cellular processes.

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