Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Ease of finding cells for SiMS imaging <s> Previous observations indicated that the lamellipodium ("leading edge") of fibroblasts contains a dense meshwork, as well as numerous bundles (microspikes) of actin filaments. Most, if not all, of the filaments have a uniform polarity, with the "barbed" end associated with the membrane. I investigated whether and how actin subunits exchange in this region by microinjecting living gerbil fibroma cells (IMR-33) with actin that had been labeled with iodoacetamidotetramethylrhodamine. After incorporation of the labeled actin into the lamellipodium, I used a laser microbeam to photobleach a 3-4-micron region at and surrounding a microspike, without disrupting the integrity of the structure. I then recorded the pattern of fluorescence recovery and analyzed it using a combination of TV image intensification and digital image processing techniques. Fluorescence recovery was first detected near the edge of the cell and then moved toward the cell's center at a constant rate of 0.79 +/- 0.31 micron/min. When only part of the lamellipodium near the edge of the cell was photobleached, the bleached spot also moved toward the cell's center and through an area unbleached by the laser beam. These results indicated that steady state incorporation of actin subunits occurred predominantly at the membrane-associated end of actin filaments, and that actin subunits in the lamellipodium underwent a constant movement toward the center of the cell. I suggest that treadmilling, possibly in combination with other molecular interactions, may provide an effective mechanism for the movement of actin subunits and the protrusion of cytoplasm in the lamellipodium of fibroblasts. <s> BIB001 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Ease of finding cells for SiMS imaging <s> Actions of cytochalasin B (CB) on cytoskeletons and motility of growth cones from cultured Aplysia neurons were studied using a rapid flow perfusion chamber and digital video light microscopy. Living growth cones were observed using differential interference contrast optics and were also fixed at various time points to assay actin filament (F-actin) and microtubule distributions. Treatment with CB reversibly blocked motility and eliminated most of the phalloidin-stainable F-actin from the leading lamella. The loss of F-actin was nearly complete within 2-3 min of CB application and was largely reversed within 5-6 min of CB removal. The loss and recovery of F-actin were found to occur with a very distinctive spatial organization. Within 20-30 s of CB application, F-actin networks receded from the entire peripheral margin of the lamella forming a band devoid of F-actin. This band widened as F-actin receded at rates of 3-6 microns/min. Upon removal of CB, F-actin began to reappear within 20-30 s. The initial reappearance of F-actin took two forms: a coarse isotropic matrix of F-actin bundles throughout the lamella, and a denser matrix along the peripheral margin. The denser peripheral matrix then expanded in width, extending centrally to replace the coarse matrix at rates again between 3-6 microns/min. These results suggest that actin normally polymerizes at the leading edge and then flows rearward at a rate between 3-6 microns/min. CB treatment was also observed to alter the distribution of microtubules, assayed by antitubulin antibody staining. Normally, microtubules are restricted to the neurite shaft and a central growth cone domain. Within approximately 5 min after CB application, however, microtubules began extending into the lamellar region, often reaching the peripheral margin. Upon removal of CB, the microtubules were restored to their former central localization. The timing of these microtubule redistributions is consistent with their being secondary to effects of CB on lamellar F-actin. <s> BIB002 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Ease of finding cells for SiMS imaging <s> Photon penetration into living tissue is highly dependent on the absorption and scattering properties of tissue components. The near-infrared region of the spectrum offers certain advantages for photon penetration, and both organic and inorganic fluorescence contrast agents are now available for chemical conjugation to targeting molecules. This review focuses on those parameters that affect image signal and background during in vivo imaging with near-infrared light and exogenous contrast agents. Recent examples of in vivo near-infrared fluorescence imaging of animals and humans are presented, including imaging of normal and diseased vasculature, tissue perfusion, protease activity, hydroxyapatite and cancer. <s> BIB003 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Ease of finding cells for SiMS imaging <s> Cell migration initiates by extension of the actin cytoskeleton at the leading edge. Computational analysis of fluorescent speckle microscopy movies of migrating epithelial cells revealed this process is mediated by two spatially colocalized but kinematically, kinetically, molecularly, and functionally distinct actin networks. A lamellipodium network assembled at the leading edge but completely disassembled within 1 to 3 micrometers. It was weakly coupled to the rest of the cytoskeleton and promoted the random protrusion and retraction of the leading edge. Productive cell advance was a function of the second colocalized network, the lamella, where actomyosin contraction was integrated with substrate adhesion. <s> BIB004 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Ease of finding cells for SiMS imaging <s> Migrating cells advance by first protruding a lamellipodium, a thin sheet of cytoplasm that consists mostly of filamentous actin (F-actin). The region immediately behind the lamellipodium is also relatively flat and has been termed the lamella ([Heath and Holifield, 1991][1]). Adhesion to the matrix <s> BIB005 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Ease of finding cells for SiMS imaging <s> In the accompanying Speckle Microscopy Update ([Vallotton and Small, 2009][1]), the authors present a critique of the `lamella hypothesis' – the proposal that the lamella extends to the leading edge of motile cells and overlaps with the lamellipodium ([Ponti et al., 2004][2]). In the 2004 paper, <s> BIB006 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Ease of finding cells for SiMS imaging <s> Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network. <s> BIB007 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Ease of finding cells for SiMS imaging <s> The retrograde actin flow, continuous centripetal movement of the cell peripheral actin networks, is widely observed in adherent cells. The retrograde flow is believed to facilitate cell migration when linked to cell adhesion molecules. In this review, we summarize our current knowledge regarding the functional relationship between the retrograde actin flow and focal adhesions (FAs). We also introduce our recent study in which single-molecule speckle (SiMS) microscopy dissected the complex interactions between FAs and the local actin flow. FAs do not simply impede the actin flow, but actively attract and remodel the local actin network. Our findings provide a new insight into the mechanisms for protrusion and traction force generation at the cell leading edge. Furthermore, we discuss possible roles of the actin flow-FA interaction based on the accumulated knowledge and our SiMS study. <s> BIB008
Slightly difficult: It demands experience to find cells expressing EGFP-actin at an optimal level. Easy: Electroporation enables the incorporation of probes into almost 100% of cells at an optimal level. The actin network at the cell periphery continuously moves toward the central region, namely the retrograde actin flow BIB001 BIB002 . The retrograde flow is believed to promote cell edge protrusion when linked to focal adhesions. Before the development of eSiMS method, detailed analysis on the retrograde flow was missing due to rapid actin turnover, the use of GFP-actin, the lack of appropriate analysis algorithms, and technical difficulties. Direct viewing of individual actin filaments by eSiMS microscopy reveals the actin flow velocities including formin-based, short-lived and locally modified actin populations BIB007 . By using eSiMS, we readdressed the remaining question in the previous quantitative fluorescent speckle microscopy (qFSM) study BIB004 which has caused intensive debate over the fidelity of qFSM BIB005 BIB006 . Our eSiMS analysis BIB007 failed to reproduce the previously proposed coexistence of actin populations with different speeds in lamellipodia BIB004 . In addition, we revealed how focal adhesions influence local retrograde flow in lamellipodia in detail by using eSiMS BIB007 BIB008 . Usage of chemically labeled probes in the eSiMS method extends availability of fluorophores to a wide-range of excitation/emission wavelengths. Near infrared (NIR) emissive dyes have advantages in the imaging of thick cells and tissues, because fluorescence imaging in the long-wavelength region facilitates low autofluorescence background, low phototoxicity to cells, and deep tissue penetration  BIB003 . In the accompanying article, we introduce a new NIR actin probe with excellent photostability which is comparable to that of DyLight 549. The new eSiMS microscopy introduced here makes it easy to use live-cell single-molecule imaging with a very high spatiotemporal resolution. With the methodological advantage, the eSiMS microscopy will help researchers extend the application of single-molecule analysis. For instance, the principles of the eSiMS method are applicable to many other proteins for monitoring various molecular dynamics in live cells. In addition, a new NIR actin probe introduced in the accompanying article will extend single-molecule analysis to monitoring actin remodeling processes in deep cellular structures with three-color imaging. The eSiMS microscopy with NIR probes will be applicable for monitoring single-molecules three-dimensionally. To carry out the three-dimensional SiMS analysis, tracking and analyzing molecular behavior, while reducing photodamage during SiMS imaging in multiple focal planes, will be some of the new challenges.