Ligand-Directed Acyl Imidazole Chemistry for Labeling of Membrane-Bound Proteins on Live Cells

2012 ◽  
Vol 134 (9) ◽  
pp. 3961-3964 ◽  
Author(s):  
Sho-hei Fujishima ◽  
Ryosuke Yasui ◽  
Takayuki Miki ◽  
Akio Ojida ◽  
Itaru Hamachi
Keyword(s):  
Live Cells ◽  
Membrane Bound ◽  
Acyl Imidazole

scholarly journals A Nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding

2020 ◽  
Author(s):  
Carl W. White ◽  
Kevin D. G. Pfleger ◽  
Stephen J. Hill
Keyword(s):  
Real Time ◽  
Genome Editing ◽  
Receptor Binding ◽  
Chemokine Receptors ◽  
Live Cells ◽  
Chemokine Cxcl12 ◽  
Membrane Bound ◽  
Low Levels ◽  
And Function ◽  
Steric Constraints

SummarySecreted chemokines are critical mediators of cellular communication that elicit intracellular signalling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. CRISPR/Cas9 genome-editing was used to tag the chemokine CXCL12 with the Nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of Nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analysed using BRET. These live cell approaches combine the sensitivity of Nanoluciferase with CRISPR/Cas9 genome-editing to detect, quantify and monitor binding of low levels of native secreted proteins in real time.

scholarly journals SSO and other putative inhibitors of FA transport across membranes by CD36 disrupt intracellular metabolism, but do not affect FA translocation

2020 ◽  
Vol 61 (5) ◽  
pp. 790-807 ◽  
Author(s):  
Anthony G. Jay ◽  
Jeffrey R. Simard ◽  
Nasi Huang ◽  
James A. Hamilton
Keyword(s):  
Biological Membranes ◽  
Lipid Vesicles ◽  
Competitive Inhibitor ◽  
Live Cells ◽  
Dual Fluorescence ◽  
Active Protein ◽  
Ph Indicator ◽  
Transporter Protein ◽  
Membrane Bound ◽  
Intracellular Metabolism

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2′,7′-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the “inhibitors” previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

scholarly journals Aptamer Directly Evolved from Live Cells Recognizes Membrane Bound Immunoglobin Heavy Mu Chain in Burkitt's Lymphoma Cells

2007 ◽  
Vol 6 (12) ◽  
pp. 2230-2238 ◽  
Author(s):  
Prabodhika Mallikaratchy ◽  
Zhiwen Tang ◽  
Sefah Kwame ◽  
Ling Meng ◽  
Dihua Shangguan ◽  
...  
Keyword(s):  
Burkitt’S Lymphoma ◽  
Burkitt's Lymphoma ◽  
Live Cells ◽  
Lymphoma Cells ◽  
Membrane Bound ◽  
Burkitt’S Lymphoma Cells

scholarly journals Local protein dynamics during microvesicle exocytosis in neuroendocrine cells

2018 ◽  
Vol 29 (15) ◽  
pp. 1891-1903 ◽  
Author(s):  
Agila Somasundaram ◽  
Justin W. Taraska
Keyword(s):  
Protein Dynamics ◽  
Spatial Dynamics ◽  
Neuroendocrine Cells ◽  
Live Cells ◽  
Rab Proteins ◽  
Vesicle Membrane ◽  
Physiological Processes ◽  
Associated Proteins ◽  
Membrane Bound ◽  
Local Protein

Calcium-triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine ACh transporter tagged to pHluorin) and the dynamics of associated proteins at the moments surrounding exocytosis. Our experiments show that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are preclustered at fusion sites and rapidly lost at fusion. The ATPase N-ethylmaleimide–sensitive factor is locally recruited at fusion. Interestingly, the endocytic Bin-Amphiphysin-Rvs domain–containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites and slow the loss of vesicle membrane-bound cargo from fusion sites. A similar effect on vesicle membrane protein dynamics was seen with the overexpression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis can regulate exocytosis of synaptic-like microvesicles. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key factors of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.

scholarly journals Activity-Based Sensing with a Metal-Directed Acyl Imidazole Strategy Reveals Cell Type-Dependent Pools of Labile Brain Copper

2020 ◽  
Author(s):  
Sumin Lee ◽  
Yik Sham Clive Chung ◽  
Pei Liu ◽  
Laura Craciun ◽  
Yuki Nishikawa ◽  
...  
Keyword(s):  
Spatial Information ◽  
Cell Types ◽  
Redox Activity ◽  
Live Cells ◽  
Cell Type ◽  
Disease States ◽  
Starting Point ◽  
Neural Signaling ◽  
The Brain ◽  
Acyl Imidazole

Copper is a required nutrient for life and particularly important to the brain and central nervous system. Indeed, copper redox activity is essential to maintaining normal physiological responses spanning neural signaling to metabolism, but at the same time copper misregulation is associat-ed with inflammation and neurodegeneration. As such, chemical probes that can track dynamic changes in copper with spatial resolution, espe-cially in loosely-bound, labile forms, are valuable tools to identify and characterize its contributions to healthy and disease states. In this report, we present an activity-based sensing (ABS) strategy for copper detection in live cells that preserves spatial information by a copper-dependent bioconjugation reaction. Specifically, we designed copper-directed acyl imidazole (CD) dyes that operate through copper-mediated activation of acyl imidazole electrophiles for subsequent labeling of proximal proteins at sites of elevated labile copper to provide a permanent stain that resists washing and fixation. To showcase the utility of this new ABS platform, we sought to characterize labile copper pools in the three main cell types in the brain: neurons, astrocytes, and microglia. Exposure of each of these cell types to physiologically relevant stimuli shows distinct changes in labile copper pools. Neurons display translocation of labile copper from somatic cell bodies to peripheral processes upon activation, whereas astrocytes and microglia exhibit global decreases and increases in intracellular labile copper pools, respectively, after exposure to inflam-matory stimuli. This work provides foundational information on cell type-dependent homeostasis of copper, an essential metal in the brain, as well as a starting point for the design of new activity-based probes for metals and other dynamic signaling and stress analytes in biology.

scholarly journals Single-Molecule Tracking of DNA Translocases inBacillus subtilisReveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA

2018 ◽  
Vol 84 (8) ◽  
pp. e02610-17 ◽  
Author(s):  
Nina El Najjar ◽  
Jihad El Andari ◽  
Christine Kaimer ◽  
Georg Fritz ◽  
Thomas C. Rösch ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Single Molecule ◽  
Average Dwell Time ◽  
Glycolytic Enzyme ◽  
Live Cells ◽  
Content Type ◽  
Dna Translocases ◽  
Membrane Bound

ABSTRACTLike many bacteria,Bacillus subtilispossesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: anftsAdeletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzymein vivo. Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slow- and fast-moving molecules in live cells.IMPORTANCEDNA translocases couple the late events of chromosome segregation to cell division and thereby play an important role in the bacterial cell cycle. The proteins fall into one of two categories, integral membrane translocases or nonintegral translocases. We show that the membrane-bound translocase SpoIIIE moves slowly throughout the cell membrane inB. subtilisand does not show a clear association with the division septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell division across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the division septum as a component of the division machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the division machinery, but it is not the only factor that binds SftA. Our work represents a detailedin vivostudy of DNA translocases at the single-molecule level.

scholarly journals Mapping the micro-proteome of the nuclear lamina and lamina-associated domains

2021 ◽  
Vol 4 (5) ◽  
pp. e202000774
Author(s):  
Xianrong Wong ◽  
Jevon A Cutler ◽  
Victoria E Hoskins ◽  
Molly Gordon ◽  
Anil K Madugundu ◽  
...  
Keyword(s):  
Large Fraction ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Mammalian Genome ◽  
Live Cells ◽  
H3k9 Methylation ◽  
Tracer System ◽  
Membrane Bound ◽  
Gene Regulatory ◽  
Regulatory Functions

The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, the so-called lamina-associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin-directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.

scholarly journals Mapping the micro-proteome of the nuclear lamina and lamin associated domains

2019 ◽  
Author(s):  
Jevon A. Cutler ◽  
Xianrong Wong ◽  
Victoria E. Hoskins ◽  
Molly Gordon ◽  
Anil K. Madugundu ◽  
...  
Keyword(s):  
Large Fraction ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Mammalian Genome ◽  
Live Cells ◽  
H3k9 Methylation ◽  
Tracer System ◽  
Membrane Bound ◽  
Gene Regulatory ◽  
Regulatory Functions

AbstractThe nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, so-called lamin associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.

scholarly journals Recent applications of N-acyl imidazole chemistry in chemical biology

2021 ◽  
Vol 85 (1) ◽  
pp. 53-60
Author(s):  
Takeharu Mino ◽  
Seiji Sakamoto ◽  
Itaru Hamachi
Keyword(s):  
Chemical Biology ◽  
Live Cells ◽  
Chemical Labeling ◽  
Solubility In Water ◽  
Native Proteins ◽  
Functional Control ◽  
Endogenous Proteins ◽  
Living Organisms ◽  
Chemical Selectivity ◽  
Acyl Imidazole

Abstract N-Acyl imidazoles are unique electrophiles that exhibit moderate reactivity, relatively long-half life, and high solubility in water. Thanks to their tunable reactivity and chemical selectivity, the application of N-acyl imidazole derivatives has launched to a number of chemical biology researches, which include chemical synthesis of peptide/protein, chemical labeling of native proteins of interest (POIs), and structural analysis and functional manipulation of RNAs. Since proteins and RNAs play pivotal roles in numerous biological events in all living organisms, the methods that enable the chemical modification of endogenously existing POIs and RNAs in live cells may offer a variety of opportunities not only for fundamental scientific study but also for biotechnology and drug development. In this review, we discuss the recent progress of N-acyl imidazole chemistry that contributes to the chemical labeling and functional control of endogenous proteins and RNAs under multimolecularly crowded biological conditions of live cells.

scholarly journals Exploring cells with targeted biosensors

2016 ◽  
Vol 149 (1) ◽  
pp. 1-36 ◽  
Author(s):  
Diana Pendin ◽  
Elisa Greotti ◽  
Konstantinos Lefkimmiatis ◽  
Tullio Pozzan
Keyword(s):  
Spatial Distribution ◽  
Temporal Pattern ◽  
Cellular Signaling ◽  
Live Cells ◽  
Signaling Networks ◽  
Form Complex ◽  
Intracellular Compartments ◽  
Membrane Bound ◽  
Cellular Compartments ◽  
Protein Sensors

Cellular signaling networks are composed of multiple pathways, often interconnected, that form complex networks with great potential for cross-talk. Signal decoding depends on the nature of the message as well as its amplitude, temporal pattern, and spatial distribution. In addition, the existence of membrane-bound organelles, which are both targets and generators of messages, add further complexity to the system. The availability of sensors that can localize to specific compartments in live cells and monitor their targets with high spatial and temporal resolution is thus crucial for a better understanding of cell pathophysiology. For this reason, over the last four decades, a variety of strategies have been developed, not only to generate novel and more sensitive probes for ions, metabolites, and enzymatic activity, but also to selectively deliver these sensors to specific intracellular compartments. In this review, we summarize the principles that have been used to target organic or protein sensors to different cellular compartments and their application to cellular signaling.

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