Abstract
Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin - Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.
Highlights
Actin polymerization is a dynamic and highly controlled process that regulates a range of cellular functions such as organelle transport, intracellular pathogen spread and cell motility
To test whether photoconverted Dendra2 - b-actin (D2BA) would incorporate into macrophage podosomes under steady-state conditions, TIRF microscopy was employed in order to minimize the contribution of the cell body and eliminate out-of-focus light
We have previously demonstrated that active WASp, a major actin polymerization activator involved in podosome formation and dynamics, is visible by TIRF in macrophage podosomes [14], we reasoned actin polymerization should be visible by TIRF
Summary
Actin polymerization is a dynamic and highly controlled process that regulates a range of cellular functions such as organelle transport, intracellular pathogen spread and cell motility. Our previously described method using EGFP-b-actin [9], reliable and accurate, can only identify barbed ends during a burst of actin polymerization, i.e., when addition of monomers at the barbed end (which induces an increase in fluorescence) is much faster than the depolymerization (which induces a decrease in fluorescence) at the pointed end of the filament. It does not extend the measurement of bactin incorporation into barbed ends when the relative contributions of polymerization and depolymerization are comparable, such as in a treadmilling filament
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