High Resolution Two-Dimensional Detection of Ncd Motility with Optical Tweezers

1997 ◽  
Vol 489 ◽  
Author(s):  
M. W. Allersma ◽  
F. Gittes ◽  
M. J. deCastro ◽  
R. J. Stewart ◽  
C. F. Schmidt
Keyword(s):  
Optical Tweezers ◽  
Focal Plane ◽  
Detection Method ◽  
Field Of View ◽  
Viscous Drag ◽  
Two Dimensional ◽  
Laser Focus ◽  
Temporal And Spatial ◽  
Elastic Constraints

AbstractThe ATP-dependent motility of the kinesin-related non claret disjunctional (ncd) mechanoenzyme was observed in an in vitro bead motility assay using optical tweezers in combination with a new two-dimensional displacement detection method. The detection technique is based on observing the far-field interference pattern formed in the back focal plane (BFP) of the microscope condenser by the illuminating laser focus and the light scattered from the trapped dielectric bead. The ability to observe the two-dimensional motion, with high temporal and spatial resolution, and in a manner largely independent of position in the microscope field-of-view, is the particular advantage of this detection method. In the assay, a fusion protein (GST-N195) of truncated ncd and glutathione-Stransferase was adsorbed to silica beads and the axial and lateral motions of the beads along the microtubule surface were observed. The average axial velocity of the ncd coated beads was 230 ± 30 nm/s (± std. dev.). Spectral analysis of bead motion showed an increase in viscous drag near the surface. Furthermore, we also found that any elastic constraints of the moving motors are much smaller than the constraints due to binding in the presence of the non-hydrolyzable nucleotide adenylylimido-diphosphate (AMP-PNP).

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

scholarly journals Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping

1988 ◽  
Vol 252 (2) ◽  
pp. 607-615 ◽  
Author(s):  
J M Tavaré ◽  
R M Denton
Keyword(s):  
Thin Layer ◽  
Insulin Receptor ◽  
Peptide Mapping ◽  
Beta Subunit ◽  
Two Dimensional ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Domain 3 ◽  
Receptor Beta

1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.

scholarly journals Force generation by protein–DNA co-condensation

2021 ◽  
Author(s):  
Thomas Quail ◽  
Stefan Golfier ◽  
Maria Elsner ◽  
Keisuke Ishihara ◽  
Vasanthanarayan Murugesan ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Self Assembly ◽  
Spider Silk ◽  
Regulatory Elements ◽  
Heterochromatin Formation ◽  
First Order Phase Transition ◽  
Dna Strand ◽  
First Order Phase

AbstractInteractions between liquids and surfaces generate forces1,2 that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water3, modulation of the material properties of spider silk4 and self-assembly of microstructures5. Recent studies have shown that cells assemble biomolecular condensates via phase separation6. In the nucleus, these condensates are thought to drive transcription7, heterochromatin formation8, nucleolus assembly9 and DNA repair10. Here we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein–DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism is a possible general regulatory principle for chromatin organization that may be relevant in vivo.

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii confers resistance to anti-microtubule herbicides

1993 ◽  
Vol 106 (1) ◽  
pp. 209-218 ◽  
Author(s):  
S.W. James ◽  
C.D. Silflow ◽  
P. Stroom ◽  
P.A. Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Resistance Mutation ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Two Dimensional ◽  
Wild Type ◽  
Tubulin Gene ◽  
Alpha Tubulin ◽  
Tubulin Genes

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii was isolated by using the amiprophos-methyl-resistant mutation apm1-18 as a background to select new mutants that showed increased resistance to the drug. The upA12 mutation caused twofold resistance to amiprophos-methyl and oryzalin, and twofold hypersensitivity to the microtubule-stabilizing drug taxol, suggesting that the mutation enhanced microtubule stability. The resistance mutation was semi-dominant and mapped to the same interval on linkage group III as the alpha 1-tubulin gene. Two-dimensional gel immunoblots of proteins in the mutant cells revealed two electrophoretically altered alpha-tubulin isoforms, one of which was acetylated and incorporated into microtubules in the axoneme. The mutant isoforms co-segregated with the drug-resistance phenotypes when mutant upA12 was backcrossed to wild-type cells. Two-dimensional gel analysis of in vitro translation products showed that the non-acetylated variant alpha-tubulin was a primary gene product. DNA sequence analysis of the alpha 1-tubulin genes from mutant and wild-type cells revealed a single missense mutation, which predicted a change in codon 24 from tyrosine in wild type to histidine in mutant upA12. This alteration in the predicted amino acid sequence corroborated the approximately +1 basic charge shift observed for the variant alpha-tubulins. The mutant allele of the alpha 1-tubulin gene was designated tua1-1.

Biotransformations and cytotoxicity of graphene and inorganic two-dimensional nanomaterials using simulated digestions coupled with a triculture in vitro model of the human gastrointestinal epithelium

2021 ◽  
Author(s):  
Lila Bazina ◽  
Dimitrios Bitounis ◽  
Xiaoqiong Cao ◽  
Glen M. DeLoid ◽  
Dorsa Parviz ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Engineered Nanomaterials ◽  
Two Dimensional ◽  
Gastrointestinal Epithelium ◽  
Vitro Model ◽  
Human Gastrointestinal Epithelium

Background: engineered nanomaterials (ENMs) have already made their way into myriad applications and products across multiple industries.

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