Figure 2.

Pearling membranes labeled with concanavalin A or PLCδ1. (A) Cells labeled with FITC–concanavalin A (green), a lectin binding preferentially to α-mannosyl residues on N-glycosylated proteins, and counterstained with Alexa Fluor 568 succinimidyl ester, a cell surface marker (red). The three panels display merged images (left), the concanavalin A label (middle), and the cell-surface label (right). (B) Cell expressing mRFP- LimEΔ (red) and GFP-PLCδ1, which recognizes PI(4,5)P₂ (green). PI(4,5)P₂ is localized to the membrane of the pearls as well as to the plasma membrane enveloping the cell body. (C) Pearl formation by protrusion of the cell surface. (D) Pearl formation by retraction of the cell. In both (C) and (D) pearl formation begins at a late stage of actin depolymerization. (E) Withdrawal of pearls during the repolymerization of actin. Cells in (C–E) were labeled similar to the cell in (B). Patches of PLCδ1 indicate local enrichment of PIP2 under conditions of intense clathrin-dependent endocytosis (2), in accord with a role of PIP2 in membrane invagination (69). Numbers indicate seconds after the addition (C and D) or the removal of 5 μM latA (E). Bars, 10 μm. The time series corresponding to (B–E) are displayed in Movies S3–S5, S7.