Elena E. Grintsevich

Cofilin-Linked Changes in Actin Filament Flexibility Promote Severing

The actin regulatory protein, cofilin, increases the bending and twisting elasticity of actin filaments and severs them. It has been proposed that filaments partially decorated with cofilin accumulate stress from thermally driven shape fluctuations at bare (stiff) and decorated (compliant) boundaries, thereby promoting severing. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. Here, we test this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale with the flexural rigidities determined from analysis of shape fluctuations. Yeast actin filaments are more compliant in bending than vertebrate actin filaments. Severing activities of cofilactin isoforms correlate with changes in filament flexibility. Vertebrate cofilin binds but does not increase the yeast actin filament flexibility, and does not sever them. Imaging of filament thermal fluctuations reveals that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully decorated filaments. These measurements support a cofilin-severing mechanism in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments, analogous to failure of some nonprotein materials.

Atomic force microscopy reveals drebrin induced remodeling of f-actin with subnanometer resolution.

We show by high-resolution atomic force microscopy analysis that drebrin A (a major neuronal actin binding protein) induced F-actin structural and mechanical remodeling involves significant changes in helical twist and filament stiffness (+55% persistence length). These results provide evidence of a unique mechanical role of drebrin in the dendrites, contribute to current molecular-level understanding of the properties of the neuronal cytoskeleton, and reflect the role of biomechanics at the nanoscale, to modulate nanofilament-structure assemblies such as F-actin.

Drebrin-Induced Stabilization of Actin Filaments

Background: Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines. Results: Drebrin protects actin filaments from depolymerization and rescues their formation in different cases of longitudinal and lateral contact perturbation. Conclusion: Drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts. Significance: We elucidate the mechanism by which drebrin governs F-actin dynamics in dendritic spines. Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines, the receptive regions of most excitatory synapses that play a crucial role in higher brain functions. Here, the structural effects of drebrin on F-actin were examined in solution. Depolymerization and differential scanning calorimetry assays show that F-actin is stabilized by the binding of drebrin. Drebrin inhibits depolymerization mainly at the barbed end of F-actin. Full-length drebrin and its C-terminal truncated constructs were used to clarify the domain requirements for these effects. The actin binding domain of drebrin decreases the intrastrand disulfide cross-linking of Cys-41 (in the DNase I binding loop) to Cys-374 (C-terminal) but increases the interstrand disulfide cross-linking of Cys-265 (hydrophobic loop) to Cys-374 in the yeast mutants Q41C and S265C, respectively. We also demonstrate, using solution biochemistry methods and EM, the rescue of filament formation by drebrin in different cases of longitudinal interprotomer contact perturbation: the T203C/C374S yeast actin mutant and grimelysin-cleaved skeletal actin (between Gly-42 and Val-43). Additionally, we show that drebrin rescues the polymerization of V266G/L267G, a hydrophobic loop yeast actin mutant with an impaired lateral interface formation between the two filament strands. Overall, our data suggest that drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts.

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments.

Mapping the Cofilin Binding Site on Yeast G-Actin by Chemical Cross-Linking

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the alpha 3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 A) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.

Drebrin inhibits cofilin-induced severing of F-actin.

Molecular cross‐talk between neuronal drebrin A and cofilin is believed to be a part of the activity‐dependent cytoskeleton‐modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimers disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1‐300) inhibits, but do not abolish cofilin‐induced severing of actin filaments. Cosedimentation experiments showed that F‐actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F‐actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F‐actin induced by the competitors binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre‐steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments.

Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1

Gap junctions are membrane specialization domains identified in most tissue types where cells abut each other. The connexin channels found in these membrane domains are conduits for direct cell-to-cell transfer of ions and molecules. Connexin43 (Cx43) is the most ubiquitous connexin, with critical roles in heart, skin, and brain. Several studies described the interaction between Cx43 and the cytoskeleton involving the actin binding proteins Zonula occludens (ZO-1) and drebrin, as well as with tubulin. However, a direct interaction has not been identified between drebrin and Cx43. In this study, co-IP and NMR experiments were used to demonstrate that the Cx43-CT directly interacts with the highly conserved N-terminus region of drebrin. Three Cx43-CT areas were found to be involved in drebrin binding, with residues 264–275 being critical for the interaction. Mimicking Src phosphorylation within this region (Y265) significantly disrupted the interaction between the Cx43-CT and drebrin. Immunofluorescence showed colocalization of Cx43, drebrin, and F-actin in astrocytes and Vero cells membrane, indicating that Cx43 forms a submembrane protein complex with cytoskeletal and scaffolding proteins. The co-IP data suggest that Cx43 indirectly interacts with F-actin through drebrin. Along with the known interaction of the Cx43-CT with ZO-1 and tubulin, the data presented here for drebrin indicate non-overlapping and separated binding sites for all three proteins for which simultaneous binding could be important in regulating cytoskeleton rearrangements, especially for neuronal migration during brain development.

INF2-Mediated Severing through Actin Filament Encirclement and Disruption

BACKGROUND INF2 is a formin protein with the unique ability to accelerate both actin polymerization and depolymerization, the latter requiring filament severing. Mutations in INF2 lead to the kidney disease focal segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot-Marie Tooth disease (CMTD). RESULTS Here, we compare the severing mechanism of INF2 with that of the well-studied severing protein cofilin. INF2, like cofilin, binds stoichiometrically to filament sides and severs in a manner that requires phosphate release from the filament. In contrast to cofilin, however, INF2 binds ADP and ADP-Pi filaments equally well. Furthermore, two-color total internal reflection fluorescence (TIRF) microscopy reveals that a low number of INF2 molecules, as few as a single INF2 dimer, are capable of severing, while measurable cofilin-mediated severing requires more extensive binding. Hence, INF2 is a more potent severing protein than cofilin. While a construct containing the FH1 and FH2 domains alone has some severing activity, addition of the C-terminal region increases severing potency by 40-fold, and we show that the WH2-resembling DAD motif is responsible for this increase. Helical 3D reconstruction from electron micrographs at 20 Å resolution provides a structure of filament-bound INF2, showing that the FH2 domain encircles the filament. CONCLUSIONS We propose a severing model in which FH2 binding and phosphate release causes local filament deformation, allowing the DAD to bind adjacent actin protomers, further disrupting filament structure.

Molecular Cooperativity of Drebrin1-300 Binding and Structural Remodeling of F-Actin

Drebrin A, an actin-binding protein, is a key regulatory element in synaptic plasticity of neuronal dendrites. Understanding how drebrin binds and remodels F-actin is important for a functional analysis of their interactions. Conventionally, molecular models for protein-protein interactions use binding parameters derived from bulk solution measurements with limited spatial resolution, and the inherent assumption of homogeneous binding sites. In the case of actin filaments, their structural and dynamic states-as well as local changes in those states-may influence their binding parameters and interaction cooperativity. Here, we probed the structural remodeling of single actin filaments and the binding cooperativity of DrebrinA(1-300) -F-actin using AFM imaging. We show direct evidence of DrebrinA(1-300)-induced cooperative changes in the helical structure of F-actin and observe the binding cooperativity of drebrin to F-actin with nanometer resolution. The data confirm at the in vitro molecular level that variations in the F-actin helical structure can be modulated by cooperative binding of actin-binding proteins.

A Nucleotide State-sensing Region on Actin

The nucleotide state of actin (ATP, ADP-Pi, or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165–172) changes from an unstructured loop to a β-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277–1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-Pi state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G- and F-actin with actin-binding proteins.