Abstract
Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”.
Highlights
In recent years, the organization, dynamics, and functions of actin filaments on and near the cytoplasmic surface of the plasma membrane (PM), often termed cortical actin filaments or the cortical actin meshwork [1,2,3,4,5], have gained extensive attention
The results indicated that 72% and 89% of Lifeact-TM molecules that entered the projected areas of actin-pl-clusters and stress fibers, respectively, underwent at least one Temporary Arrest of LateraL diffusion (TALLs) event there (Table 1), suggesting that at least 72% of the actin-pl-clusters and 89% of the stress fibers visible by total internal reflection fluorescence microscopy (TIRFM) are closely apposed to the PM cytoplasmic surface
In this study, we found that the actin-pl-clusters or the actin nodes observed here were quite different structures from those found after the latrunculin treatment (Fig 9)
Summary
The organization, dynamics, and functions of actin filaments on and near the cytoplasmic surface of the plasma membrane (PM), often termed cortical actin filaments or the cortical actin meshwork [1,2,3,4,5], have gained extensive attention. In the cortical actin meshwork, the meshwork apposed to the PM cytoplasmic surface, located within ~8 nm from the cytoplasmic surface, has been identified using three-dimensional (3D) reconstruction of electron microscopy (EM) images of PM specimens prepared by the rapid-freeze deep-etch technique [6,7]. This actin meshwork was termed the “actin-based membrane skeleton” [6,8,9], and similar meshwork structures have been observed in both the top (apical) and bottom (basal) PMs [9,10]. The rapid freezing of samples using pure copper blocks cooled with liquid helium is likely to preserve the actin meshwork structure reasonably well [6,9], this discrepancy suggests that observing the cortical actin organization in living cells would provide more accurate information
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