Molecular mechanism of actin filament elongation by formins.

Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.

Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin.

Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is the actin ADP-ribosyltransferase TccC3, whose activity leads to the clustering of the cellular cytoskeleton and ultimately cell death. Here, we show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD+ to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a mechanism of action of a bacterial toxin through specific targeting and modification of F-actin.

A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks.

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the ß tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.

Cryo-EM Resolves Molecular Recognition Of An Optojasp Photoswitch Bound To Actin Filaments In Both Switch States.

Actin is essential for key processes in all eukaryotic cells. Cellpermeable optojasps provide spatiotemporal control of the actin cytoskeleton, confining toxicity and potentially rendering F-actin druggable by photopharmacology. Here, we report cryo electron microscopy (cryo-EM) structures of both isomeric states of one optojasp bound to actin filaments. The high-resolution structures reveal for the first time the pronounced effects of photoswitching a functionalized azobenzene. By characterizing the optojasp binding site and identifying conformational changes within F-actin that depend on the optojasp isomeric state, we refine determinants for the design of functional F-actin photoswitches.

Profilin and formin constitute a pacemaker system for robust actin filament growth.

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

Structural transitions of F-actin upon ATP hydrolysis at near-atomic resolution revealed by cryo-EM.

The function of actin is coupled to the nucleotide bound to its active site. ATP hydrolysis is activated during polymerization; a delay between hydrolysis and inorganic phosphate (Pi) release results in a gradient of ATP, ADP-Pi and ADP along actin filaments (F-actin). Actin-binding proteins can recognize F-actin's nucleotide state, using it as a local 'age' tag. The underlying mechanism is complex and poorly understood. Here we report six high-resolution cryo-EM structures of F-actin from rabbit skeletal muscle in different nucleotide states. The structures reveal that actin polymerization repositions the proposed catalytic base, His161, closer to the γ-phosphate. Nucleotide hydrolysis and Pi release modulate the conformational ensemble at the periphery of the filament, thus resulting in open and closed states, which can be sensed by coronin-1B. The drug-like toxin jasplakinolide locks F-actin in an open state. Our results demonstrate in detail how ATP hydrolysis links to F-actin's conformational dynamics and protein interaction.

Parameter-Based Evaluation of Attentional Impairments in Schizophrenia and Their Modulation by Prefrontal Transcranial Direct Current Stimulation.

BACKGROUND: Attentional dysfunctions constitute core cognitive symptoms in schizophrenia, but the precise underlying neurocognitive mechanisms remain to be elucidated. METHODS: In this randomized, double-blind, sham-controlled study, we applied, for the first time, a theoretically grounded modeling approach based on Bundesen's Theory of Visual Attention (TVA) to (i) identify specific visual attentional parameters affected in schizophrenia and (ii) assess, as a proof of concept, the potential of single-dose anodal transcranial direct current stimulation (tDCS; 20 min, 2 mA) to the left dorsolateral prefrontal cortex to modulate these attentional parameters. To that end, attentional parameters were measured before (baseline), immediately after, and 24 h after the tDCS intervention in 20 schizophrenia patients and 20 healthy controls. RESULTS: At baseline, analyses revealed significantly reduced visual processing speed and visual short-term memory storage capacity in schizophrenia. A significant stimulation condition × time point interaction in the schizophrenia patient group indicated improved processing speed at the follow-up session only in the sham condition (a practice effect), whereas performance remained stable across the three time points in patients receiving verum stimulation. In healthy controls, anodal tDCS did not result in a significant change in attentional performance. CONCLUSION: With regard to question (i) above, these findings are indicative of a processing speed and short-term memory deficit as primary sources of attentional deficits in schizophrenia. With regard to question (ii), the efficacy of single-dose anodal tDCS for improving (speed aspects of visual) cognition, it appears that prefrontal tDCS (at the settings used in the present study), rather than ameliorating the processing speed deficit in schizophrenia, actually may interfere with practice-dependent improvements in the rate of visual information uptake. Such potentially unexpected effects of tDCS ought to be taken into consideration when discussing its applicability in psychiatric populations. The study was registered at http://apps.who.int/trialsearch/Trial2.aspx?TrialID=DRKS00011665.

Single-session transcranial direct current stimulation induces enduring enhancement of visual processing speed in patients with major depression.

Attentional deficits are considered key cognitive symptoms in major depressive disorder (MDD) arising from abnormal activation patterns within dorsolateral prefrontal cortex (dlPFC) alertness networks. Altering these activity patterns with transcranial direct current stimulation (tDCS) might thus ameliorate alertness-dependent cognitive deficits in MDD patients. In a double-blind, randomized, sham-controlled study, we investigated the effect of a single session of anodal tDCS (2 mA) applied to the left dlPFC on different parameters of visual attention based on Bundesen's theory of visual attention (Psychol Rev 97(4):523-547, 1990) in a group of 20 patients with MDD and a control group of 20 healthy participants. The parametric attention assessment took place before, immediately after and 24 h after tDCS intervention. It revealed a selective impairment in visual processing speed as a primary functional deficit in MDD at baseline assessment. Furthermore, a significant stimulation condition × time point interaction showed that verum tDCS over the left dlPFC resulted in a processing speed enhancement 24 h post-stimulation in MDD patients. In healthy control participants, we did not find similar tDCS-induced effects. Our results suggest that even a single session of tDCS over the dlPFC can induce enduring neurocognitive benefits that indicate an amelioration of cortical under-arousal in MDD patients in a time frame beyond that of immediate, excitability increases that are directly induced by the current.

tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast.

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct6A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct6A37 or Ψ38/39 and mcm5U34 or s2U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNALysUUU or tRNAGlnUUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm5U34 or s2U34, and this defect can be rescued by overexpression of tRNAGlnUUG Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

Effects of feedback-based visual line-orientation discrimination training for visuospatial disorders after stroke.

BACKGROUND: Patients with right or more rarely left parietotemporal lesions after stroke may have profound visuospatial disorders that impair activities of daily living (ADL) and long-term outcome. Clinical studies indicate improvements with systematic training of perception. Studies of perceptual learning in healthy persons suggest rapid improvements in perceptual learning of spatial line orientation with partial transfer to nontrained line orientations. OBJECTIVE: The authors investigated a novel feedback-based perceptual training procedure for the rehabilitation of patients after stroke. METHODS: In an uncontrolled trial, 13 participants showing profound deficits in line orientation and related visuospatial tasks within 12 to 28 weeks of onset performed repetitive feedback-based, computerized training of visual line orientation over 4 weeks of treatment. Visual line-orientation discrimination and visuospatial and visuoconstructive tasks were assessed before and after training. RESULTS: The authors found (a) rapid improvements in trained but also in nontrained spatial orientation tests in all 13 participants, partially up to a normal level; (b) stability of the obtained improvements at 2-month follow-up; (c) interocular transfer of training effects to the nontrained eye in 2 participants suggesting a central, postchiasmatic locus for this perceptual improvement; and (d) graded transfer of improvements to related spatial tasks, such as horizontal writing, analog clock reading, and visuoconstructive capacities but no transfer to unrelated measures of visual performance. CONCLUSIONS: These results suggest the potential for treatment-induced improvements in visuospatial deficits by feedback-based, perceptual orientation training as a component of rehabilitation after stroke.

Effects of lateral head inclination on multimodal spatial orientation judgments in neglect: evidence for impaired spatial orientation constancy.

Recent research revealed that patients with spatial hemineglect show deficits in the judgment of the subjective vertical and horizontal. Systematic deviations in the subjective axes have been demonstrated in the visual and tactile modality, indicating a supramodal spatial orientation deficit. Further, the magnitude of the bias was shown to be modulated by head- and body-position. The present study investigated the effect of passive lateral head inclination on the subjective visual and tactile vertical and horizontal in neglect patients, control patients with left- or right-sided brain damage without neglect and healthy controls. Subjects performed visual- and tactile-spatial judgments of axis orientations in an upright head orientation and with lateral head inclination 25 degrees in clockwise (CW) or counterclockwise (CCW) direction. Neglect patients displayed a marked variability as well as a systematic tilt in their spatial judgments. In line with a multisensory spatial orientation deficit their subjective vertical and horizontal was tilted CCW in the visual and in the tactile modality, while such a tilt was not evident in any other subject group. Furthermore, lateral head inclination had a differential effect in neglect patients, but not in control subjects. Neglect patients' judgments were modulated in the direction of the head tilt ('A-effect'). That is, a CCW inclination further increased the CCW spatial bias whereas a CW inclination decreased the spatial bias and thus led to approximately normal performance. The increased A-effect might be caused by a pathologically strong attraction of the subjective vertical by an idiotropic vector relying on the actual head orientation, as a consequence of impaired processing of gravitational information in neglect patients.

The fearful-face advantage is modulated by task demands: evidence from the attentional blink.

Fearful faces receive privileged access to awareness relative to happy and nonemotional faces. We investigated whether this advantage depends on currently available attentional resources. In an attentional blink paradigm, observers detected faces presented during the attentional blink period that could depict either a fearful or a happy expression. Perceptual load of the blink-inducing target was manipulated by increasing flanker interference. For the low-load condition, fearful faces were detected more often than happy faces, replicating previous reports. More important, this advantage for fearful faces disappeared for the high-load condition, during which fearful and happy faces were detected equally often. These results suggest that the privileged access of fearful faces to awareness does not occur mandatorily, but instead depends on attentional resources.

Systematic biases in the tactile perception of the subjective vertical in patients with unilateral neglect and the influence of upright vs. supine posture.

Patients with right hemisphere lesions often show contralesional neglect. Recent research focused on deficits beyond the typical neglect symptoms observed in the horizontal plane. Studies investigating deficits in the frontal and sagittal plane revealed impairments in the judgment of the subjective vertical. Systematic deviations in the subjective vertical have been demonstrated in the visual and tactile modality, indicating a supramodal spatial orientation deficit. Further, the magnitude of deviations appears to be manipulable by modulations of body posture. The present study investigated the subjective tactile vertical (STV) in neglect patients in the frontal and sagittal plane and its dependence on posture. Neglect patients and healthy controls performed tactile-spatial judgments of axis orientations in supine and upright posture. Neglect patients displayed a marked variability as well as a systematic tilt in their STV judgments. The STV was tilted counterclockwise in the frontal and backward in the sagittal plane. This tilt was larger in severe compared to moderate neglect patients, while it was not evident in healthy subjects. Our results support previous evidence and indicate a multisensory spatial orientation deficit in neglect patients which is related to neglect severity. Further, we found that performance of neglect patients deteriorated in supine compared to upright posture. This finding conflicts with the suggestion of a performance benefit in supine posture due to reduced (asymmetric) gravitational input. The negative effect of supine posture on the spatial bias in neglect is discussed with respect to a presumably further reduced intrinsic alertness state in the typically hypo-aroused neglect patients.