Natural and targeted isovariants of the rice actin depolymerizing factor 2 can alter its functional and regulatory binding properties.

Actin depolymerizing factors (ADFs) are ubiquitous actin-binding proteins that play essential roles in maintaining cellular actin dynamics by depolymerizing/severing F-actin. Plant ADF isoforms show functional divergence via differential biochemical and cellular properties. We have shown previously that ADF2 of rice (OsADF2) and smooth cordgrass (SaADF2) displayed contrasting biochemical properties and stress response in planta. As a proof-of-concept that amino acid variances contribute to such functional difference, single amino acid mutants of OsADF2 were generated based on its sequence differences with SaADF2. Biochemical studies showed that the single-site amino acid mutations altered actin binding, depolymerizing, and severing properties of OsADF2. Phosphosensitive mutations, such as serine-6>threonine, changed the regulatory phosphorylation efficiency of ADF2 variants. The N-terminal mutations had greater effect on the phosphorylation pattern of OsADF2, whereas C-terminal mutations affected actin binding and severing. The presence of introduced mutations in isovariants of monocot ADF suggests that these residues are significant control points regulating their functional divergence, including abiotic stress response.

Isolation and characterization of cytoplasmic cofilin-actin rods.

Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca(2+), and ATP. Cofilin-GFP-containing rods are stable in 500 mM NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.

Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex.

Capping protein (CP) is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between CP and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of CP and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility.

Actin hydrophobic loop 262-274 and filament nucleation and elongation.

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a "parked" conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by approximately 5 degrees per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by approximately 1 degrees per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.

PKCdelta and cofilin activation affects peripheral actin reorganization and cell-cell contact in cells expressing integrin alpha5 but not its tailless mutant.

Integrin-mediated cell adhesion transduces signaling activities for actin reorganization, which is crucially involved in cellular function and architectural integrity. In this study, we explored the possibility of whether cell-cell contacts might be regulated via integrin-alpha5beta1-mediated actin reorganization. Ectopic expression of integrin alpha5 in integrin-alpha5-null intestinal epithelial cells resulted in facilitated retraction, cell-cell contact loss, and wound healing depending on Src and PI3K (phosphoinositide 3-kinase) activities by a reagent that affects actin organization. However, cytoplasmic tailless integrin alpha5 (hereafter referred to as alpha5/1) expression caused no such effects but rather sustained peripheral actin fibers, regardless of Src and PI3K signaling activities. Furthermore, integrin alpha5 engagement with fibronectin phosphorylated Ser643 of PKCdelta, upstream of FAK and Src and at a transmodulatory loop with PI3K/Akt. Pharmacological PKCdelta inactivation, dominant-negative PKCdelta adenovirus or inactive cofilin phosphatase (SSH1L mutant) retrovirus infection of alpha5-expressing cells sustained peripheral actin organization and blocked the actin reorganizing-mediated loss of cell-cell contacts. Meanwhile, wild-type PKCdelta expression sensitized alpha5/1-expressing cells to the actin disruptor to induce cell scattering. Altogether, these observations indicate that integrin alpha5, but not alpha5/1, mediates PKCdelta phosphorylation and cofilin dephosphorylation, which in turn modulate peripheral actin organization presumably leading to an efficient regulation of cell-cell contact and migration.