The Switch-associated Protein 70 (SWAP-70) Bundles Actin Filaments and Contributes to the Regulation of F-actin Dynamics

Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks.     

Purification of actin from fission yeast Schizosaccharomyces pombe and characterization of functional differences from muscle actin

Fission yeast Schizosaccharomyces pombe is an important genetic model organism for studying the mechanisms of endocytosis and cytokinesis. However, most work on the biochemical properties of fission yeast actin-binding proteins has been done with skeletal muscle actin for matters of convenience. When simulations of mathematical models of the mechanism of endocytosis were compared with events in live cells, some of the reactions appeared to be much faster than observed in biochemical experiments with muscle actin. Here, we used gelsolin affinity chromatography to purify actin from fission yeast. S. pombe actin shares many properties with skeletal muscle actin but has higher intrinsic nucleotide exchange rate, faster trimer nucleus formation, faster phosphate dissociation rate from polymerized actin, and faster nucleation of actin filaments with Arp2/3 complex. These properties close the gap between the biochemistry and predictions made by mathematical models of endocytosis in S. pombe cells.     

Abl2/Abl-related Gene Stabilizes Actin Filaments,Stimulates Actin Branching by Actin-related Protein 2/3 Complex,and Promotes Actin Filament Severing by Cofilin

Both Arp2/3 complex and the Abl2/Arg nonreceptor tyrosine kinase are essential to form and maintain diverse actin-based structures in cells, including cell edge protrusions in fibroblasts and cancer cells and dendritic spines in neurons. The ability of Arg to promote cell edge protrusions in fibroblasts does not absolutely require kinase activity, raising the question of how Arg might modulate actin assembly and turnover in the absence of kinase function. Arg has two distinct actin-binding domains and interacts physically and functionally with cortactin, an activator of the Arp2/3 complex. However, it was not known whether and how Arg influences actin filament stability, actin branch formation, or cofilin-mediated actin severing or how cortactin influences these reactions of Arg with actin. Arg or cortactin bound to actin filaments stabilizes them from depolymerization. Low concentrations of Arg and cortactin cooperate to stabilize filaments by slowing depolymerization. Arg stimulates formation of actin filament branches by Arp2/3 complex and cortactin. An Arg mutant lacking the C-terminal calponin homology actin-binding domain stimulates actin branch formation by the Arp2/3 complex, indicative of autoinhibition. ArgΔCH can stimulate the Arp2/3 complex even in the absence of cortactin. Arg greatly potentiates cofilin severing of actin filaments, and cortactin attenuates this enhanced severing. The ability of Arg to stabilize filaments, promote branching, and increase severing requires the internal (I/L)WEQ actin-binding domain. These activities likely underlie important roles that Arg plays in the formation, dynamics, and stability of actin-based cellular structures.     

Rho GTPases RhoA and Rac1 Mediate Effects of Dietary Folate on Metastatic Potential of A549 Cancer Cells through the Control of Cofilin Phosphorylation

Folate, an important nutrient in the human diet, has been implicated in cancer, but its role in metastasis is not established. We have shown previously that the withdrawal of medium folate leads to the inhibition of migration and invasion of A549 lung carcinoma cells. Here we have demonstrated that medium folate regulates the function of Rho GTPases by enabling their carboxyl methylation and translocation to plasma membrane. Conversely, the lack of folate leads to the retention of these proteins in endoplasmic reticulum. Folate also promoted the switch from inactive (GDP-bound) to active (GTP-bound) GTPases, resulting in the activation of downstream kinases p21-activated kinase and LIM kinase and phosphorylation of the actin-depolymerizing factor cofilin. We have further demonstrated that in A549 cells two GTPases, RhoA and Rac1, but not Cdc42, are immediate sensors of folate status: the siRNA silencing of RhoA or Rac1 blocked effects of folate on cofilin phosphorylation and cellular migration and invasion. The finding that folate modulates metastatic potential of cancer cells was confirmed in an animal model of lung cancer using tail vein injection of A549 cells in SCID mice. A folate-rich diet enhanced lung colonization and distant metastasis to lymph nodes and decreased overall survival (35 versus 63 days for mice on a folate-restricted diet). High folate also promoted epithelial-mesenchymal transition in cancer cells and experimental mouse tumors. Our study provides experimental evidence for a mechanism of metastasis promotion by dietary folate and highlights the interaction between nutrients and metastasis-related signaling.     

Cofilin与肿瘤

Cofilin是肌动蛋白相关蛋白,对肌动蛋白动力学特性的调节很重要。近年发现Cofilin活化与肿瘤细胞的恶性侵袭性质有关。Cofilin的局部激活可以诱导片状伪足的形成,并影响肿瘤细胞运动的方向,从而增强肿瘤细胞的运动和迁移;抑制Cofilin的活性可以减少肿瘤细胞的运动和迁移。本文对Cofilin的结构、功能、调控机制和与肿瘤的关系进行综述。     

Ins and outs of ADF/cofilin activity and regulation

The actin-binding proteins of the actin-depolymerisation factor (ADF)/cofilin family were first described more than three decades ago, but research on these proteins still occupies a front role in the actin and cell migration field. Moreover, cofilin activity is implicated in the malignant, invasive properties of cancer cells. The effects of ADF/cofilins on actin dynamics are diverse and their regulation is complex. In stimulated cells, multiple signalling pathways can be initiated resulting in different activation/deactivation switches that control ADF/cofilin activity. The output of this entire regulatory system, in combination with spatial and temporal segregation of the activation mechanisms, underlies the contribution of ADF/cofilins to various cell migration/invasion phenotypes. In this framework, we describe current views on how ADF/cofilins function in migrating and invading cells.     

Slow Axonal Transport of Soluble Actin with Actin Depolymerizing Factor, Cofilin, and Profilin Suggests Actin Moves in an Unassembled Form

Abstract: We examined the axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, in the chicken sciatic nerve following injection of [³⁵S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Actin and its binding proteins were then isolated by affinity chromatography on DNase I-Sepharose and by one- and two-dimensional polyacrylamide gel electrophoresis. Fluorographic analysis showed that the specific activity of soluble actin was two to three times that of its particulate form and that soluble actin, cofilin, actin depolymerizing factor, and profilin were transported at similar rates in slow component b of axonal flow. Our data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its "inactive" phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool.     

A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface

Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.     

Identification of a cofilin-like actin-binding site on translationally controlled tumor protein (TCTP)

Translationally controlled tumor protein (TCTP) expression is suppressed during cancer cell reversion to a non-malignant phenotype. We identified a primary sequence of TCTP with homology to ADF/cofilin. We confirm that a synthetic peptide corresponding to this sequence binds specifically to actin and is displaced from actin by cofilin. TCTP peptide has higher affinity for G-actin than F-actin and does not block actin-filament depolymerization by cofilin. These results suggest that TCTP may channel active cofilin to F-actin, enhancing the cofilin-activity cycle in invasive tumor cells. Loss of TCTP may result in sequestration of active cofilin by a monomeric pool of actin.     

Cofilin aggregation blocks intracellular trafficking and induces synaptic loss in hippocampal neurons

Cofilin is an actin-binding protein and a major actin depolymerization factor in the central nervous system (CNS). Cofilin-actin aggregates are associated with neurodegenerative disorders, but how cofilin-actin aggregation induces pathological effects in the CNS remains unclear. Here, we demonstrated that cofilin rods disrupted dendritic microtubule integrity in rat hippocampal cultures. Long term time-lapse imaging revealed that cofilin rods block intracellular trafficking of both mitochondria and early endosomes. Importantly, cofilin rod formation induced a significant loss of SV2 and PSD-95 puncta as well as dendritic spines. Cofilin rods also impaired local glutamate receptor responses. We discovered an inverse relationship between the number of synaptic events and the accumulation of cofilin rods in dendrites. We also detected cofilin rods in aging rat brains in vivo. These results suggest that cofilin aggregation may contribute to neurodegeneration and brain aging by blocking intracellular trafficking and inducing synaptic loss.