Recognition-driven chemical labeling of endogenous proteins in multi-molecular crowding in live cells

2017 ◽  
Vol 53 (88) ◽  
pp. 11972-11983 ◽  
Author(s):  
Kazuma Amaike ◽  
Tomonori Tamura ◽  
Itaru Hamachi
Keyword(s):  
Protein Labeling ◽  
Live Cells ◽  
Molecular Crowding ◽  
Chemical Labeling ◽  
Endogenous Protein ◽  
Bona Fide ◽  
Endogenous Proteins

Endogenous protein labeling is one of the most invaluable methods for studying the bona fide functions of proteins in live cells.

scholarly journals Improved Split Fluorescent Proteins for Endogenous Protein Labeling

2017 ◽  
Author(s):  
Siyu Feng ◽  
Sayaka Sekine ◽  
Veronica Pessino ◽  
Han Li ◽  
Manuel D. Leonetti ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Protein Localization ◽  
Super Resolution ◽  
Screening Strategy ◽  
Protein Labeling ◽  
Cell Contact ◽  
Endogenous Protein ◽  
Endogenous Proteins

ABSTRACTSelf-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split-GFP1-10/11, as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split-sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.

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 Modular transient nanoclustering of activated β2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies

2020 ◽  
Vol 117 (48) ◽  
pp. 30476-30487 ◽  
Author(s):  
Rachel S. Gormal ◽  
Pranesh Padmanabhan ◽  
Ravikiran Kasula ◽  
Adekunle T. Bademosi ◽  
Sean Coakley ◽  
...  
Keyword(s):  
Single Molecule ◽  
Neurosecretory Cells ◽  
Single Particle Tracking ◽  
Single Domain ◽  
Live Cells ◽  
Conformational States ◽  
Superresolution Microscopy ◽  
Endogenous Protein ◽  
Endogenous Proteins ◽  
Microscopy Techniques

None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous β2-adrenoreceptor (β2-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated β2-AR(Nb80) is highly immobile and organized in nanoclusters. The Gαs−GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated β2-AR(Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.

Superfast Tetrazole–BCN Cycloaddition Reaction for Bioorthogonal Protein Labeling on Live Cells

2021 ◽  
Author(s):  
Gangam Srikanth Kumar ◽  
Stefano Racioppi ◽  
Eva Zurek ◽  
Qing Lin
Keyword(s):  
Cycloaddition Reaction ◽  
Protein Labeling ◽  
Live Cells

scholarly journals The CaMKII holoenzyme structure in activation-competent conformations

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Janette B. Myers ◽  
Vincent Zaegel ◽  
Steven J. Coultrap ◽  
Adam P. Miller ◽  
K. Ulrich Bayer ◽  
...  
Keyword(s):  
Molecular Crowding ◽  
Extended State ◽  
Dependent Protein Kinase ◽  
Subunit Exchange ◽  
Regulatory Processes ◽  
Predominant Form ◽  
Dependent Protein ◽  
Bona Fide ◽  
Additional Level ◽  
Compact Conformation

Abstract The Ca2+/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15–35 nm in diameter. While most kinase domains are ordered independently, ∼20% appear to form dimers and <3% are consistent with a compact conformation. An additional level of plasticity is revealed by a small fraction of bona-fide 14-mers (<4%) that may enable subunit exchange. Biochemical and cellular FRET studies confirm that the extended state of CaMKIIα resolved by EM is the predominant form of the holoenzyme, even under molecular crowding conditions.

scholarly journals Fluorescent reporters, calibration standards and endogenous protein labeling for quantitative single-molecule localization microscopy in cells

2021 ◽  
Author(s):  
◽  
Tim Niklas Baldering
Keyword(s):  
Single Molecule ◽  
Protein Labeling ◽  
Calibration Standards ◽  
Localization Microscopy ◽  
Fluorescent Reporters ◽  
Endogenous Protein ◽  
Single Molecule Localization Microscopy ◽  
In Cells

Die Kommunikation von Zellen mit ihrer Umgebung wird durch Rezeptorproteine arrangiert, die sich in der Plasmamembran befinden. Membranrezeptoren werden durch die Bindung von extrazellulären Liganden, Pathogenen oder Zell-Zell-Interaktionen aktiviert, wodurch die Bildung eines aktiven Zustands gefördert wird, der eine intrazelluläre Reaktion einleitet. Eine Beschreibung auf molekularer Ebene, wie sich Membranrezeptoren in Proteinanordnungen organisieren und wie diese Proteinanordnungen eine spezifische funktionelle Aufgabe ausführen, ist der Ausgangspunkt für das Verständnis der molekularen Mechanismen, die Gesundheit und Krankheit zugrunde liegen. Die Fluoreszenzmikroskopie gibt Aufschluss über die Lage von Proteinen in Zellen, und mit der Einführung der höchstauflösenden Mikroskopie wurde der Nachweis einzelner Proteingruppierungen möglich. Eine Einschränkung der meisten Methoden der höchstauflösenden Mikroskopie ist, dass einzelne Komponenten einer Proteingruppierung optisch nicht aufgelöst werden können, was an der geringen Größe und dichten Packung der Bestandteile im Vergleich zur erreichbaren räumlichen Auflösung liegt. Eine Lösung, die für Einzelmolekül-Lokalisierungsmethoden gezeigt wurde, besteht darin, zusätzliche experimentelle Informationen in die Analyse zu implementieren, also „die Aufl sungsgrenze der höchstauflösenden Mikroskopie zu umgehen". Bei der Einzelmolekül-Bildgebung kann diese zusätzliche Information zum Beispiel die Kinetik von mehrfachen und wiederkehrenden Emissionsereignissen sein, die bei einzelnen Fluorophoren beobachtet werden, was als "Blinken" bezeichnet wird. Das Ziel dieser Arbeit war die Entwicklung einer höchstauflösenden Fluoreszenzmikroskopiemethode zur Detektion von Proteinmonomeren und -dimeren in der Plasmamembran von Zellen durch die Verwendung der kinetischen Information. Im ersten Teil dieser Arbeit wurden photoschaltbare fluoreszierende Proteine als Reporter verwendet, deren photoschaltbare Kinetik mit kinetischen Gleichungen analysiert wurden. Synthetische, genetische und zelluläre Referenzproteine wurden konstruiert und dienten als Kalibrierungsreferenzen für monomere und dimere Proteine. Im zweiten Teil dieser Arbeit wurde das kinetische Modell, das zur Annäherung des Häufigkeitshistogramms von Blinkereignissen einzelner Fluorophore verwendet wird, auf Oligomere höherer Ordnung erweitert. Ein Vergleich mit einem zuvor entwickelten Modell zeigte, dass das erweiterte Modell genauere Ergebnisse für Oligomere höherer Ordnung und Mischungen verschiedener Oligomere liefert. Zusätzlich wird die Anwesenheit von unerkannten Oligomeren berücksichtigt. Die erweiterte Theorie bietet somit die Grundlage, um größere Oligomere und Mischungen unterschiedlicher Stöchiometrie mit besserer Genauigkeit zu untersuchen. Im dritten Teil dieser Arbeit wurde eine Methode zur stöchiometrischen endogenen Markierung von Proteinen verwendet, um zwei Rezeptortyrosinkinasen, MET und EGFR, mit einem photoschaltbaren fluoreszierenden Protein zu markieren. Das Vorkommen von monomerem und dimerem MET-Rezeptor wurde auf der Plasmamembran von HEK293T- Zellen mittels quantitativer höchstauflösender Mikroskopie bestimmt. Der Diffusionskoeffizient und der Diffusionsmodus des MET-Rezeptors in lebenden HEK293T-Zellen wurden mit Einzelpartikelverfolgung gemessen. Dieser Teil der Arbeit zeigte, dass die Kombination von CRISPR/Cas12a-gestützter endogener Markierung und Einzelmolekül-Lokalisierungsmikroskopie ein leistungsfähiges Werkzeug zur Untersuchung der molekularen Organisation und Dynamik von Membranproteinen ist. Im vierten Teil dieser Arbeit wurde die Einzelmoleküldatenanalyse durch ein Softwaretool beschleunigt, das eine automatisierte und unvoreingenommene Detektion von Einzelmolekül-Emissionsereignissen ermöglicht. Der Anteil von Monomeren und Dimeren von fluoreszierenden Reportern wurde durch die Implementierung eines neuronalen Netzwerks bestimmt (die Software wurde von Alon Saguy geschrieben; Gruppe von Prof. Yoav Shechtman, Technion, Israel). Der oligomere Zustand der monomeren und dimeren Referenzproteine CD86 und CTLA-4 wurde erfolgreich bestimmt. Die automatisierte Detektion einzelner Proteingruppierungen ermöglichte die Analyse von MET-mEos4b in einzelnen Zellen, wodurch die Heterogenität zwischen den Zellen bestimmt und das Expressionsniveau des Rezeptors mit der Dimerisierung korreliert werden konnte. Zusammenfassend wurden in dieser Arbeit Ergebnisse zu elementaren Aspekten hin zu einer molekularen Quantifizierung von Proteinzahlen mittels Einzelmolekül- Lokalisationsmikroskopie generiert, die fluoreszierende Reporter, stöchiometrische Markierung von zellulären Proteinen und Bildanalyse umfassen. Das Potential dieser Entwicklungen wurde anhand der Beobachtung der Liganden-induzierten Verschiebung von monomeren zu dimeren MET-Rezeptoren in einzelnen HEK293T-Zellen gezeigt.

scholarly journals Phaseolin type and heat treatment influence the biochemistry of protein digestion in the rat intestine

2008 ◽  
Vol 99 (3) ◽  
pp. 531-539 ◽  
Author(s):  
Carlos A. Montoya ◽  
Pascal Leterme ◽  
Stephen Beebe ◽  
Wolfgang B. Souffrant ◽  
Daniel Mollé ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Western Blotting ◽  
Sole Source ◽  
Protein Band ◽  
Molecular Weights ◽  
Endogenous Protein ◽  
Anionic Trypsin ◽  
Endogenous Proteins ◽  
Protein Components

The study aimed to investigate thein vivodigestion ofPhaseolus vulgarisphaseolin types differing in their subunit pattern composition. Diets contained either casein as the sole source of protein or a mixture (1:1) of casein and pure Sanilac (S), Tendergreen (T) or Inca (I) phaseolin either unheated or heated. Rats were fed for 11 d with the experimental diets. Their ileal content and mucosa were collected and prepared for electrophoresis, Western blotting, densitometry and MS. Differences in digestion among native phaseolin types were observed for intact phaseolin at molecular weights (MW) of 47–50·5 kDa and for an undigested fragment at MW of 19–21·5 kDa in ileal digesta. In both cases, the concentration of these protein bands was lower for I phaseolin than for S or T phaseolin (P < 0·05). In the mucosa, the concentration of a protein band at MW of 20·5–21·5 kDa was lower for S phaseolin as compared to T or I phaseolin (P < 0·001). The presence of phaseolin subunits and their fragments was confirmed by Western blotting. MS analysis revealed the presence of undigested α and β subunit fragments from phaseolin and endogenous proteins (anionic trypsin I and pancreatic α-amylase) in ileal digesta. Thermal treatment improved digestion (P < 0·01), acting on both dietary and endogenous protein components. In conclusion, this study provides evidence for differences in intestinal digestion among phaseolin types, S phaseolin being more resistant and I phaseolin more susceptible. These differences were affected by the origin of the phaseolin subunit precursor. Heat treatment enhanced phaseolin digestion.

Potassium channel KCNJ15 is required for histamine-stimulated gastric acid secretion

2015 ◽  
Vol 309 (4) ◽  
pp. C264-C270 ◽  
Author(s):  
Jianye Yuan ◽  
Wensheng Liu ◽  
Serhan Karvar ◽  
Susan S. Baker ◽  
Wenjun He ◽  
...  
Keyword(s):  
Acid Secretion ◽  
Gastric Acid Secretion ◽  
Gastric Acid ◽  
Apical Membrane ◽  
Imaging System ◽  
Parietal Cells ◽  
Live Cells ◽  
Protein Levels ◽  
Endogenous Protein ◽  
Luminal Side

Gastric acid secretion is mediated by the K+-dependent proton pump (H+,K+-ATPase), which requires a continuous supply of K+ at the luminal side of the apical membrane. Several K+ channels are implicated in gastric acid secretion. However, the identity of the K+ channel(s) responsible for apical K+ supply is still elusive. Our previous studies have shown the translocation of KCNJ15 from cytoplasmic vesicles to the apical membrane on stimulation, indicating its involvement in gastric acid secretion. In this study, the stimulation associated trafficking of KCNJ15 was observed in a more native context with a live cell imaging system. KCNJ15 molecules in resting live cells were scattered in cytoplasm but exhibited apical localization after stimulation. Furthermore, knocking down KCNJ15 expression with a short hairpin RNA adenoviral construct abolished histamine-stimulated acid secretion in rabbit primary parietal cells. Moreover, KCNJ15, like H+,K+-ATPase, was detected in all of the parietal cells by immunofluorescence staining, whereas only about half of the parietal cells were positive for KCNQ1 under the same condition. Consistently, the endogenous protein levels of KCNJ15, analyzed by Western blotting, were higher than those of KCNQ1 in the gastric mucosa of human, mouse, and rabbit. These results provide evidence for a major role of KCNJ15 in apical K+ supply during stimulated acid secretion.

scholarly journals Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

2014 ◽  
Vol 25 (22) ◽  
pp. 3610-3618 ◽  
Author(s):  
Robert Mahen ◽  
Birgit Koch ◽  
Malte Wachsmuth ◽  
Antonio Z. Politi ◽  
Alexis Perez-Gonzalez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Quantitative Imaging ◽  
Live Cells ◽  
Single Molecule Spectroscopy ◽  
Mitotic Kinase ◽  
Endogenous Proteins

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

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