Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> VERTEBRATE non-muscle cells are known to contain considerable amounts of actin and myosin1–3, but the mechanisms underlying their motility have yet to be elucidated. Various theories have been proposed1,4–7 to explain the well known phenomena of cell migration, membrane ruffling and unidirectional growth processes, different emphasis being placed on the involvement of membrane components and contractile filament assemblies. These hypotheses have been limited, however, by the lack of information about the detailed structural organisation of the motile regions of the cytoplasm. As one approach to this problem we have developed procedures for the direct observation of the filamentous components of the lamella regions of cultured cells in the electron microscope8,9. The method, similar to that adopted by Brown et al.10, consists of the negative-staining of cells grown directly on electron microscope grids after removal of the cell membranes with Triton X-100. By this means, we demonstrate here a single polarity of actin at the leading edge of cultured cells, and consider its implications with regard to cell motility. <s> BIB001 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <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> BIB002 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <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> BIB003 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> We have studied the interaction of CapZ, a barbed-end actin capping protein from the Z line of skeletal muscle, with actin. CapZ blocks actin polymerization and depolymerization (i.e., it "caps") at the barbed end with a Kd of approximately 0.5-1 nM or less, measured by three different assays. CapZ inhibits the polymerization of ATP-actin onto filament ends with ATP subunits slightly less than onto ends with ADP subunits, and onto ends with ADP-BeF3- subunits about as much as ends with ADP subunits. No effect of CapZ is seen at the pointed end by measurements either of polymerization from acrosomal processes or of the critical concentration for polymerization at steady state. CapZ has no measureable ability to sever actin filaments in a filament dilution assay. CapZ nucleates actin polymerization at a rate proportional to the first power of the CapZ concentration and the 2.5 power of the actin concentration. No significant binding is observed between CapZ and rhodamine-labeled actin monomers by fluorescence photobleaching recovery. These new experiments are consistent with but do not distinguish between three models for nucleation proposed previously (Cooper & Pollard, 1985). As a prelude to the functional studies, the purification protocol for CapZ was refined to yield 2 mg/kg of chicken breast muscle in 1 week. The activity is stable in solution and can be lyophilized. The native molecular weight is 59,600 +/- 2000 by equilibrium ultracentrifugation, and the extinction coefficient is 1.25 mL mg-1 cm-1 by interference optics. Polymorphism of the alpha and beta subunits has been detected by isoelectric focusing and reverse-phase chromatography. CapZ contains no phosphate (less than 0.1 mol/mol). <s> BIB004 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> Cap Z is a widely distributed, highly conserved, heterodimeric protein that binds to the barbed ends of actin filaments, but does not sever filaments. In chicken, two variant cDNAs (alpha 1 and alpha 2) encoding proteins homologous to the alpha subunit of Cap Z have been described versus one for the beta subunit. To establish the effect of each subunit and of the two potential heterodimers (alpha 1 beta and alpha 2 beta) on actin and to explore the functional domains of the proteins, RNA transcripts derived from these cDNAs were studied using in vitro translation. Sequential deletion mutants at the carboxyl and amino termini of the alpha subunit and at the amino terminus of the beta subunit were constructed, and the ability of each mutant to recombine with its heterologous subunit was assessed by gel filtration. The interaction of the individual subunits, heterodimers, and mutants with actin was studied using cosedimentation and quantitative binding assays. This study demonstrates that 1) both alpha 1 beta and alpha 2 beta heterodimers assemble in vitro after translation and bind selectively to the barbed ends of actin filaments; 2) the affinity of alpha 1 beta heterodimers for actin (KD = 1.6 x 10(-10) M) is approximately 4-fold higher than that of alpha 2 beta heterodimers (KD = 6.3 x 10(-10) M); 3) the amino-terminal 40% of the alpha 1 subunit (amino acids 1-115) and the amino-terminal 31% of the beta subunit (amino acids 1-86) are not required for high affinity binding of Cap Z to the barbed ends of actin filaments; and 4) the carboxyl-terminal 55 amino acids of the alpha subunit appear to be required for binding to actin, as are the carboxyl-terminal 15 amino acids of the beta subunit. <s> BIB005 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation-release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid-region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton. <s> BIB006 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on-rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro. <s> BIB007 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <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> BIB008 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <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> BIB009 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <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> BIB010 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> 4-[(4-Methyl-1-piperazinyl)methyl]- N -[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate (STI571) is the first successful target-based drug with excellent potency against chronic myelogenous leukemia. Studies on this compound have illuminated potentials and problems of kinase inhibitors in the treatment of cancer. As found in crystal structures, STI571-bound Abelson kinase (abl) is believed to form closed conformation with N-terminal regulatory domains. Here we present evidence of distinct STI571-induced modulation of abl functions using high-resolution live-cell imaging approaches. Within lamellipodia of fibroblast cells, STI571 was found to induce rapid translocation of abl to the lamellipodium tip. Quantitative analysis yielded 0.81 and 1.8 μM for EC 50 values of STI571-induced cell edge translocation of abl-KD-green fluorescent protein (GFP) and wild-type abl-GFP, respectively. It also revealed adverse response of drug-resistant abl-T334I to STI571, suggesting that drug binding to abl-GFP triggers translocation. N -myristoylation and the src homology 3 (SH3) domain were required for this translocation, whereas disruption of intramolecular interactions of these motifs enhanced cell-edge association of abl. An intact C-terminal last exon region in abl, but not its F-actin binding, was required for efficient cell-edge translocation. Moreover, single-molecule observation revealed an STI571-induced rapid increase in slow diffusive species of abl in both the tip and the body region of lamellipodia. These results suggest that although activated abl translocates to the cell edge at its open state, STI571 can also bind and lock abl in the open and membrane-tethered conformation as long as the SH3 domain and the C-terminal region are intact. High-resolution imaging can be a powerful tool for elucidating inhibitor modulation of abl functions under intracellular environment. <s> BIB011 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> BACKGROUND ::: The identification of somatic mutations in the gene encoding the serine-threonine protein kinase B-RAF (BRAF) in the majority of melanomas offers an opportunity to test oncogene-targeted therapy for this disease. ::: ::: ::: METHODS ::: We conducted a multicenter, phase 1, dose-escalation trial of PLX4032 (also known as RG7204), an orally available inhibitor of mutated BRAF, followed by an extension phase involving the maximum dose that could be administered without adverse effects (the recommended phase 2 dose). Patients received PLX4032 twice daily until they had disease progression. Pharmacokinetic analysis and tumor-response assessments were conducted in all patients. In selected patients, tumor biopsy was performed before and during treatment to validate BRAF inhibition. ::: ::: ::: RESULTS ::: A total of 55 patients (49 of whom had melanoma) were enrolled in the dose-escalation phase, and 32 additional patients with metastatic melanoma who had BRAF with the V600E mutation were enrolled in the extension phase. The recommended phase 2 dose was 960 mg twice daily, with increases in the dose limited by grade 2 or 3 rash, fatigue, and arthralgia. In the dose-escalation cohort, among the 16 patients with melanoma whose tumors carried the V600E BRAF mutation and who were receiving 240 mg or more of PLX4032 twice daily, 10 had a partial response and 1 had a complete response. Among the 32 patients in the extension cohort, 24 had a partial response and 2 had a complete response. The estimated median progression-free survival among all patients was more than 7 months. ::: ::: ::: CONCLUSIONS ::: Treatment of metastatic melanoma with PLX4032 in patients with tumors that carry the V600E BRAF mutation resulted in complete or partial tumor regression in the majority of patients. (Funded by Plexxikon and Roche Pharmaceuticals.) <s> BIB012 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> Inhibitors of RAF enzymes can suppress or activate the same signalling pathway. The details of how this happens provide a cautionary note for those targeting the pathway for anticancer drug discovery. <s> BIB013 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> Many animal cells initiate crawling by protruding lamellipodia, consisting of a dense network of actin filaments, at their leading edge. We imaged XTC cells that exhibit flat lamellipodia on poly-L-lysine-coated coverslips. Using active contours, we tracked the leading edge and measured the total amount of F-actin by summing the pixel intensities within a 5-μm band. We observed protrusion and retraction with period 130-200 s and local wavelike features. Positive (negative) velocities correlated with minimum (maximum) integrated actin concentration. Approximately constant retrograde flow indicated that protrusions and retractions were driven by fluctuations of the actin polymerization rate. We present a model of these actin dynamics as an excitable system in which a diffusive, autocatalytic activator causes actin polymerization; F-actin accumulation in turn inhibits further activator accumulation. Simulations of the model reproduced the pattern of actin polymerization seen in experiments. To explore the model's assumption of an autocatalytic activation mechanism, we imaged cells expressing markers for both F-actin and the p21 subunit of the Arp2/3 complex. We found that integrated Arp2/3-complex concentrations spike several seconds before spikes of F-actin concentration. This suggests that the Arp2/3 complex participates in an activation mechanism that includes additional diffuse components. Response of cells to stimulation by fetal calf serum could be reproduced by the model, further supporting the proposed dynamical picture. <s> BIB014 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <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> BIB015 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Application of SiMS Microscopy in Cell Biology, Biophysics and Pharmacology <s> Capping protein (CP) binds the fast growing barbed end of the actin filament and regulates actin assembly by blocking the addition and loss of actin subunits. Recent studies provide new insights into how CP and barbed-end capping are regulated. Filament elongation factors, such as formins and ENA/VASP (enabled/vasodilator-stimulated phosphoprotein), indirectly regulate CP by competing with CP for binding to the barbed end, whereas other molecules, including V-1 and phospholipids, directly bind to CP and sterically block its interaction with the filament. In addition, a diverse and unrelated group of proteins interact with CP through a conserved 'capping protein interaction' (CPI) motif. These proteins, including CARMIL (capping protein, ARP2/3 and myosin I linker), CD2AP (CD2-associated protein) and the WASH (WASP and SCAR homologue) complex subunit FAM21, recruit CP to specific subcellular locations and modulate its actin-capping activity via allosteric effects. <s> BIB016
Direct visualization of single-molecule proteins in live cells has revealed unexpected molecular behaviors that are not consistent with prior knowledge from in vitro biochemical studies. For examples, at steady state in vitro, actin assembles at the barbed end (plus end) and depolymerizes at the pointed end (minus end), which is referred to as "treadmilling". Lamellipodia, the sheet-like structure which cells extend during migration, contain a highly dense actin meshwork  . In lamellipodia, actin filaments are mostly oriented with their barbed ends facing the cell edge BIB001 , and the actin meshwork is constantly conveyed to the cell center by the retrograde actin flow BIB002 BIB003 . These observations led researchers to hypothesize that actin undergoes treadmilling in a manner that actin polymerizes at the leading edge and depolymerizes at the base of lamellipodia BIB006 . On the other hand, observations of single-molecule actin speckles revealed that filament turnover in the lamellipodial actin network is very rapid, as nearly one-third of filaments have short lifetimes of less than 10 s, and actin turnover distributes broadly throughout lamellipodia BIB008 BIB015 . As discussed in our recent publication  , these observations suggest that treadmiling does not solely account for the actin filament turnover in vivo. Capping protein (CP) regulates actin polymerization by binding the barbed end of an actin filament, which blocks assembly and disassembly of actin subunits BIB016 . In vitro, CP tightly binds to the barbed end with a Kd of 0.06-1 nM BIB004 BIB005 BIB007 . Surprisingly in lamellipodia, SiMS analyses revealed that the dissociation of EGFP-tagged CP (EGFP-CP) occurs at 0.58 s −1 , which is 100-times faster than the dissociation of EGFP-CP from the barbed end at 0.005 s −1 in vitro BIB009 . Attenuation of the fast dissociation of CP upon blockade of the actin depolymerizing activity suggests that frequent filament severing might take place in the lamellipodial actin networks. Evidence from SiMS analysis on the cofilin cofactor AIP1 BIB010 also supports the existence of a non-treadmilling actin turnover mechanism by which a substantial amount of actin might disassemble near the barbed end of the filament  .  Direct visualization of single-molecule proteins in live cells has revealed unexpected molecular behaviors that are not consistent with prior knowledge from in vitro biochemical studies. For examples, at steady state in vitro, actin assembles at the barbed end (plus end) and depolymerizes at the pointed end (minus end), which is referred to as "treadmilling". Lamellipodia, the sheet-like structure which cells extend during migration, contain a highly dense actin meshwork  . In lamellipodia, actin filaments are mostly oriented with their barbed ends facing the cell edge BIB001 , and the actin meshwork is constantly conveyed to the cell center by the retrograde actin flow BIB002 BIB003 . These observations led researchers to hypothesize that actin undergoes treadmilling in a manner that actin polymerizes at the leading edge and depolymerizes at the base of lamellipodia BIB006 . On the other hand, observations of single-molecule actin speckles revealed that filament turnover in the lamellipodial actin network is very rapid, as nearly one-third of filaments have short lifetimes of less than 10 s, and actin turnover distributes broadly throughout lamellipodia BIB008 BIB015 . As discussed in our recent publication  , these observations suggest that treadmiling does not solely account for the actin filament turnover in vivo. Capping protein (CP) regulates actin polymerization by binding the barbed end of an actin filament, which blocks assembly and disassembly of actin subunits BIB016 . In vitro, CP tightly binds to the barbed end with a Kd of 0.06-1 nM BIB004 BIB005 BIB007 . Surprisingly in lamellipodia, SiMS analyses revealed that the dissociation of EGFP-tagged CP (EGFP-CP) occurs at 0.58 s −1 , which is 100-times faster than the dissociation of EGFP-CP from the barbed end at 0.005 s −1 in vitro BIB009 . Attenuation of the fast dissociation of CP upon blockade of the actin depolymerizing activity suggests that frequent filament severing might take place in the lamellipodial actin networks. Evidence from SiMS analysis on the cofilin cofactor AIP1 BIB010 also supports the existence of a non-treadmilling actin turnover mechanism by which a substantial amount of actin might disassemble near the barbed end of the filament  . SiMS data have been used in quantitative mathematical models to investigate how complex actin dynamics is coordinated in vivo. Ryan et al. proposed a model postulating that a diffusive, autocatalytic activator promotes actin polymerization; filamentous actin accumulation in turn inhibits further activator accumulation BIB014 . Simulations of the model reproduced the pattern of actin polymerization seen in SiMS experiments in the cycle of lamellipodial protrusion and retraction. The Arp2/3 complex, which is the key nucleator of branched actin meshwork in lamellipodia  , accumulates several seconds before actin filament accumulation BIB014 . This observation suggests that the Arp2/3 complex may participate in an activation mechanism. SiMS analysis is often capable of revealing the rapid response of molecules to pharmacological treatment. Vemurafenib is a BRAF (v-raf murine sarcoma viral oncogene homolog B1) enzyme inhibitor for the treatment of patients who have metastatic melanoma. The compound was developed to inhibit BRAF V600E , a cancer-associated mutant of BRAF. However, vemurafenib can paradoxically activate the Ras/RAF/MAPK pathway via activation of CRAF (v-raf-1 murine leukemia viral oncogene homolog 1) through the formation of dimeric RAF complexes BIB012 BIB013 . Such unexpected allosteric effects of target-based drugs have attracted substantial attention, because these allosteric effects may change the therapeutic effects of the drugs or cause unexpected side effects. Prior to the findings with vemurafenib, our study employing SiMS imaging approach reported that imatinib, a target-based drug against chronic myelogenous leukemia, induces allosteric effects on its target molecule, Abelson kinase (c-abl) BIB011 . SiMS imaging revealed that rapid cell-edge translocation of c-abl was induced by imatinib. We compared imatinib-induced molecular behaviors of c-abl mutant proteins, which provided clues to solving the conformational changes of c-abl upon imatinib binding BIB011 . As shown in this example, direct viewing of molecular behavior would facilitate solving the action of target-based drugs unambiguously.