Figure 1. APC directly nucleates actin assembly. (A) Schematic of APC, and Coomassie-stained gel of purified APC polypeptides. (B) GFP fluorescence and rhodamine-phalloidin staining of serum-starved NIH3T3 cells. Arrowheads, cells microinjected with EGFP-APC-B plasmid. Arrow, F-actin accumulation at cell–cell junction. Bar, 10 µm. Right panel shows linescan quantification of F-actin at cell–cell junctions in the absence (b, red line) or presence (d, red line) of GFP-APC-B. (C) Quantification of F-actin levels in cells (n > 50 cells). (D) Assembly of actin (2 µM; 5% pyrene labeled) induced by 0–200 nM APC-B. (E) Electron micrographs of actin polymerized for 30 s in the presence or absence of 50 nM APC-C. Bar, 100 nm. (F) TIRF microscopy of actin (1 µM; 30% labeled) assembled in the presence and absence of 2 nM APC-B, visualized 2–3 min after initiation of polymerization. Bar, 5 µm. (G) Severing and depolymerization of 2 µM preformed actin filaments was induced by Cof1 but not APC-B upon addition of 3 µM Vitamin-D binding protein (a monomer sequestering factor) to induce depolymerization. (H) 100 nM cytochalasin D blocks the assembly of actin (2 µM) stimulated by 20 nM APC-B. (I) APC-B does not protect filament barbed ends against 100 nM CapZ (CP) in seeded filament elongation assay.
Figure 1.

APC directly nucleates actin assembly. (A) Schematic of APC, and Coomassie-stained gel of purified APC polypeptides. (B) GFP fluorescence and rhodamine-phalloidin staining of serum-starved NIH3T3 cells. Arrowheads, cells microinjected with EGFP-APC-B plasmid. Arrow, F-actin accumulation at cell–cell junction. Bar, 10 µm. Right panel shows linescan quantification of F-actin at cell–cell junctions in the absence (b, red line) or presence (d, red line) of GFP-APC-B. (C) Quantification of F-actin levels in cells (n > 50 cells). (D) Assembly of actin (2 µM; 5% pyrene labeled) induced by 0–200 nM APC-B. (E) Electron micrographs of actin polymerized for 30 s in the presence or absence of 50 nM APC-C. Bar, 100 nm. (F) TIRF microscopy of actin (1 µM; 30% labeled) assembled in the presence and absence of 2 nM APC-B, visualized 2–3 min after initiation of polymerization. Bar, 5 µm. (G) Severing and depolymerization of 2 µM preformed actin filaments was induced by Cof1 but not APC-B upon addition of 3 µM Vitamin-D binding protein (a monomer sequestering factor) to induce depolymerization. (H) 100 nM cytochalasin D blocks the assembly of actin (2 µM) stimulated by 20 nM APC-B. (I) APC-B does not protect filament barbed ends against 100 nM CapZ (CP) in seeded filament elongation assay.

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