CD2AP inhibits actin polymerization and depolymerization by capping actin barbed ends. (a) Pyrene-actin spectroscopy assay showing a dose-dependent decrease in spontaneous actin polymerization rate by CD2AP. Pyrene fluorescence was measured at 30-s intervals to obtain the trace. A representative trace from four experiments is shown. (b) Two-color actin single filament imaging showing addition of rhodamine-labeled (red) monomeric actin to preformed rhodamine green-labeled (green) actin filaments. (c) Two-color actin single filament imaging showing inhibition of red actin addition by preincubating green actin filaments with CD2AP. (d) Two-color actin single filament imaging showing addition of red actin after preincubating green actin filaments with the C-terminal half of CD2AP. (e) Two-color actin filament imaging of limulus parallel actin bundles showing inhibition of red actin addition to the fast-growing barbed ends by preincubating green actin filaments with CD2AP. (f) Pyrene-actin spectroscopy assay in the presence of monomer sequestration protein, vitamin D–binding protein (VDBP), showing suppression of actin depolymerization by CD2AP. Pyrene fluorescence was measured at 30-s intervals to obtain the trace. A representative trace from two experiments is shown. (g) Single filament imaging showing actin depolymerization upon dilution into buffer. (h) Single filament imaging showing inhibition of actin depolymerization by preincubating filaments with CD2AP.