scholarly journals A general fluorescent light-up probe for staining and quantifying protein

2019 ◽  
Vol 6 (8) ◽  
pp. 190580 ◽  
Author(s):  
Jiawei Zou ◽  
Gangyi Chen ◽  
Feng Du ◽  
Yi Yuan ◽  
Xin Huang ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Visual Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fluorescent Light ◽  
Cell Penetration ◽  
Cellular Processes ◽  
Low Toxicity ◽  
Protein Rich

Proteins are the primary functional agents in all cellular processes, facilitating various functions such as enzymes and structure-forming or signal-transducing molecules. In this work, we report a fluorescent dye, PyMDI-Zn, which could specifically bind with proteins and provide a red-shifted fluorescent emission. The visual analysis of protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis could be realized in 5 min by using PyMDI-Zn as a light-up dye. Based on its cell penetration and low toxicity, PyMDI-Zn could also be applied to locate protein-rich regions and organelles in live cell imaging. Moreover, the direct protein quantitation can be realized based on PyMDI-Zn, providing a method of screening for food adulteration by nitrogen-rich compounds.

Indolizino[3,2-c]quinolines as environment-sensitive fluorescent light-up probes for targeted live cell imaging

2017 ◽  
Vol 252 ◽  
pp. 340-352 ◽  
Author(s):  
Soonbum Kwon ◽  
Dah In Kwon ◽  
Youngeun Jung ◽  
Ju Hee Kim ◽  
Yeongcheol Lee ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Fluorescent Light

scholarly journals Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation

2018 ◽  
Author(s):  
Jan Huebinger ◽  
Jessica Spindler ◽  
Kristin J. Holl ◽  
Björn Koos
Keyword(s):  
Technological Progress ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cultured Cells ◽  
Chemical Fixation ◽  
Cellular Processes ◽  
Cytoplasmic Proteins ◽  
Resolution Imaging ◽  
Spatio Temporal ◽  
Key Parameter

AbstractTo understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes along with long acquisition times and high phototoxicity. Physiological live cell imaging is therefore often unfeasible and chemical fixation is employed. However, fixation methods have not been rigorously reviewed to preserve patterns at the resolution at which they can be nowadays imaged. A key parameter for this is the time span until fixation is completed. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from the cells. Other small aldehydes like glyoxal or acrolein showed inferior results. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely antagonized by supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

scholarly journals Live cell imaging of the hyperthermophilic archaeon Sulfolobus acidocaldarius identifies complementary roles for two ESCRTIII homologues in ensuring a robust and symmetric cell division

2020 ◽  
Author(s):  
Andre Arashiro Pulschen ◽  
Delyan R. Mutavchiev ◽  
Kim Nadine Sebastian ◽  
Jacques Roubinet ◽  
Marc Roubinet ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Halophilic Archaea ◽  
Live Cell ◽  
Tight Coupling ◽  
Dna Compaction ◽  
Cellular Processes ◽  
Daughter Cells

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details.

scholarly journals Caught in the Act: Live-Cell Imaging of Plant Meiosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Maria Ada Prusicki ◽  
Martina Balboni ◽  
Kostika Sofroni ◽  
Yuki Hamamura ◽  
Arp Schnittger
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Ex Vivo ◽  
Live Cell ◽  
Cellular Processes ◽  
Short Period ◽  
Molecular Forces ◽  
Cellular Components ◽  
Plant Meiosis ◽  
Imaging Conditions

Live-cell imaging is a powerful method to obtain insights into cellular processes, particularly with respect to their dynamics. This is especially true for meiosis, where chromosomes and other cellular components such as the cytoskeleton follow an elaborate choreography over a relatively short period of time. Making these dynamics visible expands understanding of the regulation of meiosis and its underlying molecular forces. However, the analysis of meiosis by live-cell imaging is challenging; specifically in plants, a temporally resolved understanding of chromosome segregation and recombination events is lacking. Recent advances in live-cell imaging now allow the analysis of meiotic events in plants in real time. These new microscopy methods rely on the generation of reporter lines for meiotic regulators and on the establishment of ex vivo culture and imaging conditions, which stabilize the specimen and keep it alive for several hours or even days. In this review, we combine an overview of the technical aspects of live-cell imaging in plants with a summary of outstanding questions that can now be addressed to promote live-cell imaging in Arabidopsis and other plant species and stimulate ideas on the topics that can be addressed in the context of plant meiotic recombination.

scholarly journals Archaeal imaging: leading the hunt for new discoveries

2018 ◽  
Vol 29 (14) ◽  
pp. 1675-1681 ◽  
Author(s):  
Alexandre W. Bisson-Filho ◽  
Jenny Zheng ◽  
Ethan Garner
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Structural Information ◽  
Eukaryotic Cell ◽  
Live Cell ◽  
Biological Processes ◽  
Cellular Processes ◽  
Archaeal Species ◽  
Eukaryotic Organisms ◽  
Cellular Components

Since the identification of the archaeal domain in the mid-1970s, we have collected a great deal of metagenomic, biochemical, and structural information from archaeal species. However, there is still little known about how archaeal cells organize their internal cellular components in space and time. In contrast, live-cell imaging has allowed bacterial and eukaryotic cell biologists to learn a lot about biological processes by observing the motions of cells, the dynamics of their internal organelles, and even the motions of single molecules. The explosion of knowledge gained via live-cell imaging in prokaryotes and eukaryotes has motivated an ever-improving set of imaging technologies that could allow analogous explorations into archaeal biology. Furthermore, previous studies of essential biological processes in prokaryotic and eukaryotic organisms give methodological roadmaps for the investigation of similar processes in archaea. In this perspective, we highlight a few fundamental cellular processes in archaea, reviewing our current state of understanding about each, and compare how imaging approaches helped to advance the study of similar processes in bacteria and eukaryotes.

scholarly journals Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast

2005 ◽  
Vol 16 (4) ◽  
pp. 1756-1768 ◽  
Author(s):  
Isabelle Loïodice ◽  
Jayme Staub ◽  
Thanuja Gangi Setty ◽  
Nam-Phuong T. Nguyen ◽  
Anne Paoletti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Microtubule Organization ◽  
Cellular Processes ◽  
Spindle Midzone ◽  
Fluorescence Live Cell Imaging

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Δ mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.

scholarly journals New proline-rich proteins in isolated insect Z-discs

1980 ◽  
Vol 191 (2) ◽  
pp. 333-339 ◽  
Author(s):  
G M Sainsbury ◽  
B Bullard
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Flight Muscle ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Ionic Strength ◽  
Polyacrylamide Gel ◽  
Protein Rich

Z-discs were isolated from Lethocerus (waterbug) flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. Sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis confirmed a previous report that major Z-disc proteins had subunit mol.wts of 200 000, 180 000, 105 000, 95 000, 42 000 and 35 000. A protein of subunit mol.wt 25 000 was found in once-washed Z-discs but was degraded or was removed by successive washes. In addition, a protein of high molecular weight (less than 300 000) was found in Z-discs. Proteins of subunit mol.wts. 42 000, 35 000 and 25 000 were individually sliced from SDS/polyacrylamide gels and eluted. Amino acid analysis showed that the 35 000-subunit-mol.wt. protein was not, as was previously suggested, tropomyosin, but was a distinct Z-disc protein rich in proline. Calculations based on the amino acid analysis showed that this protein contained substantial hydrophobic regions. Preliminary investigations into the isoelectric point and a method of isolation of the 35 000-subunit-mol.wt. Z-disc protein are described. This protein was found in slices cut from SDS/polyacrylamide-gel electrophoretograms of whole myofibrils. The protein of 42 000 subunit mol.wt. was shown by amino acid analysis to be actin and the 25 000-subunit-mol.wt. Z-disc protein was proline-rich.

scholarly journals cNap1 bridges centriole contact sites to maintain centrosome cohesion

2021 ◽  
Author(s):  
Robert Mahen
Keyword(s):  
Cell Migration ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Spatial Proximity ◽  
Contact Site ◽  
Physical Force ◽  
Cellular Processes ◽  
Contact Sites ◽  
Reduced Viscosity ◽  
Membrane Bound

Centrioles are non-membrane bound organelles that participate in fundamental cellular processes through their ability to form physical contacts with other structures. During interphase, two mature centrioles can associate to form a single centrosome - a phenomenon known as centrosome cohesion. Centrosome cohesion is important for processes such as cell migration, and yet how it is maintained is unclear. Current models indicate that pericentriolar fibres termed rootlets, also known as the centrosome linker, entangle to maintain centriole proximity. Here, I uncover a new centriole-centriole contact site and mechanism of centrosome cohesion, based on coalescence of the proximal centriole component cNap1. Using live-cell imaging of endogenously tagged cNap1, I show that proximal centrioles form dynamic contacts in response to physical force from the cytoskeleton. Expansion microscopy reveals that cNap1 bridges between these contact sites, physically linking proximal centrioles on the nanoscale. When ectopically tethered to organelles such as lysosomes, cNap1 forms viscous and cohesive condensates that promote organelle spatial proximity. Conversely, cNap1 mutants with reduced viscosity are unable to maintain centrosome cohesion. These results define a previously unrecognised mechanism of centrosome cohesion by cNap1 assemblies at the proximal centriole and illustrate how a non-membrane bound organelle forms dynamic organelle contact sites.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

The Recovery of Factor VIII from Fresh-Frozen, Indated and Outdated Human Plasma

1976 ◽  
Vol 36 (01) ◽  
pp. 071-077 ◽  
Author(s):  
Daniel E. Whitman ◽  
Mary Ellen Switzer ◽  
Patrick A. McKee
Keyword(s):  
Factor Viii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
The United States ◽  
Fresh Frozen Plasma ◽  
Red Cross ◽  
Random Samples ◽  
Fresh Frozen ◽  
Factor Viii Concentrates ◽  
Frozen Plasma

SummaryThe availability of factor VIII concentrates is frequently a limitation in the management of classical hemophilia. Such concentrates are prepared from fresh or fresh-frozen plasma. A significant volume of plasma in the United States becomes “indated”, i. e., in contact with red blood cells for 24 hours at 4°, and is therefore not used to prepare factor VIII concentrates. To evaluate this possible resource, partially purified factor VIII was prepared from random samples of fresh-frozen, indated and outdated plasma. The yield of factor VIII protein and procoagulant activity from indated plasma was about the same as that from fresh-frozen plasma. The yield from outdated plasma was substantially less. After further purification, factor VIII from the three sources gave a single subunit band when reduced and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results indicate that the approximately 287,000 liters of indated plasma processed annually by the American National Red Cross (ANRC) could be used to prepare factor VIII concentrates of good quality. This resource alone could quadruple the supply of factor VIII available for therapy.

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