Cholesterol asymmetry at the tip of filopodia during cell adhesion

Abstract

During adhesion, cells develop filopodia to facilitate the attachment to the extracellular matrix. The small guanosine triphosphate (GTP)-binding protein, Cdc42, plays a central role in the formation of filopodia. It has been reported that Cdc42 activity is regulated by cholesterol (Chol). We examined Chol distribution in filopodia using Chol-binding domain 4 (D4) fragment of bacterial toxin, perfringolysin O that senses high membrane concentration of Chol. Our results indicate that fluorescent D4 was enriched at the tip of the outer leaflet of filopodia in the initiation phase of cell adhesion. This enrichment was accompanied by a defect of D4 labeling in the inner leaflet. Steady phase adhered cell experiment indicated that both Cdc42 and ATP-binding cassette transporter, ABCA1, were involved in the binding of D4 to the cell surface. Depletion of Chol activated Cdc42. Our results suggest that asymmetric distribution of Chol at the tip of filopodia induces activation of Cdc42, and thus, facilitates filopodia formation. K E Y W O R D S ABCA1, Cdc42, cholesterol probe, lipid asymmetry 6186 | KISHIMOTO eT al.

Active cholesterol 20 years on

“…However, other methods have shown that most of the sterol is in that leaflet 40 . Indeed, endogenously expressed fluorescent cytolysins were found to light up the cytoplasmic face of the plasma membrane bilayer, signifying that its cholesterol exceeded ~30 mol% and was therefore not far from that in the outer leaflet 144,163,171,172 . While the extreme transbilayer asymmetry reported by Liu et al and Zhang et al is therefore at odds with other reports, those studies nevertheless showed that the active transport of cholesterol plays a role in Wnt and Hedgehog signaling 115,170 …”

“…Fluorescent cytolysin derivatives have been used to probe the sidedness of plasma membrane bilayer cholesterol 115,170,171 . Quantifying the cholesterol level in each leaflet is challenging, because the sterol dependence of toxin binding is thresholded and must be calibrated with synthetic vesicles.…”

Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane

“…To express GFP-D4H and GFPenvy-D4H in Escherichia coli , the D4H (D4 D434S ) mutant fragment was generated by the standard two-step PCR mutagenesis technique using pColdI-mCherry-D4 as a template ( Kishimoto et al. , 2020 ).…”

Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane

“…To express GFP-D4H and GFPenvy-D4H in Escherichia coli , the D4H (D4 D434S ) mutant fragment was generated by the standard two-step PCR mutagenesis technique using pColdI-mCherry-D4 as a template [121]. The GFP and D4H fragments were inserted into pColdI (Takara bio, Shiga, Japan) to construct pColdI-GFP-D4H.…”

“…Recombinant GFP-D4H and GFPenvy-D4H proteins were prepared from Escherichia coli as described previously [121]. The protein concentrations were determined by BCA assay.…”