DNM1L-Variants-Scripts

R-scripts & ImageJ macros used in the submitted manuscript "Novel DNM1L variants impair mitochondrial dynamics through divergent mechanisms"

R-scripts for biophysical studies:

Contributed by K. Nolden

1. 20200417_Drp1_variant_additional_boxplots

• Calculating and plotting Kcat,K0.5, Kcat/K0.5, and vmax for the following:
• Drp1 WT, L230dup, G363D, G401S, and R710G GTPase results
• Experiment initially performed with recombinant L230dup
• However, later MS + SDS-PAGE confirmed protein was truncated and not usable

2. 20210608_Drp1_variants_analysis.R

• Script for analyzing and visualizing SEC-MALS data
• Data collected on 20210608
• Drp1 constructs dialyzed overnight into SEC-MALS buffer
• Exact buffer match to running buffer used
• 6xHis tag present

3. 20210804_CD_Drp1_UK.R

• Analyzing circular dichroism data
• experiment: CD of WT Drp1 + UK pathological variants
• data collected on 20210804
• data corrected for empty cuvette + buffer signal
• scaled in Excel prior to loading to provide comparable baselines
• MRE calculated for each sample at each wavelength evaluated

4. UK_Drp1_WT_variants_TSA_merged.R

• Plotting combined Thermal Shift Assay (TSA) data
• Amplification data = Temperature vs Fluorescence
• Sypro-Orange thermofluor TSA on Drp1 WT and UK variants +/- 100 or 500 uM GDP or GTP
• Data collected on 20210514 and 20210602
• 1st derivative of fluorescence with respect to temperature used to determine
• melting temperature (Tm)

R-scripts for analysing co-localization data:

1. Drp1_coloc_analysis.R

• Extract Pearson's R values from ImageJ Output
• for DRP1-TOM20 colocalization experiments using patient-derived
• fibroblasts immunostained for DRP1, TOM20, and DAPI
• performed by Ollie of UK team
• Two data sets per sample: C1, C2, P1, P2, P3, and P4
• C1: pediatric control
• C2: adult control
• P1: Drp1 G401S variant
• P2: Drp1 G363D variant
• P3: Drp1 L230dup variant
• P4: Drp1 R710G variant

2. Drp1_coloc_analysis_PMP.R

• Extract Pearson's R values from ImageJ Output
• for DRP1-PMP70 colocalization experiments using patient-derived
• fibroblasts immunostained for DRP1, TOM20, and DAPI
• performed by Ollie of UK team
• Two replicates per sample: C1, C2, P1, P2, P3, and P4
• Collected in same experiment/data set
• C1: pediatric control
• C2: adult control
• P1: Drp1 G401S variant
• P2: Drp1 G363D variant
• P3: Drp1 L230dup variant
• P4: Drp1 R710G variant