Structure of the Lifeact-F-actin complex.

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

Brain ischemic insult induces cofilin rod formation leading to synaptic dysfunction in neurons.

Ischemic stroke not only induces neuron death in the infarct area but also structural and functional damage of the surviving neurons in the surrounding peri-infarct area. In the present study, we first identified cofilin rod, a pathological rod-like aggregation, formed in neurons of in vivo ischemic stroke animal model and induced neuronal impairment. Cofilin rods formed only on the ipsilateral side of the middle cerebral artery occlusion and reperfusion (MCAO-R) rat brain and showed the highest density in peri-infarct area. Our real-time live cell imaging, immunostaining and patch clamp studies showed that cofilin rod formation in neurons led to dendritic mitochondrial transportation failure, as well as impairment of synaptic structure and functions. Overexpression of LIM kinase or activation of its upstream regulator Rho, suppressed ischemia-induced cofilin rod formation and showed protective effect on synaptic function and structure impairment in both cultured neurons and MCAO-R rat model. In summary, our results demonstrate a novel mechanism of ischemic stroke-induced neuron injury in peri-infarct area and provide a potential target for the protection of neuronal structure and function against brain ischemia insult.

Cofilin-actin rod formation in neuronal processes after brain ischemia.

Functional impairment after brain ischemia results in part from loss of neuronal spines and dendrites, independent of neuronal death. Cofilin-actin rods are covalently linked aggregates of cofilin-1 and actin that form in neuronal processes (neurites) under conditions of ATP depletion and oxidative stress, and which cause neurite degeneration if not disassembled. ATP depletion and oxidative stress occur with differing severity, duration, and time course in different ischemic conditions. Here we evaluated four mouse models of brain ischemia to define the conditions that drive formation of cofilin-actin rods. Three of the models provide early reperfusion: transient middle cerebral artery occlusion (MCAo), transient bilateral common carotid artery occlusion (CCAo), and cardiac arrest / cardiopulmonary resuscitation (CA/CPR). Early reperfusion restores ATP generating capacity, but also induces oxidative stress. The fourth model, photothrombotic cortical infarction, does not provide reperfusion. Cofilin-actin rods were formed in each of these models, but with differing patterns. Where acute reperfusion occurred, rod formation was maximal within 4 hours after reperfusion. Where infarction occurred, rods continued to form for at least 24 hours after ischemic onset, and extended into the adjacent non-ischemic tissue. Interventions that limit cofilin-actin rod formation may help to preserve integrity of neuronal processes in permanent ischemia.

Mapping cofilin-actin rods in stressed hippocampal slices and the role of cdc42 in amyloid-beta-induced rods.

Dissociated hippocampal neurons exposed to a variety of degenerative stimuli form neuritic cofilin-actin rods. Here we report on stimulus driven regional rod formation in organotypic hippocampal slices. Ultrastructural analysis of rods formed in slices demonstrates mitochondria and vesicles become entrapped within some rods. We developed a template for combining and mapping data from multiple slices, enabling statistical analysis for the identification of vulnerable sub-regions. Amyloid-beta (Abeta) induces rods predominantly in the dentate gyrus region, and Abeta-induced rods are reversible following washout. Rods that persist 24 h following transient (30 min) ATP-depletion are broadly distributed, whereas rods formed in response to excitotoxic glutamate localize within and nearby the pyramidal neurons. Time-lapse imaging of cofilin-GFP-expressing neurons within slices shows neuronal rod formation begins rapidly and peaks by 10 min of anoxia. In approximately 50% of responding neurons, Abeta-induced rod formation acts via cdc42, an upstream regulator of cofilin. These new observations support a role for cofilin-actin rods in stress-induced disruption of cargo transport and synaptic function within hippocampal neurons and suggest both cdc42-dependent and independent pathways modulate cofilin activity downstream from Abeta.