In vivo dynamics of the cortical actin network revealed by fast-scanning atomic force microscopy

Abstract

Together with lamellipodia and stress fibers, a dynamic network of actin filaments in the cell cortex plays a major role in the maintenance of cell morphology and motility. In contrast to lamellipodia, which have been well studied in various motile cells, the dynamics of actin filaments in the cell cortex have not yet been clarified due to a lack of proper imaging techniques. Here, we utilized high-speed atomic force microscopy for live-cell imaging and analyzed cortical actin dynamics in living cells. We successfully measured the polymerization rate and the frequency of filament synthesis in living COS-7 cells, and examined the associated effects of various inhibitors and actin-binding proteins. Actin filaments are synthesized beneath the plasma membrane and eventually descend into the cytoplasm. The inhibitors, cytochalasin B inhibited the polymerization, while jasplakinolide, inhibited the turnover of actin filaments as well as descension of the newly synthesized filaments, suggesting that actin polymerization near the membrane drives turnover of the cortical actin meshwork. We also determined how actin turnover is maintained and regulated by the free G-actin pool and G-actin binding proteins such as profilin and thymosin β4, and found that only a small amount of free G-actin was present in the cortex. Finally, we analyzed several different cell types, and found that the mesh size and the orientation of actin filaments were highly divergent, indicating the involvement of various actin-binding proteins in the maintenance and regulation of cortical actin architecture in each cell type.

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

“…The analysis of the CCP lifetime in the presence of actin inhibitors revealed an inhibitory effect of the cortical actin network on the progress of CME. Cytochalasin B, an inhibitor of actin polymerization, and CK666, an inhibitor of the Arp2/3 complex, which binds to F-actin and generates a branching point, both shortened the CCP lifetime, whereas Jasplakinolide, which inhibits actin depolymerization and stabilizes the cortical actin network 31 , prolonged the lifetime (Fig. 6a, Supplementary Table 1, Supplementary Movie S5-7).…”

“…Actin and actin-related proteins are also known to contribute to CCP assembly, although their exact role is not fully understood 15,30 . We previously observed and reported the dynamic turnover of the cortical actin network 31 ; actin filaments are polymerized near the plasma membrane and descend into the cytoplasm. Therefore, we first examined the effect of actin inhibitors on the CME process.…”

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

“…Actin and actin-related proteins are also known to contribute to CCP assembly, although their exact role is not fully understood [ 14 , 35 ]. We previously observed and reported the dynamic turnover of the cortical actin network [ 36 ]; actin filaments are polymerized near the plasma membrane and descend into the cytoplasm. Therefore, we first examined the effect of actin inhibitors on the CME process.…”

“…The analysis of the CCP lifetime in the presence of actin inhibitors revealed an inhibitory effect of the cortical actin network on the progress of CME. Cytochalasin B (an inhibitor of actin polymerization) and CK666 (an inhibitor of the Arp2/3 complex, which binds to F-actin and generates a branching point) both shortened the CCP lifetime, whereas jasplakinolide—which inhibits actin depolymerization and stabilizes the cortical actin network [ 36 ]—prolonged the lifetime ( Fig 6A , S2 Table , S5 – S7 Movies). In the presence of cytochalasin B, both the growing and stable phases shortened from 45 ± 34 s to 7 ± 12 s and from 88 ± 29 s to 59 ± 33 s, respectively, whereas there was little effect on the duration of the closing step (31 ± 9 to 22 ± 7 s) ( Fig 6B , S3 Table , S8 Movie ).…”

Multiple Mechanisms Driving F-actin-Dependent Transport of Organelles to and From Secretory Sites in Bovine Chromaffin Cells

“…In order to test if these local changes are associated with the activity of the F-actin cytoskeleton, we evaluated the effect of chemicals that specifically interact with F-actin or its molecular motor, myosin II. We first incubated cells expressing EGFP-LifeAct for 15 min with the F-actin stabilizer jasplakinolide (1 μM) at room temperature (Bubb et al, 1994 ; Zhang et al, 2017 ). When the F-actin dynamics were assessed by confocal microscopy, the changes measured in expansions (Figure 4K ) and retractions (Figure 4L ) that were induced by stimulation (Figures 4C,D ), and are absent in control non-stimulated cells (Figures 4A,B ), were almost totally abolished in the presence of this compound, as shown by the variations in distance relative to the initial profile (see white line in Figures 4E,F ).…”