Journal: Discover Nano

Article Title: Impact of temporal resolution in single particle tracking analysis

doi: 10.1186/s11671-024-04029-1

Figure Lengend Snippet: Diffusivity distributions determined starting from exact tracks. Tracks simulated using different diffusion coefficients D S (as reported at the top of each graph) and time steps Δt (as reported in the legends) were analysed to extract the reported diffusivity (D) distributions as calculated from the first two points of the MSD function. Simulations were performed on a square area of 256 μm 2 with reflective boundaries at a density of 0.3 particles/μm 2 . The different temporal resolutions were obtained by appropriate sampling on the same simulated tracks, stopping at 500 time steps in each case. 385 tracks from 5 different simulations were analysed for each case

Article Snippet: MSD analysis and calculation of the short-term diffusion coefficient D were performed in MATLAB as previously described [ , , , ].

Techniques: Diffusion-based Assay, Sampling

Journal: Discover Nano

Article Title: Impact of temporal resolution in single particle tracking analysis

doi: 10.1186/s11671-024-04029-1

Figure Lengend Snippet: Single particle tracking experiment on p75 NTR receptors fluorescently labelled on the membrane of living SK-N-BE(2) neuroblastoma cells and comparison with simulated results. A Experimental images from the movies recorded by TIRF microscopy; on each image we report the integration time; frame time was 25 ms longer due to readout time. Images are shown in the same fixed grayscale; scale bar: 5 μm. B Corresponding measured diffusivity distributions. Mean particle density was 0.3 µm −2 . Data were obtained from 2180 to 3050 tracks for each case. C Diffusivity distribution derived from detection, tracking, and MSD analysis on simulated movies with integration, frame times as written in the legend. Particle density was 0.3 µm −2 , D S was 0.75 μm 2 /s (equal to D P for all curves but the red one for which it is 0.8 μm 2 /s). 385 tracks from 5 different simulations were used for each case. In both B and C 130-frames movies were analysed for each temporal resolution. We calculated the uncertainties in the distributions for both the experimental and simulation cases; some examples are reported in Fig.

Article Snippet: MSD analysis and calculation of the short-term diffusion coefficient D were performed in MATLAB as previously described [ , , , ].

Techniques: Single-particle Tracking, Membrane, Comparison, Microscopy, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs

doi: 10.1093/nar/gkae011

Figure Lengend Snippet: Spd1 carries not one, but two PIP motifs. ( A ) Alignment of 16 different PIP-degron SLiMs from Fungi and Animalia illustrates a remarkable conservation. The PIP-degron of Spd1 is permutated almost beyond recognition and lacks the two canonical aromatic residues. ( B ) Secondary chemical shift of C α (upper panel) and C’ (lower panel) nuclei of Spd1 in solutions calculated from assignment in the absence and presence of 8 M urea. Transient helices are indicated by TH1 through TH4. ( C ) 1 H-– 15 N-HSQC spectra of 15 N-labelled Spd1 in the absence (black) and presence (red) of equimolar concentration of S. pombe PCNA. Signals from representative residues that disappear from the spectrum in the presence of PCNA are highlighted to the right. ( D ) Chemical shift perturbations (upper panel) and intensity ratios (lower panel) of 15 N-Spd1 upon addition of PCNA. Two regions are affected, the known PIP-degron N-terminally, and 14 residues C-terminal to that, a hitherto unidentified PIP-box. Green squares: unassigned/proline residues, blue squares: overlapping residues and red squares: disappearing NMR signals.

Article Snippet: 15 N-edited 1D 1 H NMR spectra of 50 μM 15 N-labelled Spd1 were recorded at 25°C on a Bruker 600 MHz spectrometer equipped with a QCI-cryoprobe and Z-field gradient with varying gradient strengths from 2.4 to 47.2 G/cm, a diffusion time Δ of 100 ms and gradient length δ of 4 ms. Translational diffusion coefficients D for Spd1 (50 μM) were determined by fitting the peak intensity decay of the amide resonances in Dynamics Center v2.5.6 (Bruker, Switzerland) using the Stejskal-Tanner equation ( ).

Techniques: Concentration Assay

Journal: Nucleic Acids Research

Article Title: Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs

doi: 10.1093/nar/gkae011

Figure Lengend Snippet: The structure of the PCNA-Spd1 27-46 complex reveals non-canonical binding. ( A ) Ribbon diagram of the homotrimeric PCNA sliding clamp showing the position of the PIP-degron peptide (red). The peptide is extended except for residues Leu33 to Val36, which forms a 3 10 -helical turn in the bound state. ( B ) Close up of the Spd1 peptide in the PIP binding pockets. The conserved Gln sits in the Q-pocket on the right, where it makes the only intermolecular hydrogen bond in the complex. The hydrophobic pocket to the left harbours Leu33, Val36 and Met38. ( C ) LigPlot representation of the interactions between the peptide and PCNA, highlighting that, apart from the hydrogen bond in the Q-pocket, all interactions are mediated by van der Waals’ contacts.

Article Snippet: 15 N-edited 1D 1 H NMR spectra of 50 μM 15 N-labelled Spd1 were recorded at 25°C on a Bruker 600 MHz spectrometer equipped with a QCI-cryoprobe and Z-field gradient with varying gradient strengths from 2.4 to 47.2 G/cm, a diffusion time Δ of 100 ms and gradient length δ of 4 ms. Translational diffusion coefficients D for Spd1 (50 μM) were determined by fitting the peak intensity decay of the amide resonances in Dynamics Center v2.5.6 (Bruker, Switzerland) using the Stejskal-Tanner equation ( ).

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs

doi: 10.1093/nar/gkae011

Figure Lengend Snippet: The PIP-degron and PIP-box explore the same binding site on PCNA. (A ) Assigned 1 H, 15 N-HSQC spectrum of 15 N, 13 C, 2 H-labeled S. pombe PCNA (see also Fig. S2). ( B ) Chemical shift perturbation of PCNA by the addition of full-length Spd1 (50 μM). The changes in peak position for residues with red bars are shown in detail in C). ( C ) Chemical shift perturbations of three representative residues from the three most affected regions of PCNA in the presence of either of the two peptides, Spd1 27-46 (blue) or Spd1 49-68 (red) or full-length Spd1 (purple). ( D ) Determination of the dissociation constant, K d , from changes in chemical shifts for the Spd1-peptides, Spd1 49-68 including the PIP-box (upper panels) and Spd1 27–46 including the PIP-degron (lower panels).

Article Snippet: 15 N-edited 1D 1 H NMR spectra of 50 μM 15 N-labelled Spd1 were recorded at 25°C on a Bruker 600 MHz spectrometer equipped with a QCI-cryoprobe and Z-field gradient with varying gradient strengths from 2.4 to 47.2 G/cm, a diffusion time Δ of 100 ms and gradient length δ of 4 ms. Translational diffusion coefficients D for Spd1 (50 μM) were determined by fitting the peak intensity decay of the amide resonances in Dynamics Center v2.5.6 (Bruker, Switzerland) using the Stejskal-Tanner equation ( ).

Techniques: Binding Assay, Labeling

Journal: Nucleic Acids Research

Article Title: Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs

doi: 10.1093/nar/gkae011

Figure Lengend Snippet: The PIP-box and the PIP-degron of Spd1 act individually and with balanced affinities. ( A ) Sequences of PIP regions of Spd1 variants. ( B ) NMR peak intensity plots of Spd1 WT, Spd1-2G1, Spd1-2G2 and Spd1-4G from titrations with PCNA. Red dots on x-axis indicate signals that disappear. ( C ) Serial dilutions of strains with the indicated genotypes spotted on plates either untreated or treated with thiamine and incubated at the indicated temperature for three days. All strains also harbour the cdt2-TR and the rad3-TS alleles. ( D ) ITC analysis of PCNA binding to the 2F2 49-68 peptide. ( E ) Western Blot of total extracts from growing cells with the indicated genotypes. The spd1 gene was tagged with the VC155-tag in its C-terminal in a wild-type background. The blot was probed with anti-GFP and anti-PCNA antibodies. The picture below shows Ponceau staining of the gel.

Article Snippet: 15 N-edited 1D 1 H NMR spectra of 50 μM 15 N-labelled Spd1 were recorded at 25°C on a Bruker 600 MHz spectrometer equipped with a QCI-cryoprobe and Z-field gradient with varying gradient strengths from 2.4 to 47.2 G/cm, a diffusion time Δ of 100 ms and gradient length δ of 4 ms. Translational diffusion coefficients D for Spd1 (50 μM) were determined by fitting the peak intensity decay of the amide resonances in Dynamics Center v2.5.6 (Bruker, Switzerland) using the Stejskal-Tanner equation ( ).

Techniques: Incubation, Binding Assay, Western Blot, Staining

Journal: Nucleic Acids Research

Article Title: Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs

doi: 10.1093/nar/gkae011

Figure Lengend Snippet: Consequences of PCNA-interaction mutants for in vivo function of Spd1. ( A ) Interaction of Spd1 with PCNA studied by BiFC. All strains harbour the cdt2-TR allele. Cells were incubated at 30°C in the presence of thiamine for three hrs prior to photography. ( B ) Localization of GFP-Suc22 in cells of the indicated spd1 genotypes. ( C ) Serial dilutions of strains with the indicated genotypes were spotted on plates either with or without thiamine and incubated at the indicated temperature for three days. All strains also harbour the cdt2-TR and the rad3-TS alleles.

Article Snippet: 15 N-edited 1D 1 H NMR spectra of 50 μM 15 N-labelled Spd1 were recorded at 25°C on a Bruker 600 MHz spectrometer equipped with a QCI-cryoprobe and Z-field gradient with varying gradient strengths from 2.4 to 47.2 G/cm, a diffusion time Δ of 100 ms and gradient length δ of 4 ms. Translational diffusion coefficients D for Spd1 (50 μM) were determined by fitting the peak intensity decay of the amide resonances in Dynamics Center v2.5.6 (Bruker, Switzerland) using the Stejskal-Tanner equation ( ).

Techniques: In Vivo, Incubation

Journal: Nucleic Acids Research

Article Title: Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs

doi: 10.1093/nar/gkae011

Figure Lengend Snippet: In silico analysis of Spd1 binding to PCNA reveals compacton formation and different ensembles depending on the active SLiM. (A ) Schematic overview of the fully disordered, 124 amino acid residues Spd1 protein. ( B ) The probability of finding the PIP-box SLiM at different distances from the PIP binding pocket of subunits A (blue line) or B (orange line). ( C ) Schematic representation of the principle plotted in B) and D). ( D ) The probability of finding the PIP-degron as a function of distance to the PIP binding pockets on subunits A (blue) and B (orange). ( E ) 100 trajectories of the simulation of the PIP-degron bound state superposed with Spd1 in brown, yellow (compacton 1) and red (compacton 2) and PCNA in gray and cyan. The C-terminal disordered tail (cyan) from the C-subunit of PCNA interacts with the compactons. The compactons are red and yellow according to A). ( F ) Like E, only with Spd1 bound to PCNA via the PIP-box SLiM. The C-terminal compactons (red and yellow according to A)) are more distinct, the PCNA C-terminal tail (cyan) interacts exclusively with compacton 1. The N-terminal Spd1 chain covers the central opening in the PCNA toroid.

Article Snippet: 15 N-edited 1D 1 H NMR spectra of 50 μM 15 N-labelled Spd1 were recorded at 25°C on a Bruker 600 MHz spectrometer equipped with a QCI-cryoprobe and Z-field gradient with varying gradient strengths from 2.4 to 47.2 G/cm, a diffusion time Δ of 100 ms and gradient length δ of 4 ms. Translational diffusion coefficients D for Spd1 (50 μM) were determined by fitting the peak intensity decay of the amide resonances in Dynamics Center v2.5.6 (Bruker, Switzerland) using the Stejskal-Tanner equation ( ).

Techniques: In Silico, Binding Assay

Journal: Chemistry (Weinheim an der Bergstrasse, Germany)

Article Title: Size Determination of Organic Cages by Diffusion NMR Spectroscopy.

doi: 10.1002/chem.202303318

Figure Lengend Snippet: Figure 1. a) Flow chart for the determination of diffusion coefficients D and solvodynamic diameters ds from 1H-DOSY NMR measurements, b) cut-out of 1H NMR spectrum (400 MHz, CDCl3, rt) of AnBu

Article Snippet: The diffusion coefficients D were calculated with the T1/T2 relaxation module in TopSpin 4.0 (Bruker) using mono-exponential fitting with the Stejskal-Tanner Equation (1).

Techniques: Diffusion-based Assay