Cells treated with scrambled sgRNA (i.e. control)

Introduction

We created this dataset to provide sample movies for
the paper, “Functional characterization of 67 endocytic accessory
proteins using multiparametric quantitative analysis of CCP dynamics”, https://doi.org/10.1073/pnas.2020346117.
In this paper, CRISPR-mediated protein knock-down was used to study the
functional role of 67 proteins during clathrin-mediated endocytosis. Due to the
extremely large data volume, we only provide a small subset of movies for 2
conditions. The full dataset (or part of it) is available upon request. Please
contact Marcel Mettlen ([e-mail]).

The movies provided are fluorescent images of ARPE19 cells
expressing eGFP-CLC treated with sgRNA against CALM. The corresponding scramble
controls are also included. Readers are referred to the software pipeline
cmeAnalysis to analyze these datasets. The pipeline and instructions are available
on GitHub: https://github.com/DanuserLab/cmeAnalysis

Methods
and Materials

For a detailed description
of cell generation, sgRNA production and cell transduction, readers are referred
to the main publication and the supporting material (10.1073/pnas.2020346117​.)

TIR-FM imaging

On the day of TIR-FM imaging, cells were checked for
appropriate spreading and density. Fresh media was added at least 30 min before
imaging each slide-mounted coverslip. To minimize experiment-to-experiment
variation during image acquisition, all microscope components were primed by a
30-min mock acquisition: pre-determined laser and camera settings were used for
acquisition without a sample on stage. These settings were defined prior to the
screen to allow for sufficient signal intensity over background while keeping
photobleaching <10% over the course of the acquisition time. It is important
to image with the highest possible intensity, while still avoiding
photobleaching. During imaging, cells were maintained at 37°C in the same
culture media.

Two TIR-FM systems were used for acquisition of live
cell data: #1 fully motorized Nikon Eclipse Ti-E inverted microscope with
integrated second-generation Perfect Focus System, 60x Nikon 1.49NA TIRF DIC
objective, coupled to an Andor “Diskovery TIRF/ Borealis widefield illuminator”
equipped with an additional 1.8x tubelens (yielding a final magnification of
108x), motorized laser incident angle adjustment, set to 80 nm penetration
depth, and an Andor laser launch. Images were acquired with a PCO-Edge 16 bit,
2560x2160px sCMOS camera. Temperature was maintained via an OKO lab custom
built full body environmental chamber with temperature control and CO2 stage
incubator operated by Bold Line controller and OKO-Touch with SmartBox for data
logging. Components were controlled via MetaMorph v.7.7 (Molecular Devices).
TIR-FM system #2 is closely related, but uses an Andor Zyla 4.2 16 bit,
2048x2048 px sCMOS camera.

Time-lapse movies for all conditions were acquired for
7.5 min with a 1 sec interval between consecutive frames. Given the storage
size of a single movie (up to 3.6Gb), image stacks were split into two along
the largest xy dimension using a custom-written Fiji macro, yielding a total of
22-24 datasets per condition.