Journal: bioRxiv

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

doi: 10.1101/2021.01.31.428633

Figure Lengend Snippet: A) Overview of recovery conditions. Following AY9944 sterol depletion, HEK293T hCLTA-Tq2 EN cells were exposed to cholesterol-loaded MβCD (MβCD-Chol, and chased in LPDS media) [Top] , supplemented continuously with lipoprotein-rich 15% FBS in the presence of AY9944 [Middle] , or incubated continuously in 7.5% LPDS media in the absence of AY9944 to allow endogenous cholesterol synthesis [Bottom] . B) Mid-plane live-cell confocal microscopy of HEK293T hCLTA-Tq2 EN following addition of transferrin-conjugated to AF-555 (Tfn-555) during recovery as described in (A). Representative images from different fields of view shown. Tfn and clathrin distributions in normal and arrested CME [Inset] . Scale bar = 10 μm. C) Corresponding cellular sterol profiles quantified by GC/MS (Mean ± SD). N = 3 biological replicates from independent experiments. Dotted and dashed lines represent total sterol abundance of untreated FBS and LPDS cultured cells, respectively.

Article Snippet: HEK293T cells were then incubated with transferrin (Tfn) conjugated to Alexa FluorTM 555 (25 ug/mL, Invitrogen) for 30 mins at 37°C, followed by rinsing in ice-cold PBS, and fixation in 4% paraformaldehyde for 20 min. For high-content imaging analysis, 24 well glass bottom plates (Cellvis) were imaged using CX7 Rescue High-Content Screening (HCS) Navigator software (PerkinElmer, Waltham, MA) and standard HCS imaging protocol.

Techniques: Incubation, Confocal Microscopy, Gas Chromatography-Mass Spectrometry, Cell Culture

Journal: bioRxiv

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

doi: 10.1101/2021.01.31.428633

Figure Lengend Snippet: The ability of structurally diverse sterols to rescue CME inhibition due to sterol depletion by AY9944 treatment was evaluated by direct delivery of sterols to the PM via MβCD carrier. A) Summary of physical properties and phase separation behavior of sterols. Topological Polar Surface Area (TPSA) computed surface sum over all polar atoms. Ordered lipid ( L o ) domain (raft) stabilizing (+) or disrupting (−) sterols indicated relative to cholesterol (++). Refer to Table S2 for additional details. B) Sterol profiles of AY9944 treated HEK293T cells following 1h incubation with sterol loaded MβCD (Mean ± SD). N = 4 independent biological replicates from two MβCD-sterol preparations. C) Tfn uptake relative to controls cultured in 7.5% LPDS for 48 h (Mean ± SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA (F(12, 51) = 11.23, p < 0.0001) and Dunnett’s test versus LPDS control (N = 5 biological replicates from 3 independent experiments, ~1,500 cells per replicate). Cholesterol, desmosterol, 7DHC, and lathosterol support formation of L o domains in model membranes as designated. D) Representative confocal images taken mid-plane following 30 min incubation with AF-555 conjugated transferrin (Tfn). Scale bar = 20 μm. (c), Commercially available, pre-loaded MβCD-Chol.

Article Snippet: HEK293T cells were then incubated with transferrin (Tfn) conjugated to Alexa FluorTM 555 (25 ug/mL, Invitrogen) for 30 mins at 37°C, followed by rinsing in ice-cold PBS, and fixation in 4% paraformaldehyde for 20 min. For high-content imaging analysis, 24 well glass bottom plates (Cellvis) were imaged using CX7 Rescue High-Content Screening (HCS) Navigator software (PerkinElmer, Waltham, MA) and standard HCS imaging protocol.

Techniques: Inhibition, Incubation, Cell Culture, Control