Antiviral responses must be tightly regulated to rapidly defend against infection while minimizing inflammatory damage. Type 1 interferons (IFN-I) are crucial mediators of antiviral responses1 and their transcription is regulated by a variety of transcription factors2; principal amongst these is the family of interferon regulatory factors (IRFs)3. The IRF gene regulatory networks are complex and contain multiple feedback loops. The tools of systems biology are well suited to elucidate the complex interactions that give rise to precise coordination of the interferon response. Here we have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. Genome-wide location analysis combined with gene deletion studies identified the Irf7 gene as a critical target of FOXO3. FOXO3 was identified as a negative regulator of Irf7 transcription and we have further demonstrated that FOXO3, IRF7 and IFN-I form a coherent feed-forward regulatory circuit. Our data suggest that the FOXO3-IRF7 regulatory circuit represents a novel mechanism for establishing the requisite set points in the interferon pathway that balances the beneficial effects and deleterious sequelae of the antiviral response.
Functional neurosurgical techniques provide a unique opportunity to explore patterns of interaction between the cerebral cortex and basal ganglia in patients with Parkinson's disease (PD). Previous work using simultaneous magnetoencephalographic (MEG) and local field potential (LFP) recordings from the region of the subthalamic nucleus (STNr) has characterised resting patterns of connectivity in the alpha and beta frequency bands and their modulation by dopaminergic medication. Recently we have also characterised the effect of movement on patterns of gamma band coherence between the STNr and cortical sites. Here we specifically investigate how the prominent coherence between the STNr and temporal cortex in the alpha band is modulated by movement both on and off dopaminergic medication in patients following the insertion of Deep Brain Stimulation (DBS) electrodes. We show that movement is associated with a suppression of local alpha power in the temporal cortex and STNr that begins about 2 s prior to a self-paced movement and is independent of dopaminergic status. In contrast, the peak reduction in coherence between these sites occurs after movement onset and is more marked in the on than in the off dopaminergic medication state. The difference in alpha band coherence on and off medication was found to correlate with the drug related improvement in clinical parameters. Overall, the movement-related behaviour of activities in the alpha band in patients with PD serves to highlight the role of dopamine in modulating large-scale, interregional synchronisation.
► We studied subthalamo-cortical coherence in the alpha band in Parkinson's patients. ► A decrease in coherence occurred with movement facilitated by dopamine. ► This effect correlated with clinical improvement.
Oscillations; Human; Intracranial recordings
Functional neurosurgery has afforded the opportunity to assess interactions between populations of neurons in the human cerebral cortex and basal ganglia in patients with Parkinson’s disease (PD). Interactions occur over a wide range of frequencies, and the functional significance of those above 30 Hz is particularly unclear. Do they improve movement and, if so, in what way? We acquired simultaneously magnetoencephalography (MEG) and direct recordings from the subthalamic nucleus (STN) in 17 PD patients. We examined the effect of synchronous and sequential finger movements and of the dopamine prodrug levodopa on induced power in the contralateral primary motor cortex (M1) and STN and on the coherence between the two structures. We observed discrete peaks in M1 and STN power over 60-90 Hz and 300-400 Hz. All these power peaks increased with movement and levodopa treatment. Only STN activity over 60-90 Hz was coherent with activity in M1. Directionality analysis showed that STN gamma activity at 60-90 Hz tended to drive gamma activity in M1. The effects of levodopa on both local and distant synchronisation over 60-90 Hz correlated with the degree of improvement in bradykinesia-rigidity, as did local STN activity at 300-400 Hz. Despite this, there were no effects of movement type, nor interactions between movement type and levodopa in the STN, nor in the coherence between STN and M1. We conclude that synchronisation over 60-90 Hz in the basal ganglia cortical network is prokinetic, but likely through a modulatory effect rather than any involvement in explicit motor processing.
Oscillatory activity in the beta frequency band has been shown to be modulated during the preparation and execution of voluntary movements at both cortical and subcortical levels. The exaggeration of beta activity in the basal ganglia of patients with Parkinson's disease has heightened interest in this phenomenon. However, the precise function, if any, subserved by modulations in beta activity remains unclear. Here we test the hypothesis that beta reactivity can be dissociated from processing of specific actions and can index the salience of cues with respect to future behavior in a way that might help prospectively prioritize resources. To this end we used an experimental paradigm designed to dissociate salient warning cues from processing of specific motor or cognitive actions. We recorded local field potential activity from the subthalamic nucleus of humans undergoing functional neurosurgery for the treatment of Parkinson's disease, while the same patients were on or off the dopamine prodrug levodopa. In this way we demonstrate that beta reactivity is indeed dependent on the salience of cues with respect to future motor and cognitive action and is promoted by dopamine. The loss of normal beta encoding of saliency may underlie some of the motor and cognitive features of basal ganglia disorders such as Parkinson's disease.
In Kilner et al. [Kilner, J.M., Kiebel, S.J., Friston, K.J., 2005. Applications of random field theory to electrophysiology. Neurosci. Lett. 374, 174–178.] we described a fairly general analysis of induced responses—in electromagnetic brain signals—using the summary statistic approach and statistical parametric mapping. This involves localising induced responses—in peristimulus time and frequency—by testing for effects in time–frequency images that summarise the response of each subject to each trial type. Conventionally, these time–frequency summaries are estimated using post‐hoc averaging of epoched data. However, post‐hoc averaging of this sort fails when the induced responses overlap or when there are multiple response components that have variable timing within each trial (for example stimulus and response components associated with different reaction times). In these situations, it is advantageous to estimate response components using a convolution model of the sort that is standard in the analysis of fMRI time series. In this paper, we describe one such approach, based upon ordinary least squares deconvolution of induced responses to input functions encoding the onset of different components within each trial. There are a number of fundamental advantages to this approach: for example; (i) one can disambiguate induced responses to stimulus onsets and variably timed responses; (ii) one can test for the modulation of induced responses—over peristimulus time and frequency—by parametric experimental factors and (iii) one can gracefully handle confounds—such as slow drifts in power—by including them in the model. In what follows, we consider optimal forms for convolution models of induced responses, in terms of impulse response basis function sets and illustrate the utility of deconvolution estimators using simulated and real MEG data.
► We propose a new approach to analysis of induced responses in M/EEG. ► The General Linear Model is used to model continuous power as in fMRI 1st-level. ► The results can be presented as conventional time–frequency images. ► Our method is better for experiments with variable timing and overlapping events.
EEG; MEG; ERSP; Induced responses; Time–frequency analysis; General linear model; Convolution; Statistical parametric mapping
A multimodal neuroimaging study of virtual spatial navigation extends the role of the hippocampal theta rhythm to human memory and self-directed learning.
The hippocampus is crucial for episodic or declarative memory and the theta rhythm has been implicated in mnemonic processing, but the functional contribution of theta to memory remains the subject of intense speculation. Recent evidence suggests that the hippocampus might function as a network hub for volitional learning. In contrast to human experiments, electrophysiological recordings in the hippocampus of behaving rodents are dominated by theta oscillations reflecting volitional movement, which has been linked to spatial exploration and encoding. This literature makes the surprising cross-species prediction that the human hippocampal theta rhythm supports memory by coordinating exploratory movements in the service of self-directed learning. We examined the links between theta, spatial exploration, and memory encoding by designing an interactive human spatial navigation paradigm combined with multimodal neuroimaging. We used both non-invasive whole-head Magnetoencephalography (MEG) to look at theta oscillations and Functional Magnetic Resonance Imaging (fMRI) to look at brain regions associated with volitional movement and learning. We found that theta power increases during the self-initiation of virtual movement, additionally correlating with subsequent memory performance and environmental familiarity. Performance-related hippocampal theta increases were observed during a static pre-navigation retrieval phase, where planning for subsequent navigation occurred. Furthermore, periods of the task showing movement-related theta increases showed decreased fMRI activity in the parahippocampus and increased activity in the hippocampus and other brain regions that strikingly overlap with the previously observed volitional learning network (the reverse pattern was seen for stationary periods). These fMRI changes also correlated with participant's performance. Our findings suggest that the human hippocampal theta rhythm supports memory by coordinating exploratory movements in the service of self-directed learning. These findings directly extend the role of the hippocampus in spatial exploration in rodents to human memory and self-directed learning.
Neural activity both within and across brain regions can oscillate in different frequency ranges (such as alpha, gamma, and theta frequencies), and these different ranges are associated with distinct functions. In behaving rodents, for example, theta rhythms (4–12 Hz) in the hippocampus are prominent during the initiation of movement and have been linked to spatial exploration. Recent evidence in humans, however, suggests that the human hippocampus is involved in guiding self-directed learning. This suggests that the human hippocampal theta rhythm supports memory by coordinating exploratory movements in the service of self-directed learning. In this study, we tested whether there is a human analogue for the movement-initiation-related theta rhythm found in the rodent hippocampus by using a virtual navigation paradigm, combined with non-invasive recordings and functional imaging techniques. Our recordings showed that, indeed, theta power increases are linked to movement initiation. We also examined the relationship to memory encoding, and we found that hippocampal theta oscillations related to pre-retrieval planning predicted memory performance. Imaging results revealed that periods of the task showing movement-related theta also showed increased activity in the hippocampus, as well as other brain regions associated with self-directed learning. These findings directly extend the role of the hippocampal theta rhythm in rodent spatial exploration to human memory and self-directed learning.
Statistical parametric mapping (SPM) locates significant clusters based on a ratio of signal to noise (a ‘contrast’ of the parameters divided by its standard error) meaning that very low noise regions, for example outside the brain, can attain artefactually high statistical values. Similarly, the commonly applied preprocessing step of Gaussian spatial smoothing can shift the peak statistical significance away from the peak of the contrast and towards regions of lower variance. These problems have previously been identified in positron emission tomography (PET) (Reimold et al., 2006) and voxel-based morphometry (VBM) (Acosta-Cabronero et al., 2008), but can also appear in functional magnetic resonance imaging (fMRI) studies. Additionally, for source-reconstructed magneto- and electro-encephalography (M/EEG), the problems are particularly severe because sparsity-favouring priors constrain meaningfully large signal and variance to a small set of compactly supported regions within the brain. (Acosta-Cabronero et al., 2008) suggested adding noise to background voxels (the ‘haircut’), effectively increasing their noise variance, but at the cost of contaminating neighbouring regions with the added noise once smoothed. Following theory and simulations, we propose to modify – directly and solely – the noise variance estimate, and investigate this solution on real imaging data from a range of modalities.
► Statistical parametric mapping judges significance with a signal-to-noise ratio. ► Low noise, e.g. outside the brain, can yield artefactually high statistical values. ► Spatial smoothing can shift peaks substantially towards regions of low variance. ► Source-reconstructed M/EEG data exhibits the problem particularly severely. ► The problem can be addressed by modifying the noise variance estimate.
EEG, electroencephalography; fMRI, functional magnetic resonance imaging; FWHM, full-width at half-maximum; GM, grey matter; MEG, magnetoencephalography; MIP, maximum intensity projection; MNI, Montreal Neurological Institute; ResMS, residual mean squares; SPM, statistical parametric mapping; PET, positron emission tomography; VBM, voxel-based morphometry; SPM; Low variance; VBM; MEG; EEG; Source reconstruction
Optimal decision-making is guided by evaluating the outcomes of previous decisions. Prediction errors are theoretical teaching signals which integrate two features of an outcome: its inherent value and prior expectation of its occurrence. To uncover the magnetic signature of prediction errors in the human brain we acquired magnetoencephalographic (MEG) data while participants performed a gambling task. Our primary objective was to use formal criteria, based upon an axiomatic model (Caplin and Dean, 2008a), to determine the presence and timing profile of MEG signals that express prediction errors. We report analyses at the sensor level, implemented in SPM8, time locked to outcome onset. We identified, for the first time, a MEG signature of prediction error, which emerged approximately 320 ms after an outcome and expressed as an interaction between outcome valence and probability. This signal followed earlier, separate signals for outcome valence and probability, which emerged approximately 200 ms after an outcome. Strikingly, the time course of the prediction error signal, as well as the early valence signal, resembled the Feedback-Related Negativity (FRN). In simultaneously acquired EEG data we obtained a robust FRN, but the win and loss signals that comprised this difference wave did not comply with the axiomatic model. Our findings motivate an explicit examination of the critical issue of timing embodied in computational models of prediction errors as seen in human electrophysiological data.
► We identified, for the first time, an MEG signature of a human prediction error. ► The waveform resembled the Feedback-Related Negativity (FRN) signal in EEG. ► MEG effects of probability and valence were emerged before the prediction error signals, 200 ms after the outcome. ► The EEG data revealed classic FRN which was modulated by probability.
Decision-making; Prediction error; Reward; MEG; Feedback-related negativity; Error-related negativity
Cortico-basal ganglia-thalamocortical circuits are severely disrupted by the dopamine depletion of Parkinson's disease (PD), leading to pathologically exaggerated beta oscillations. Abnormal rhythms, found in several circuit nodes are correlated with movement impairments but their neural basis remains unclear. Here, we used dynamic causal modelling (DCM) and the 6-hydroxydopamine-lesioned rat model of PD to examine the effective connectivity underlying these spectral abnormalities. We acquired auto-spectral and cross-spectral measures of beta oscillations (10–35 Hz) from local field potential recordings made simultaneously in the frontal cortex, striatum, external globus pallidus (GPe) and subthalamic nucleus (STN), and used these data to optimise neurobiologically plausible models. Chronic dopamine depletion reorganised the cortico-basal ganglia-thalamocortical circuit, with increased effective connectivity in the pathway from cortex to STN and decreased connectivity from STN to GPe. Moreover, a contribution analysis of the Parkinsonian circuit distinguished between pathogenic and compensatory processes and revealed how effective connectivity along the indirect pathway acquired a strategic importance that underpins beta oscillations. In modelling excessive beta synchrony in PD, these findings provide a novel perspective on how altered connectivity in basal ganglia-thalamocortical circuits reflects a balance between pathogenesis and compensation, and predicts potential new therapeutic targets to overcome dysfunctional oscillations.
Parkinson's disease is a progressive age-related neurodegenerative disorder that severely disrupts movement. The major pathology in Parkinson's disease is the degeneration of a group of neurons that contain a chemical known as dopamine. Treatment of Parkinsonism includes pharmacological interventions that aim to replace dopamine and more recently, implanted devices that aim to restore movement through electrical stimulation of the brain's movement circuits. Understanding the electrical properties that emerge as a result of depleted dopamine may reveal new avenues for developing these technologies. By combining a novel model-based approach with multi-site electrophysiological recordings from an animal model of Parkinson's disease we provide empirical evidence for a link between abnormal electrical activity in the Parkinsonian brain and its physiological basis. We have examined the connections along the brain's motor circuits, and found an abnormality in inter-area connections in a particular neural pathway, a pathway critically dependent on dopamine. The scheme makes strong and testable predictions about which neural pathways are significantly altered in the pathological state and so represent empirically motivated therapeutic targets.
We address the problem of controlling false positive rates in mass-multivariate tests for electromagnetic responses in compact regions of source space. We show that mass-univariate thresholds based on sensor level multivariate thresholds (approximated using Roy's union–intersection principle) are unduly conservative. We then consider a Bonferroni correction for source level tests based on the number of unique lead-field extrema. For a given source space, the sensor indices corresponding to the maxima and minima (for each dipolar lead field) are listed, and the number of unique extrema is given by the number of unique pairs in this list. Using a multivariate beamformer formulation, we validate this heuristic against empirical permutation thresholds for mass-univariate and mass-multivariate tests (of induced and evoked responses) for a variety of source spaces, using simulated and real data. We also show that the same approximations hold when dealing with a cortical manifold (rather than a volume) and for mass-multivariate minimum norm solutions. We demonstrate that the mass-multivariate framework is not restricted to tests on a single contrast of effects (cf, Roy's maximum root) but also accommodates multivariate effects (cf, Wilk's lambda).
► We aim to estimate the number of independent tests made in MEG source space. ► We trial a heuristic based on the number of unique lead field extrema. ► We compare Bonferroni corrected tests using this heuristic to permutation methods. ► The heuristic performs well for both mass-univariate and mass-multivariate tests.
SPM is a free and open source software written in MATLAB (The MathWorks, Inc.). In addition to standard M/EEG preprocessing, we presently offer three main analysis tools: (i) statistical analysis of scalp-maps, time-frequency images, and volumetric 3D source reconstruction images based on the general linear model, with correction for multiple comparisons using random field theory; (ii) Bayesian M/EEG source reconstruction, including support for group studies, simultaneous EEG and MEG, and fMRI priors; (iii) dynamic causal modelling (DCM), an approach combining neural modelling with data analysis for which there are several variants dealing with evoked responses, steady state responses (power spectra and cross-spectra), induced responses, and phase coupling. SPM8 is integrated with the FieldTrip toolbox , making it possible for users to combine a variety of standard analysis methods with new schemes implemented in SPM and build custom analysis tools using powerful graphical user interface (GUI) and batching tools.
We review recent methodological developments within a parametric empirical Bayesian (PEB) framework for reconstructing intracranial sources of extracranial electroencephalographic (EEG) and magnetoencephalographic (MEG) data under linear Gaussian assumptions. The PEB framework offers a natural way to integrate multiple constraints (spatial priors) on this inverse problem, such as those derived from different modalities (e.g., from functional magnetic resonance imaging, fMRI) or from multiple replications (e.g., subjects). Using variations of the same basic generative model, we illustrate the application of PEB to three cases: (1) symmetric integration (fusion) of MEG and EEG; (2) asymmetric integration of MEG or EEG with fMRI, and (3) group-optimization of spatial priors across subjects. We evaluate these applications on multi-modal data acquired from 18 subjects, focusing on energy induced by face perception within a time–frequency window of 100–220 ms, 8–18 Hz. We show the benefits of multi-modal, multi-subject integration in terms of the model evidence and the reproducibility (over subjects) of cortical responses to faces.
source reconstruction; bioelectromagnetic signals; data fusion; neuroimaging
Motivation: Histone acetylation (HAc) is associated with open chromatin, and HAc has been shown to facilitate transcription factor (TF) binding in mammalian cells. In the innate immune system context, epigenetic studies strongly implicate HAc in the transcriptional response of activated macrophages. We hypothesized that using data from large-scale sequencing of a HAc chromatin immunoprecipitation assay (ChIP-Seq) would improve the performance of computational prediction of binding locations of TFs mediating the response to a signaling event, namely, macrophage activation.
Results: We tested this hypothesis using a multi-evidence approach for predicting binding sites. As a training/test dataset, we used ChIP-Seq-derived TF binding site locations for five TFs in activated murine macrophages. Our model combined TF binding site motif scanning with evidence from sequence-based sources and from HAc ChIP-Seq data, using a weighted sum of thresholded scores. We find that using HAc data significantly improves the performance of motif-based TF binding site prediction. Furthermore, we find that within regions of high HAc, local minima of the HAc ChIP-Seq signal are particularly strongly correlated with TF binding locations. Our model, using motif scanning and HAc local minima, improves the sensitivity for TF binding site prediction by ∼50% over a model based on motif scanning alone, at a false positive rate cutoff of 0.01.
Availability: The data and software source code for model training and validation are freely available online at http://magnet.systemsbiology.net/hac.
Contact: email@example.com; firstname.lastname@example.org
Supplementary information: Supplementary data are available at Bioinformatics online.
Insight into how brain structures interact is critical for understanding the principles of functional brain architectures and may lead to better diagnosis and therapy for neuropsychiatric disorders. We recorded, simultaneously, magnetoencephalographic (MEG) signals and subcortical local field potentials (LFP) in a Parkinson's disease (PD) patient with bilateral deep brain stimulation (DBS) electrodes in the subthalamic nucleus (STN). These recordings offer a unique opportunity to characterize interactions between the subcortical structures and the neocortex. However, high-amplitude artefacts appeared in the MEG. These artefacts originated from the percutaneous extension wire, rather than from the actual DBS electrode and were locked to the heart beat. In this work, we show that MEG beamforming is capable of suppressing these artefacts and quantify the optimal regularization required. We demonstrate how beamforming makes it possible to localize cortical regions whose activity is coherent with the STN-LFP, extract artefact-free virtual electrode time-series from regions of interest and localize cortical areas exhibiting specific task-related power changes. This furnishes results that are consistent with previously reported results using artefact-free MEG data. Our findings demonstrate that physiologically meaningful information can be extracted from heavily contaminated MEG signals and pave the way for further analysis of combined MEG-LFP recordings in DBS patients.
The innate immune system is a two-edged sword; it is absolutely required for host defense against infection but, uncontrolled, can trigger a plethora of inflammatory diseases. Here we used systems biology approaches to predict and validate a gene regulatory network involving a dynamic interplay between the transcription factors NF-κB, C/EBPδ, and ATF3 that controls inflammatory responses. We mathematically modeled transcriptional regulation of Il6 and Cebpd genes and experimentally validated the prediction that the combination of an initiator (NF-κB), an amplifier (C/EBPδ) and an attenuator (ATF3) forms a regulatory circuit that discriminates between transient and persistent Toll-like receptor 4-induced signals. Our results suggest a mechanism that enables the innate immune system to detect the duration of infection and to respond appropriately.
The aim of this paper is to describe a simple procedure for
electromagnetic (EEG or MEG) source reconstruction, in the context of group
studies. This entails a simple extension of existing source reconstiruction
techniques based upon the inversion of hierarchical models. The extension
ensures that evoked or induced responses are reconstructed in the same subset of
sources, over subjects. Effectively, the procedure aligns the deployment of
reconstructed activity over subjects and increases, substantially, the detection
of differences between evoked or induced responses at the group or
Hierarchical Bayes; Source reconstruction; EEG; MEG; Restricted maximum likelihood; Automatic relevance determination
Macrophages are versatile immune cells that can detect a variety of pathogen-associated molecular patterns through their Toll-like receptors (TLRs). In response to microbial challenge, the TLR-stimulated macrophage undergoes an activation program controlled by a dynamically inducible transcriptional regulatory network. Mapping a complex mammalian transcriptional network poses significant challenges and requires the integration of multiple experimental data types. In this work, we inferred a transcriptional network underlying TLR-stimulated murine macrophage activation. Microarray-based expression profiling and transcription factor binding site motif scanning were used to infer a network of associations between transcription factor genes and clusters of co-expressed target genes. The time-lagged correlation was used to analyze temporal expression data in order to identify potential causal influences in the network. A novel statistical test was developed to assess the significance of the time-lagged correlation. Several associations in the resulting inferred network were validated using targeted ChIP-on-chip experiments. The network incorporates known regulators and gives insight into the transcriptional control of macrophage activation. Our analysis identified a novel regulator (TGIF1) that may have a role in macrophage activation.
Macrophages play a vital role in host defense against infection by recognizing pathogens through pattern recognition receptors, such as the Toll-like receptors (TLRs), and mounting an immune response. Stimulation of TLRs initiates a complex transcriptional program in which induced transcription factor genes dynamically regulate downstream genes. Microarray-based transcriptional profiling has proved useful for mapping such transcriptional programs in simpler model organisms; however, mammalian systems present difficulties such as post-translational regulation of transcription factors, combinatorial gene regulation, and a paucity of available gene-knockout expression data. Additional evidence sources, such as DNA sequence-based identification of transcription factor binding sites, are needed. In this work, we computationally inferred a transcriptional network for TLR-stimulated murine macrophages. Our approach combined sequence scanning with time-course expression data in a probabilistic framework. Expression data were analyzed using the time-lagged correlation. A novel, unbiased method was developed to assess the significance of the time-lagged correlation. The inferred network of associations between transcription factor genes and co-expressed gene clusters was validated with targeted ChIP-on-chip experiments, and yielded insights into the macrophage activation program, including a potential novel regulator. Our general approach could be used to analyze other complex mammalian systems for which time-course expression data are available.
Cytokinesis, the final stage of eukaryotic cell division, ensures the production of two daughter cells. It requires fine coordination between the plasma membrane and cytoskeletal networks, and it is known to be regulated by several intracellular proteins, including the small GTPase Rho and its effectors. In this study we provide evidence that the protein Nir2 is essential for cytokinesis. Microinjection of anti-Nir2 antibodies into interphase cells blocks cytokinesis, as it results in the production of multinucleate cells. Immunolocalization studies revealed that Nir2 is mainly localized in the Golgi apparatus in interphase cells, but it is recruited to the cleavage furrow and the midbody during cytokinesis. Nir2 colocalizes with the small GTPase RhoA in the cleavage furrow and the midbody, and it associates with RhoA in mitotic cells. Its N-terminal region, which contains a phosphatidylinositol transfer domain and a novel Rho-inhibitory domain (Rid), is required for normal cytokinesis, as overexpression of an N-terminal-truncated mutant blocks cytokinesis completion. Time-lapse videomicroscopy revealed that this mutant normally initiates cytokinesis but fails to complete it, due to cleavage furrow regression, while Rid markedly affects cytokinesis due to abnormal contractility. Rid-expressing cells exhibit aberrant ingression and ectopic cleavage sites; the cells fail to segregate into daughter cells and they form a long unseparated bridge-like cytoplasmic structure. These results provide new insight into the cellular functions of Nir2 and introduce it as a novel regulator of cytokinesis.
Cell morphogenesis requires dynamic reorganization of the actin cytoskeleton, a process that is tightly regulated by the Rho family of small GTPases. These GTPases act as molecular switches by shuttling between their inactive GDP-bound and active GTP-bound forms. Here we show that Nir2, a novel protein related to Drosophila retinal degeneration B (RdgB), markedly affects cell morphology through a novel Rho-inhibitory domain (Rid) which resides in its N-terminal region. Rid exhibits sequence homology with the Rho-binding site of formin-homology (FH) proteins and leads to an apparent loss of F-actin staining when ectopically expressed in mammalian cells. We also show that Rid inhibits Rho-mediated stress fiber formation and lysophosphatidic acid-induced RhoA activation. Biochemical studies demonstrated that Nir2, via Rid, preferentially binds to the inactive GDP-bound form of the small GTPase Rho. Microinjection of antibodies against Nir2 into neuronal cells markedly attenuates neurite extension, whereas overexpression of Nir2 in these cells attenuates Rho-mediated neurite retraction. These results implicate Nir2 as a novel regulator of the small GTPase Rho in actin cytoskeleton reorganization and cell morphogenesis.