Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Principles of Single-Molecule Speckle (SiMS) Microscopy <s> Lamellipodia are thin, veil-like extensions at the edge of cells that contain a dynamic array of actin filaments. We describe an approach for analyzing spatial regulation of actin polymerization and depolymerization in vivo in which we tracked single molecules of actin fused to the green fluorescent protein. Polymerization and the lifetime of actin filaments in lamellipodia were measured with high spatial precision. Basal polymerization and depolymerization occurred throughout lamellipodia with largely constant kinetics, and polymerization was promoted within one micron of the lamellipodium tip. Most of the actin filaments in the lamellipodium were generated by polymerization away from the tip. <s> BIB001 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Principles of Single-Molecule Speckle (SiMS) Microscopy <s> Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays. <s> BIB002 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Principles of Single-Molecule Speckle (SiMS) Microscopy <s> mDia1 belongs to the formin family of proteins that share FH1 and FH2 domains. Although formins play a critical role in the formation of many actin-based cellular structures, the physiological regulation of formin-mediated actin assembly within the cell is still unknown. Here we show that cells possess an acute actin polymer restoration mechanism involving mDia1. By using single-molecule live-cell imaging, we found that several treatments including low-dose G-actin-sequestering drugs and unpolymerizable actin mutants activate mDia1 to initiate fast directional movement. The FH2 region, the core domain for actin nucleation, is sufficient to respond to latrunculin B (LatB) to increase its actin nucleation frequency. Simulation analysis revealed an unexpected paradoxical effect of LatB that leads to a several fold increase in free G-actin along with an increase in total G-actin. These results indicate that in cells, the actin nucleation frequency of mDia1 is enhanced not only by Rho, but also strongly through increased catalytic efficiency of the FH2 domain. Consistently, frequent actin nucleation by mDia1 was found around sites of vigorous actin disassembly. Another major actin nucleator, the Arp2/3 complex, was not affected by the G-actin increase induced by LatB. Taken together, we propose that transient accumulation of G-actin works as a cue to promote mDia1-catalyzed actin nucleation to execute rapid reassembly of actin filaments. <s> BIB003 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Principles of Single-Molecule Speckle (SiMS) Microscopy <s> The mechanism of lamellipod actin turnover is still under debate. To clarify the intracellular behavior of the recently-identified actin disruption mechanism, we examined kinetics of AIP1 using fluorescent single-molecule speckle microscopy. AIP1 is thought to cap cofilin-generated actin barbed ends. Here we demonstrate a reduction in actin-associated AIP1 in lamellipodia of cells overexpressing LIM-kinase. Moreover, actin-associated AIP1 was rapidly abolished by jasplakinolide, which concurrently blocked the F-actin-cofilin interaction. Jasplakinolide also slowed dissociation of AIP1, which is analogous to the effect of this drug on capping protein. These findings provide in vivo evidence of the association of AIP1 with barbed ends generated by cofilin-catalyzed filament disruption. Single-molecule observation found distribution of F-actin-associated AIP1 throughout lamellipodia, and revealed even faster dissociation of AIP1 than capping protein. The estimated overall AIP1-associated actin disruption rate, 1.8 µM/s, was one order of magnitude faster than Arp2/3 complex-catalyzed actin nucleation in lamellipodia. This rate does not suffice the filament severing rate predicted in our previous high frequency filament severing-annealing hypothesis. Our data together with recent biochemical studies imply barbed end-preferred frequent filament disruption. Frequent generation of AIP1-associated barbed ends and subsequent release of AIP1 may be the mechanism that facilitates previously observed ubiquitous actin polymerization throughout lamellipodia. <s> BIB004 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Principles of Single-Molecule Speckle (SiMS) Microscopy <s> M6a is a four-transmembrane protein that is abundantly expressed in the nervous system. Previous studies have shown that over-expression of this protein induces various cellular protrusions, such as neurites, filopodia, and dendritic spines. In this detailed characterization of M6a-induced structures, we found their varied and peculiar characteristics. Notably, the M6a-induced protrusions were mostly devoid of actin filaments or microtubules and exhibited free random vibrating motion. Moreover, when an antibody bound to M6a, the membrane-wrapped protrusions were suddenly disrupted, leading to perturbation of the surrounding membrane dynamics involving phosphoinositide signaling. During single-molecule analysis, M6a exhibited cytoskeleton-independent movement and became selectively entrapped along the cell perimeter in an actin-independent manner. These observations highlight the unusual characteristics of M6a, which may have a significant yet unappreciated role in biological systems. <s> BIB005 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Principles of Single-Molecule Speckle (SiMS) Microscopy <s> The SCAR/WAVE complex drives lamellipodium formation by enhancing actin nucleation by the Arp2/3 complex. Phosphoinositides and Rac activate the SCAR/WAVE complex, but how SCAR/WAVE and Arp2/3 complexes converge at sites of nucleation is unknown. We analyzed the single-molecule dynamics of WAVE2 and p40 (subunits of the SCAR/WAVE and Arp2/3 complexes, respectively) in XTC cells. We observed lateral diffusion of both proteins and captured the transition of p40 from diffusion to network incorporation. These results suggest that a diffusive 2D search facilitates binding of the Arp2/3 complex to actin filaments necessary for nucleation. After nucleation, the Arp2/3 complex integrates into the actin network and undergoes retrograde flow, which results in its broad distribution throughout the lamellipodium. By contrast, the SCAR/WAVE complex is more restricted to the cell periphery. However, with single-molecule imaging, we also observed WAVE2 molecules undergoing retrograde motion. WAVE2 and p40 have nearly identical speeds, lifetimes and sites of network incorporation. Inhibition of actin retrograde flow does not prevent WAVE2 association and disassociation with the membrane but does inhibit WAVE2 removal from the actin cortex. Our results suggest that membrane binding and diffusion expedites the recruitment of nucleation factors to a nucleation site independent of actin assembly, but after network incorporation, ongoing actin polymerization facilitates recycling of SCAR/WAVE and Arp2/3 complexes. <s> BIB006 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Principles of Single-Molecule Speckle (SiMS) Microscopy <s> Physical force evokes rearrangement of the actin cytoskeleton. Signalling pathways such as tyrosine kinases, stretch-activated Ca(2+) channels and Rho GTPases are involved in force sensing. However, how signals are transduced to actin assembly remains obscure. Here we show mechanosensitive actin polymerization by formins (formin homology proteins). Cells overexpressing mDia1 increased the amount of F-actin on release of cell tension. Fluorescence single-molecule speckle microscopy revealed rapid induction of processive actin assembly by mDia1 on cell cortex deformation. mDia1 lacking the Rho-binding domain and other formins exhibited mechanosensitive actin nucleation, suggesting Rho-independent activation. Mechanosensitive actin nucleation by mDia1 required neither Ca(2+) nor kinase signalling. Overexpressing LIM kinase abrogated the induction of processive mDia1. Furthermore, s-FDAPplus (sequential fluorescence decay after photoactivation) analysis revealed a rapid actin monomer increase on cell cortex deformation. Our direct visualization of the molecular behaviour reveals a mechanosensitive actin filament regeneration mechanism in which G-actin released by actin remodelling plays a pivotal role. <s> BIB007 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Principles of Single-Molecule Speckle (SiMS) Microscopy <s> The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs. <s> BIB008
SiMS microscopy is an approach to monitor complex behaviors of proteins in living cells with considerably high resolution. In the original SiMS analysis, which was first introduced in 2002 BIB001 , individual EGFP-actin molecules were visualized in cells expressing a very low level of EGFP-actin under the control of a defective cytomegalovirus (CMV) promoter. EGFP-actin either co-assembles with filamentous actin (F-actin) or exists as monomeric actin (G-actin) in cells. When the expression level of EGFP-actin is sufficiently low, signals from EGFP-actin assembled with F-actin add up in a small spot on the charged-coupled device (CCD) with long exposure time up to 2 s. In contrast, signals from a freely diffusing EGFP-actin in the monomeric form are blurred on the image. The diffusion constant of actin monomer is 3-6 µm 2 ·s −1  . Monomeric probes travel through a path of a few micro meters during the 2-s exposure time. Therefore, only EGFP-actin in the F-actin state can be visualized as single-molecules ( Figure 1 ). With this simple principle, cytoskeletal proteins and their associating proteins are visualized as single-molecules in a state specific manner  BIB002 BIB001 BIB007 BIB003 BIB006 BIB004 BIB008 . The SiMS method also enables tracking of the membrane-bound proteins along the plasma membrane BIB005 . Sensors 2017, 17, 1585 3 of 9 visualized as single-molecules ( Figure 1 ). With this simple principle, cytoskeletal proteins and their associating proteins are visualized as single-molecules in a state specific manner  BIB002 BIB001 BIB007 BIB003 BIB006 BIB004 BIB008 . The SiMS method also enables tracking of the membrane-bound proteins along the plasma membrane BIB005 . 