connected components function  (Amira Pharmaceuticals)

Journal: bioRxiv

Article Title: The structure and global distribution of the endoplasmic reticulum network is actively regulated by lysosomes

doi: 10.1101/2020.01.15.907444

Figure Lengend Snippet: (A) Representative time-lapse images showing breakage of the connection between the growing tip of a newly-formed ER tubule (magenta) and a lysosome (green) in EGFP-VAPA(KD/MD)-expressing cells. See table S2 for the compromised contact sites upon VAPA(KD/MD) overexpression. See Movie S5. (B) Average velocities (black spots) of initially ER-tethered lysosomes that become detached (red arrow) from their associated ER growing tips. Detachment events are ER-lysosome connection breakages in EGFP-VAPA(KD/MD)-expressing cells as in (A). **= p<0.01, **** = p<0.0001 (Tukey’s one-way ANOVA). Velocities of 22 events from three independent experiments were quantified. See table S3. (C) Left: Diagram depicting the regions defined as perinuclear and peripheral regions for the following quantification of lysosome distribution, same definition for ( E ). Right: Percentage of the ER comprising tubules upon knockdown of lysosome motion adaptors. Data are shown as ± SEM. **** = p<0.0001 (Tukey’s one-way ANOVA). Data from 20 cells from 3 independent experiments were analyzed for each condition. See table S4. (D) Representative images showing the distribution of lysosomes and ER tubules in control cells and in cells treated with siRNAs. (E) i : Diagram depicting individual components of the chemogenetic system. ii : Quantification of lysosome intensity change and percentage of the ER comprising tubules after 1 hr inducer treatment. Data are shown as ± SEM. **** = p<0.0001 (Student’s t test). For lysosome intensity analysis, N = 10, for ER tubule percentage, N = 20. iii : Representative images showing the distribution of lysosomes and ER tubules in control cells and in cells treated with inducers. (F) Optogenetic assay for repositioning of LAMP1. ( G-J ): Live-cell imaging ( G ), representative zoom-ins ( H ), representative kymograph ( I ) and quantification ( J ) of LAMP1-mCherry-iLID and YFP-SEC61B in COS-7 cells expressing opto-kinesin before or during activation. White arrows indicate lysosome pulling ER tubule (yellow asterisk). Quantification shows mean (± S.E.M.) normalized peripheral SEC61 and LAMP1 intensity of 8 cells. Blue box indicates illumination with blue light. Scale bars represent 1 μm in ( A ) ( G ) ( H ) and ( I ), 5 μm in ( D ) and ( E ). s, seconds.

Article Snippet: These three features were labelled using MATLAB’s connected components functions.

Techniques: Expressing, Over Expression, Knockdown, Control, Live Cell Imaging, Activation Assay