Convection-Induced Biased Distribution of Actin Probes in Live Cells.

Fluorescent markers that bind endogenous target proteins are frequently employed for quantitative live-cell imaging. To visualize the actin cytoskeleton in live cells, several actin-binding probes have been widely used. Among them, Lifeact is the most popular probe with ideal properties, including fast exchangeable binding kinetics.

Multitarget super-resolution microscopy with high-density labeling by exchangeable probes.

We have developed a multitarget super-resolution microscopy technique called image reconstruction by integrating exchangeable single-molecule localization (IRIS). IRIS uses protein fragment-based probes that directly associate with and dissociate from their targets over durations on the order of tens of milliseconds. By integrating single-molecule localization and sequential labeling, IRIS enables unprecedented labeling density along multiple cellular structures.

The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy.

New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales.

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.

Molecular viewing of actin polymerizing actions and beyond: combination analysis of single-molecule speckle microscopy with modeling, FRAP and s-FDAP (sequential fluorescence decay after photoactivation).

Live-cell single-molecule imaging is a powerful tool to elucidate the in vivo biochemistry of cytoskeletal proteins. However, it is often somewhat difficult to interpret how a bulk population of the observed molecule might behave as a whole. We review our recent studies in which the combination of image analysis with modeling and bulk kinetics measurements such as FRAP (fluorescence recovery after photobleaching) clarified basic problems in the regulation of actin remodeling pathways.

CaMKIIβ-mediated LIM-kinase activation plays a crucial role in BDNF-induced neuritogenesis.

LIM-kinase 1 (LIMK1) regulates actin cytoskeletal reorganization by phosphorylating and inactivating actin-depolymerizing factor and cofilin. We examined the role of LIMK1 in brain-derived neurotrophic factor (BDNF)-induced neuritogenesis in primary-cultured rat cortical neurons. Knockdown of LIMK1 or expression of a kinase-dead LIMK1 mutant suppressed BDNF-induced enhancement of primary neurite formation.

F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins.

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.

Distributed actin turnover in the lamellipodium and FRAP kinetics.

Studies of actin dynamics at the leading edge of motile cells with single-molecule speckle (SiMS) microscopy have shown a broad distribution of EGFP-actin speckle lifetimes and indicated actin polymerization and depolymerization over an extended region. Other experiments using FRAP with the same EGFP-actin as a probe have suggested, by contrast, that polymerization occurs exclusively at the leading edge. We performed FRAP experiments on XTC cells to compare SiMS to FRAP on the same cell type.

Live-cell imaging of G-actin dynamics using sequential FDAP.

Various microscopic techniques have been developed to understand the mechanisms that spatiotemporally control actin filament dynamics in live cells. Kinetic data on the processes of actin assembly and disassembly on F-actin have been accumulated. However, the kinetics of cytoplasmic G-actin, a key determinant for actin polymerization, has remained unclear because of a lack of appropriate methods to measure the G-actin concentration quantitatively.

Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments.

The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells.

LIM kinase has a dual role in regulating lamellipodium extension by decelerating the rate of actin retrograde flow and the rate of actin polymerization.

Lamellipodium extension is crucial for cell migration and spreading. The rate of lamellipodium extension is determined by the balance between the rate of actin polymerization and the rate of actin retrograde flow. LIM kinase 1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein.

Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension.

To understand the intracellular role of G-actin concentration in stimulus-induced actin assembly and lamellipodium extension during cell migration, we developed a novel technique for quantifying spatiotemporal changes in G-actin concentration in live cells, consisting of sequential measurements of fluorescent decay after photoactivation (FDAP) of Dronpa-labeled actin. Cytoplasmic G-actin concentrations decreased by ∼40% immediately after cell stimulation and thereafter the cell area extended. The extent of stimulus-induced G-actin loss and cell extension correlated linearly with G-actin concentration in unstimulated cells, even at concentrations much higher than the critical concentration of actin filaments, indicating that cytoplasmic G-actin concentration is a critical parameter for determining the extent of stimulus-induced G-actin assembly and cell extension.

The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress.

Mutations in BRCA1 are associated with a high risk of breast and ovarian cancer. BRCA1 participates in the DNA damage response and acts as a ubiquitin ligase. However, its regulation remains poorly understood.

An unbiased screen identifies DEP-1 tumor suppressor as a phosphatase controlling EGFR endocytosis.

Background: The epidermal growth factor (EGF) stimulates rapid tyrosine phosphorylation of the EGF receptor (EGFR). This event precedes signaling from both the plasma membrane and from endosomes, and it is essential for recruitment of a ubiquitin ligase, CBL, that sorts activated receptors to endosomes and degradation. Because hyperphosphorylation of EGFR is involved in oncogenic pathways, we performed an unbiased screen of small interfering RNA (siRNA) oligonucleotides targeting all human tyrosine phosphatases.

Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers.

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity.