Journal: eLife

Article Title: Controlling motor neurons of every muscle for fly proboscis reaching

doi: 10.7554/eLife.54978

Figure Lengend Snippet:

Article Snippet: Antibody , rat mAb anti-FLAG , Novus Biologicals , Cat# NBP1-06712; RRID: AB_1625981 , (1:200) , .

Techniques: Muscles, Construct, Software

Journal: Cell host & microbe

Article Title: Single Particle Imaging of Polarized Hepatoma Organoids upon Hepatitis C Virus Infection Reveals an Ordered and Sequential Entry Process

doi: 10.1016/j.chom.2018.02.005

Figure Lengend Snippet: (A–D) Huh-7.5 organoids were incubated with 15 mM Erlotinib (Erl) or DMSO for 1 hr, infected with DiD-HCV for 1 hr at 4C, shifted to 37°C for the indicated times, fixed, and probed for claudin-1 (A) or core (C). (A) Tight junction region is shown in the inset. Left: claudin (green), Right: DiD-HCV (red). (B) Quantitation of (A). (C) Arrows indicate DiD-HCV particles enlarged in insets; dashed arrows and arrowhead represent colocalization with core. Left: core (green); right: DiD-HCV (red). Lack of DiD-HCV colocalization with core antibody, seen in DMSO treated cluster at 360 min post shift, indicates an uncoating event. (D) Quantitation of (C). (E and F) Huh-7.5, shEGFR, or shEGFR+EGFR organoids were infected with DiD-HCV as above, fixed at indicated times, and stained for ZO-1. (E) Tight junction region is shown in the inset. Left: ZO-1 (green); right: DiD-HCV (red). (F) Quantitation of (E). n = total DiD signal, mean ± SD. **p < 0.01, ***p < 0.001. See also Figure S5.

Article Snippet: Inhibitor concentrations were as follows: 10 mM Cytochalasin D (Sigma), 15 mM Erlotinib (Santa Cruz) and 20 mM Ammonium Chloride (Fisher).

Techniques: Incubation, Infection, Quantitation Assay, Staining

Journal: Cell host & microbe

Article Title: Single Particle Imaging of Polarized Hepatoma Organoids upon Hepatitis C Virus Infection Reveals an Ordered and Sequential Entry Process

doi: 10.1016/j.chom.2018.02.005

Figure Lengend Snippet: Huh-7.5 organoids were incubated with either erlotinib or vehicle control (VC) for 1 hr, if indicated, infected with DiD-HCV for 1 hr at 4C, shifted to 37°C for stated times, fixed, and probed for clathrin light chain (clathrin LC) (A and C) or Rab5a (E), both in green. Arrows mark areas enlarged in insets; dashed arrows and arrowheads denote colocalization with labeled host factor. (C) Organoids seeded with Huh-7.5 or Huh-7.5-shEGFR cells were infected with DiD-HCV as above, fixed, and stained for clathrin LC. (B, D, and F) Quantitation of (A), (C), and (E), respectively. n = total DiD signal; error bars, SD. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S6.

Article Snippet: Inhibitor concentrations were as follows: 10 mM Cytochalasin D (Sigma), 15 mM Erlotinib (Santa Cruz) and 20 mM Ammonium Chloride (Fisher).

Techniques: Incubation, Control, Infection, Labeling, Staining, Quantitation Assay

Journal: Cell host & microbe

Article Title: Single Particle Imaging of Polarized Hepatoma Organoids upon Hepatitis C Virus Infection Reveals an Ordered and Sequential Entry Process

doi: 10.1016/j.chom.2018.02.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Inhibitor concentrations were as follows: 10 mM Cytochalasin D (Sigma), 15 mM Erlotinib (Santa Cruz) and 20 mM Ammonium Chloride (Fisher).

Techniques: Control, Virus, Recombinant, Cell Recovery, Cell Viability Assay, Plasmid Preparation, shRNA, Software

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) An overview of the subunit architecture of human condensin I, with a table of equivalent subunits in the condensin and cohesin complexes. ( B ) ATPase assay of human condensin I pentamer (CI), tetramers lacking HAWK domains and reconstituted pentamers. Q refers to complexes with a mutation in the Q-loop, which cannot bind ATP. The lower panel shows SDS-PAGE analysis of the ATPase reaction mixture after completion of the assay. Purine nucleoside phosphorylase (PNP) enzyme used in the ATPase assay is indicated as a loading control. Experimental sample sizes for each lane: 1. n = 9, 2. n = 8, 3. n = 6, 4–10. n = 4, and 11–12. n = 3, where each technical replicate was performed with a distinct protein aliquot at a different time. P values determined using unpaired two-tailed t tests with Welch’s correction to account for unequal variance. ( C ) Fold-stimulation of the ATPase rate when 50 bp dsDNA is titrated, determined by dividing the ATPase rate at each DNA concentration, by the rate without DNA, for each DNA concentration, n = 3 technical replicates were performed with a distinct protein aliquot. ( D ) Gel shift assay of condensin I pentamer and tetramers, respectively, binding to 50 bp dsDNA labelled with Cy5. In each case, column value and error bars indicate, mean and s.e.m., respectively. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: ATPase Assay, Mutagenesis, SDS Page, Control, Two Tailed Test, Concentration Assay, Gel Shift, Binding Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) Comparison of two different batches of recombinant CIWT pentamer, and CIΔG and CIΔD2 tetramers. Trend of tetrameric complexes being significantly more active than pentameric condensin I is consistent. Data from three technical replicates. Error bars represent s.e.m. P values indicated are from unpaired, two-tailed t test with Welch’s correction. ( B ) Mass photometry data of condensin I tetramers, CIΔG, with and without NCAPG being added to reconstitute the pentamer. ( C ) Mass photometry data of condensin I tetramers, CIΔD2, with and without NCAPD2 being added to reconstitute the pentamer. ( D – F ) Mass photometry data of condensin I pentamers, CID2ΔC, CIHΔN and CIHΔN,D2ΔC, respectively, consistent with pentameric mass and confirming they are the major species present. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Comparison, Recombinant, Two Tailed Test

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) ATPase assay of condensin I mutants, with mutations illustrated on the right. Mutation CIΔG indicate tetrameric condensin I lacking NCAPG. CIΔSB indicates deletion of the NCAPH region where NCAPG binds. Gp1 and Gp2 indicates mutations in NCAPG homologous to patch1 and patch2 where Ycg1 and SMC2 interact in yeast condensin. CIGΔC indicates deletion of NCAPG C-terminal disordered region, CINΔH indicates deletion of NCAPH N-terminal disordered region and CID2ΔC indicates deletion of NCAPD2 C-terminal disordered region. CIQHΔN, CIQD2ΔC and CIQHΔN,D2ΔC indicating ATPase dead Q-loop mutant controls. The lower panel is SDS page analysis of the ATPase reaction mixture after completion of the assay. Purine nucleoside phosphorylase (PNP) enzyme used in the ATPase assay is indicated as a loading control. Experimental sample size for each condition: 1. n = 17, 2–3. n = 8, 4–5. n = 5, 6–9. n = 4, 10. n = 6, 11–13. n = 5, 14. n = 3, 15. n = 4, 16. n = 5 and 17. n = 3, where each technical replicate was performed using a distinct protein aliquot, collected at a different time. P values shown are comparisons to CIWT, determined using unpaired two-tail t tests with Welch’s correction to account for unequal variance. In each case, column value and error bars indicate mean and s.e.m., respectively. ( B ) AlphaFold2 model of NCAPG with the C-terminal end of NCAPD2, with NCAPD2 coloured with AlphaFold2 (Jumper et al, ; preprint: Evans et al, ) pLDDT confidence score. ( C ) Surface charge of the NCAPG/D2 interface. ( D ) Fluorescence polarisation binding assay of 5-FAM NCAPD2 1376-1398 . Error bars indicate mean ± s.d., data from three technical replicates, performed with distinct protein aliquots, read three times. Solid line indicates data fit to determine Kd. “NCAPGap” indicates NCAPG with acid patch mutation (D136K, D137K, D141K). ( E – G ) Equivalent data as ( B – D ) for NCAPH. Fluorescence polarisation in ( G ) was performed with 5-FAM NCAPH 55-77 . .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: ATPase Assay, Mutagenesis, SDS Page, Control, Fluorescence, Binding Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) AlphaFold2 model of NCAPG with the C-terminal end of KIF4A, with KIF4A coloured with AlphaFold2 (Jumper et al, ; preprint: Evans et al, ) pLDDT confidence score. ( B ) Surface charge of the NCAPG/KIF4A interface. ( C ) Overlay of NCAPG showing binding of KIF4A peptide (blue) overlaps with binding of NCAPD2 and NCAPH (pink and green, respectively). ( D ) Fluorescence polarisation binding assay of 5-FAM labelled KIF4A. “NCAPGap” indicates NCAPG with acid patch mutation (D136K, D137K, D141K). Solid line indicates Kd fit, Kd value shown ± standard error. ( E ) Competition FP assay with 100 nM 5-FAM NCAPD2 1376-1398 , 6 μM NCAPG with 10 μM KIF4A, KIF4A 1206-1228 , WT or Mut (mutant) peptide. ( F ) Competition FP assay with 100 nM 5-FAM NCAPH 55-77 , 4 μM CINΔH,D3ΔC with 100 μM KIF4A, KIF4A 1206-1228 , WT or Mut peptide. For FP data in ( D – F ), error bars indicate mean ± s.d., and data were derived from three technical replicates performed with distinct protein aliquots, read three times. P values determined using unpaired, two-tailed t tests. ( G ) ATPase assay of WT and pentameric condensin I lacking the C-terminal region of NCAPD2 and/or the N-terminal region of NCAPH, in the presence and absence of 50 μM KIF4A 1206-1228 , WT or Mut peptide. ATPase assays from 3 repeats, error bars indicate s.e.m., P values determined with unpaired, two-tailed t test with Welch’s correction. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Binding Assay, Fluorescence, Mutagenesis, FP Assay, Derivative Assay, Two Tailed Test, ATPase Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) Schematic of the single-molecule loop extrusion assay under constant buffer flow and corresponding snapshots of a loop formed by CIWT in the presence of KIF4A 1206-1228 peptide. ( B ) Fraction of DNA strands with at least one looping event during a 1000 s acquisition time, in the presence of 1 nM human CIWT and CIΔG ( n = 4 and 10 experiments, respectively). ( C ) Fraction of DNA strands with at least one looping event during a 1000 s acquisition time, in the presence of 0.5 nM condensin I WT and WT supplied with 1000x KIF4A 1206-1228 peptide ( n = 3 experiments). ( B , C ) Mean ± s.d. is shown, the total number of DNA tethers analysed is indicated by n tot and P values calculated with Wilcoxon rank-sum tests. ( D ) Example snapshots, the corresponding ( E ) kymograph and ( F ) time trace of DNA lengths for a loop extrusion event by CIΔG without flow. ( G ) Box-whisker-plots showing the loop extrusion rates of CIWT, CIΔG and CIWT in the presence of KIF4A 1206-1228 peptide. Calculated from n tot = 55 looping events, from replicates detailed in ( B , C ). For the box plots, the central line denotes the median, the box limit denotes the 25th–75th percentile and the whiskers denote minimum and maxima. The P values were calculated using Welch’s t test. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Whisker Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) Current models (Shaltiel et al, ; Dekker et al, ) for DNA loop extrusion by condensin complexes require loops to be initiated by DNA being “threaded” through the NCAPH Kleisin. ( B ) In the presence of interactions between NCAPD2/H and NCAPG, the threading step could be blocked. ( C ) The interaction with KIF4A could allow DNA threading and loop initiation. ( D ) Loss of NCAPG could also reduce inhibition of DNA threading, but may result in reduced DNA-binding affinity and slippage of NCAPG/H DNA anchor.

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Inhibition, Binding Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A , B ), AlphaFold2 model of Ycg1 with Lrs4 and Sgo, respectively, with insert of electrostatic potential. ( C ) Sequence alignment of SLiMs that bind the equivalent site of NCAPG and Ycg1. ( D , E ) Fluorescence polarisation binding data of Lrs4 and Sgo peptides with yeast condensin complex. Error bars indicate mean ± s.d., data from three technical replicates performed with distinct protein aliquots, read three times. ( F ) A general model of the role SLiM HAWK association has in regulating condensin and cohesin activity. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Sequencing, Fluorescence, Binding Assay, Activity Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Recombinant, Residue, Mutagenesis, Sequencing, Software