IQ Domain Research Articles

End-to-End Learning From Spectrum Data: A Deep Learning Approach for Wireless Signal Identification in Spectrum Monitoring Applications

This paper presents end-to-end learning from spectrum data-an umbrella term for new sophisticated wireless signal identification approaches in spectrum monitoring applications based on deep neural networks. End-to-end learning allows to: 1) automatically learn features directly from simple wireless signal representations, without requiring design of hand-crafted expert features like higher order cyclic moments and 2) train wireless signal classifiers in one end-to-end step which eliminates the need for complex multi-stage machine learning processing pipelines. The purpose of this paper is to present the conceptual framework of end-to-end learning for spectrum monitoring and systematically introduce a generic methodology to easily design and implement wireless signal classifiers. Furthermore, we investigate the importance of the choice of wireless data representation to various spectrum monitoring tasks. In particular, two case studies are elaborated: 1) modulation recognition and 2) wireless technology interference detection. For each case study three convolutional neural networks are evaluated for the following wireless signal representations: temporal IQ data, the amplitude/phase representation, and the frequency domain representation. From our analysis, we prove that the wireless data representation impacts the accuracy depending on the specifics and similarities of the wireless signals that need to be differentiated, with different data representations resulting in accuracy variations of up to 29%. Experimental results show that using the amplitude/phase representation for recognizing modulation formats can lead to performance improvements up to 2% and 12% for medium to high SNR compared to IQ and frequency domain data, respectively. For the task of detecting interference, frequency domain representation outperformed amplitude/phase and IQ data representation up to 20%.

CAMTA-Mediated Regulation of Salicylic Acid Immunity Pathway Genes in Arabidopsis Exposed to Low Temperature and Pathogen Infection.

Arabidopsis thaliana calmodulin binding transcription activator (CAMTA) factors repress the expression of genes involved in salicylic acid (SA) biosynthesis and SA-mediated immunity in healthy plants grown at warm temperature (22°C). This repression is overcome in plants exposed to low temperature (4°C) for more than a week and in plants infected by biotrophic and hemibiotrophic pathogens. Here, we present evidence that CAMTA3-mediated repression of SA pathway genes in nonstressed plants involves the action of an N-terminal repression module (NRM) that acts independently of calmodulin (CaM) binding to the IQ and CaM binding (CaMB) domains, a finding that is contrary to current thinking that CAMTA3 repression activity requires binding of CaM to the CaMB domain. Induction of SA pathway genes in response to low temperature did not occur in plants expressing only the CAMTA3-NRM region of the protein. Mutational analysis provided evidence that the repression activity of the NRM was suppressed by action of the IQ and CaMB domains responding to signals generated in response to low temperature. Plants expressing the CAMTA3-NRM region were also impaired in defense against the bacterial hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000. Our results indicate that the regulation of CAMTA3 repression activity by low temperature and pathogen infection involves related mechanisms, but with distinct differences.

The WW domain of the scaffolding protein IQGAP1 is neither necessary nor sufficient for binding to the MAPKs ERK1 and ERK2

Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (Kd ) for this interaction of 8 μm These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding.

Identifying Key Domains in Endothelial IQGAP1 Critical for Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. The majority of leukocytes undergo transendothelial migration (TEM) by using a unique endothelial cell membrane system referred to as the lateral border recycling compartment (LBRC). IQ‐domain GTPase‐activating protein 1 (IQGAP1) was identified as an LBRC‐associated protein and subsequently found to be essential for TEM. IQGAP1 is a scaffolding protein comprised of six major interaction domains. It has been reported to interact productively with over 135 cellular proteins. In endothelial cells IQGAP1 partially localizes to cell borders and knocking down its expression prevents TEM. However, it is not known which of the many proteins it is known to interact with carries out its role in TEM. Since these proteins bind to specific IQGAP1 domains, as a first step toward understanding the mechanism by which IQGAP1 regulates TEM, we explored which of its six protein interactions domains were critical for its function. We knocked down IQGAP1 in endothelial cells and re‐expressed truncated forms of IQGAP1 with specific regions deleted to identify the regions (and thereby the potential binding partners) that are critical for TEM. We show that the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM.Human endothelial cells in which >80% of endogenous IQGAP1 was depleted by shRNA did not support TEM; re‐expression of wild‐type IQGAP1 restored TEM to control levels. Re‐expression of IQGAP1 with a GFP‐tagged construct after knockdown also showed IQGAP1 enrichment around transmigrating leukocytes. Re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM. Constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.After identifying the IQ domain as having a critical role in TEM, we generated specific point mutations in this domain within the full‐length construct. In particular, the isoleucine (I)‐glutamine (Q) motif after which the IQ domain is named has been identified in the literature as having an essential role in IQGAP1 function and we are currently testing whether they are also important in TEM. Using these constructs in future studies will help identify the role of the IQ domain in TEM and its interacting partner(s). Identifying the binding partners of IQGAP1 that are critical for inflammation will provide clues to its mechanism of action and potential therapeutic targets for anti‐inflammatory therapy.Support or Funding InformationFunded by R01 HL046849, R01 HL064774, Alpha Omega Alpha Carolyn Kuckein Student Research Fellowship

The role of IQGAP1 in transendothelial migration: from in vitro identification to in vivo validation

Although inflammation is the body's stereotyped response to tissue damage, the clinical outcomes of this complex process, whether beneficial or detrimental, are rooted in the recruitment and subsequent function of the leukocytes at site of insult. It therefore follows that the regulation of leukocyte recruitment represents an acutely tractable target for therapeutic intervention. In order to reach the target tissue, leukocytes must undergo a carefully choreographed series of sequential intercellular interactions with the endothelium which culminate in transendothelial migration (TEM). As the leukocyte prepares to traverse the endothelial cell layer requisite protein‐protein interactions between the two cells directs the recruitment of the endothelial cell sub‐junctional Lateral Border Recycling Compartment (LBRC). Several of the endothelial proteins crucial for this process reside in the LBRC and its function in TEM is required for efficient leukocyte efflux.To identify additional proteins that are involved in LBRC function in an unbiased approach, we adapted a biochemical purification method known as the diaminobenzidine‐induced density shift to specifically label and recover membrane fractions enriched the LBRC proteins. Through this process we identified IQ domain containing GTPase Activating Protein 1 (IQGAP1) as a previously unknown LBRC associated proteins. IQGAP1 is a cytoplasmic multidomain scaffolding protein that has been shown in other systems to integrate and propagate complex signaling pathways. Genetic ablation of IQGAP1 in endothelial cell cultures using shRNA transfection substantially reduced their ability to support efficient monocyte TEM. Re‐expression of full‐length IQGAP1 rescued this defect, confirming its essential function in this process. Interestingly, re‐expression of IQGAP1 constructs that lack the actin‐binding or IQ‐motif domains were not able to rescue the TEM defect shRNA mediated reduction of IQGAP1 expression did not affect the physical amount of LBRC membrane that could be isolated from endothelial cells suggesting instead that IQGAP1 plays a role in some signaling aspect or other function of the LBRC. To extend these studies in vivo we created bone marrow chimeras (i.e. wild type leukocytes in IQGAP1 knockout animals) and investigated the requirement of endothelial cell IQGAP1 in inflammation. We used two separate mouse models; croton oil dermatitis visualized with conventional confocal microscopy and IL‐1β induced inflammation in the cremaster muscle visualized by 4D intravital microscopy. In both models the ability of neutrophils to extravasate across the endothelium into inflamed tissues was significantly inhibited in animals that lacked IQGAP1. Together with our in vitro studies, these findings provide important insight into the critical role of endothelial cell IQGAP1 in leukocyte diapedesis.Support or Funding InformationThis research was supported by F32 AI084454 to DPS and R21 HL102519, R01 HL046849, and R37 HL064774 to WAM.

Natural Variation in the Promoter of GSE5 Contributes to Grain Size Diversity in Rice

The utilization of natural genetic variation greatly contributes to improvement of important agronomic traits in crops. Understanding the genetic basis for natural variation of grain size can help breeders develop high-yield rice varieties. In this study, we identify a previously unrecognized gene, named GSE5, in the qSW5/GW5 locus controlling rice grain size by combining the genome-wide association study with functional analyses. GSE5 encodes a plasma membrane-associated protein with IQ domains, which interacts with the rice calmodulin protein, OsCaM1-1. We found that loss of GSE5 function caused wide and heavy grains, while overexpression of GSE5 resulted in narrow grains. We showed that GSE5 regulates grain size predominantly by influencing cell proliferation in spikelet hulls. Three major haplotypes of GSE5 (GSE5, GSE5DEL1+IN1, and GSE5DEL2) in cultivated rice were identified based on the deletion/insertion type in its promoter region. We demonstrated that a 950-bp deletion (DEL1) in indica varieties carrying the GSE5DEL1+IN1 haplotype and a 1212-bp deletion (DEL2) in japonica varieties carrying the GSE5DEL2 haplotype associated with decreased expression of GSE5, resulting in wide grains. Further analyses indicate that wild rice accessions contain all three haplotypes of GSE5, suggesting that the GSE5 haplotypes present in cultivated rice are likely to have originated from different wild rice accessions during rice domestication. Taken together, our results indicate that the previously unrecognized GSE5 gene in the qSW5/GW5 locus, which is widely utilized by rice breeders, controls grain size, and reveal that natural variation in the promoter region of GSE5 contributes to grain size diversity in rice.

Unmasking the Molecular Determinants Important for Ca2+ -Dependent Regulation of CaV2.2

(CDI) and facilitation (CDF), respectively, which contribute to short-term synaptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) binding to sites in the C-terminal domain (CT) of the Cav2.1 α1 subunit, including a consensus CaM-binding IQ-domain. Cav2.2 (N-type) channels display CDI but not CDF but the underlying mechanism that blocks CDF in Cav2.2 is unknown. Here, we tested the hypothesis that Cav2.2 does not undergo CDF since it lacks essential molecular determinants for CDF that are present in Cav2.1. We find that alternative splicing of exons in the proximal and distal CT, which regulates CDF of Cav2.1, has no effect on CDF of Cav2.2. However, replacement of the entire CT of Cav2.2 with that of Cav2.1 produces robust CDF of the chimeric channel. Further analyses reveal that transfer of the Cav2.1 EF-hand, Pre-IQ- IQ domains, and a downstream CaM-binding domain (CBD) are sufficient to support CDF in chimeric Cav2.2 channels. Our results highlight the importance of the CT in distinguishing Ca2+ feedback regulation of Cav2.2 and Cav2.1, and underscore how molecular distinctions may underlie the unique contributions of these channels in regulating neurotransmitter release.

IQGAP1 Binds to Yes-associated Protein (YAP) and Modulates Its Transcriptional Activity

During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration, and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. In this study, we report that IQGAP1 binds to YAP and modulates its activity. IQGAP1 and YAP co-immunoprecipitated from cells. In vitro analysis with pure proteins demonstrated a direct interaction between IQGAP1 and YAP. Analysis with multiple fragments of each protein showed that the interaction occurs via the IQ domain of IQGAP1 and the TEAD-binding domain of YAP. The interaction between IQGAP1 and YAP has functional effects. Knock-out of endogenous IQGAP1 significantly increased the formation of nuclear YAP-TEAD complexes. Transcription assays were performed with IQGAP1-null mouse embryonic fibroblasts and HEK293 cells with IQGAP1 knockdown by CRISPR/Cas9. Quantification demonstrated that YAP-TEAD-mediated transcription in cells lacking IQGAP1 was significantly greater than in control cells. These data reveal that IQGAP1 binds to YAP and modulates its co-transcriptional function, suggesting that IQGAP1 participates in Hippo signaling.

Role of IQGAP1 in endothelial barrier enhancement caused by OxPAPC.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via enhancement of both the peripheral actin cytoskeleton and cell junctions mediated by Rac1 and Cdc42 GTPases. This study evaluated the role for the multifunctional Rac1/Cdc42 effector and regulator, IQGAP1, as a molecular transducer of the OxPAPC-mediated EC barrier enhancing signal. IQGAP1 knockdown in endothelial cells by gene-specific siRNA abolished OxPAPC-induced enlargement of VE-cadherin-positive adherens junctions, suppressed peripheral accumulation of actin polymerization regulators, namely cortactin, N-WASP and Arp3, and attenuated remodeling of the peripheral actin cytoskeleton. Inhibition of OxPAPC-induced barrier enhancement by IQGAP1 knockdown was due to suppressed Rac1 and Cdc42 activation. Expression of an IQGAP1 truncated mutant showed that the GTPase regulatory domain (GRD) of IQGAP1 was essential for the OxPAPC-induced membrane localization of cortactin, adherens junction proteins VE-cadherin and p120-catenin as well as for EC permeability response. IQGAP1knockdown attenuated the protective effect of OxPAPC against thrombin-induced cell contraction, cell junction disruption and EC permeability. These results demonstrate for the first time the role of IQGAP1 as a critical transducer of OxPAPC-induced Rac1/Cdc42 signaling to the actin cytoskeleton and adherens junctions which promotes cortical cytoskeletal remodeling and EC barrier protective effects of oxidized phospholipids.

The relationship between Autism Spectrum Quotient (AQ) and uneven intellectual development in school-age children

AbstractPrevious studies have found correlations between uneven intellectual development and autistic symptom severity but thus far each study has looked only at specific types of discrepancy score and results have been inconsistent. This study used the Autism Spectrum Quotient (AQ) and the British Ability Scales (second edition) to look for a correlation between autistic-like traits and an overall index of unevenness based on three types of discrepancy score, namely: discrepancies between IQ subscales; discrepancies between IQ domain scores; and, discrepancies between overall IQ and word-level literacy skills. The AQ scale was designed to measure autistic-like symptoms both in those with an Autism Spectrum Disorder and at all levels across the general population. The study sample was therefore not restricted to children with an ASD. The 106 school age participants had a mean IQ of 97 (SD 19.6) and a mean AQ (child version) score of 69.6 (SD 24.6). After controlling for IQ, each of the three intellectual ...

Cardiomyopathy Mutation in Ventricular Essential Light Chain of Cardiac Myosin Alters Structural and Functional Interaction with Actin

We have used site-directed time-resolved FRET (TR-FRET) to determine the effect of pathological mutations in human ventricular essential light chain (hVELC) of cardiac myosin on interaction with actin. The hVELC modulates function of actomyosin, and the proposed mechanisms involve interaction of its N-terminus with actin and its C-terminal region with the IQ domain of myosin. Several mutations in hVELC are associated with familial hypertrophic cardiomyopathy (FHC), which disturbs morphology of the heart and can lead to sudden death. We focus on the cardiomyopathy mutation E56G, which was shown to affect binding of hVELC to the IQ domain. We have introduced this mutation in a single cysteine hVELC construct (A16C) and exchanged both ELCs (A16C and A16C-E56G) onto bovine cardiac S1. This mutation altered actin-activated S1 ATPase by increasing both Vmax and KATPase, suggesting different structural transitions from the weakly to strongly bound states in the ATPase cycle. Using a donor fluorescent probe IAEDANS on actin (at C374) and an acceptor probe Dabcyl maleimide on the N-lobe (A16C) of hVELC, we used TR-FRET to measure the distance between probes on actin and ELC for both proteins. Our previous studies showed that this distance is a sensitive measure of the weak-to-strong transition in the actomyosin complex. Current results indicate that E56G has no significant effect on the structure of strongly bound actomyosin, but fundamentally changes distribution of structural states in the presence of ATP. We conclude that the primary pathogenic effect of the E56G mutation is on the distribution of actomyosin structural states, affecting contractility and remodeling of cardiac muscle, with disastrous results.

Inactivation of Cav1.1 Channels in Adult Skeletal Muscle: Effects of a C-Terminal Pre-IQ Mutation

Calcium-dependent inactivation (CDI) has been documented for the great majority of Cav1 channels but whether native Cav1.1 of adult skeletal muscle exhibits this feature remains controversial. In CDI, Ca2+ ions can bind to specific sites in the C-terminal IQ domain, a process that requires the Ca2+-binding protein calmodulin (CaM). In addition to CaM, other Ca2+-binding proteins could in principle regulate CDI. Here we sought to explore whether S100A1, a Ca2+-binding protein highly expressed in striated muscle that fine tunes SR Ca2+ release, is also involved in the regulation of CDI. The binding of S100A1 to two different Cav1.1 C-terminal regions (pre-IQ and IQ) was determined by isothermal titration calorimetry. Under Ca2+-loaded conditions, S100A1 binds with higher affinity to the pre-IQ region in the skeletal isoform (Cav1.1) than in the cardiac isoform (CaV1.2). Then, to test for a functional role of the pre-IQ region in adult muscle fibers, we expressed EGFP-Cav1.1 wild type (used as control) or EGFP-Cav1.1 pre-IQ (I or W→A) mutant, which should not bind S100A1, via electroporation and used whole-cell voltage clamp Ca2+ current recordings to evaluate Cav1.1 activation and inactivation properties. The pre-IQ mutation did not modify any Cav1.1 activation parameters; however, the fractional inactivation during a 300 ms pulse was significantly increased. Our results highlight a Ca2+/S100A1 binding site in the C-terminal of the Cav1.1 channel that could be critical for tuning CDI in Cav1.1. While not directly critical for excitation-contraction coupling, recent evidence indicates that Ca2+ entry through Cav1.1 is important for the long-term contractile and metabolic function of skeletal muscle; therefore this S100A1 binding site may impact muscle fiber plasticity. Supported by NIH-NIAMS (R37-R055099).

Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels

Calmodulin (CaM), a Ca2+-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca2+-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca2+-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca2+/CaM. Ca2+/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca2+/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca2+ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca2+/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.

Preliminary neurocognitive outcomes in Jeavons syndrome.

Jeavons syndrome (JS, eyelid myoclonia with absences [EMA]) consists of a triad of symptoms including eyelid myoclonia that may be accompanied by absence seizures, eye closure-induced EEG paroxysms or seizures, and photosensitivity. The age of onset ranges between 2 and 14 years with symptoms peaking between 6 and 8 years of age. Though investigation of the clinical, EEG, and neurological features of JS has occurred, neurocognitive functioning has not been well-delineated despite suggestion that a subtype of the syndrome is characterized in part by cognitive impairment. The purpose of this study was to define neurocognitive functioning in a more detailed manner by examining global IQ and relevant neurocognitive domains (i.e., verbal and nonverbal reasoning, attention, executive functioning, memory) in pediatric patients. The sample (N=6, 4 females) ranged in age from 8 to 15 years (M=11, SD=2.82). All participants completed neuropsychological evaluations. Statistical analyses revealed performance that was below average on measures of global IQ, processing speed and rote, verbal learning coupled with average nonverbal reasoning, and sustained attention. There was also evidence of impaired higher-level verbal reasoning. While global IQ ranged from low average to borderline impaired, no participant could be accurately described as impaired or having intellectual disability (ID) given the consistently average performance noted on some higher-order tasks including nonverbal reasoning.

A reversal of the Flynn effect for spatial perception in German-speaking countries: Evidence from a cross-temporal IRT-based meta-analysis (1977–2014)

Generational IQ changes (the Flynn effect) have been shown to be predominantly positive but differentiated according to IQ domains and countries. However, evidence from recent studies points towards a decrease of the Flynn effect globally or even a reversal in some countries. In the present meta-analysis, we show an inverse u-shaped trajectory of IQ test performance changes in a large number of samples (k=96; N=13,172) on a well-known test for spatial perception (the three-dimensional cubes test, 3DC) in German-speaking countries over 38years (1977–2014). Assessment of both item response theory-based measures as well as more standard measures of classical test theory showed initial increases and a subsequent decrease of performance when controlling for age, sample type (general population vs. mixed samples vs. university students) and sex. Our results suggest saturation and diminishing returns of IQ increasing factors (e.g., life history speed) whilst negative associations of IQ changes with psychometric g may have led to the observed IQ score decrease in more recent years.

Abstract 37: Nrip Deficiency Leads to Dilated Cardiomyopathy

Previously, we demonstrate a gene, nuclear receptor interaction protein (NRIP, also named DCAF6 or IQWD1) as a Ca2+- dependent calmodulin binding protein that can activate calcineurin phosphatase activity. Here, we found that α-actinin-2 (ACTN2), is one of NRIP-interacting proteins from the yeast two-hybrid system using NRIP as a prey. We further confirmed the direct bound between NRIP and ACTN2 using in vitro protein-protein interaction and in vivo co-immunoprecipitation assays. To further map the binding domain of each protein, the results showed the IQ domain of NRIP responsible for ACTN2 binding, and EF hand motif of ACTN2 responsible for NRIP bound. Due to ACTN2 is a biomarker of muscular Z-disc complex; we found the co-localization of NRIP and ACTN2 in cardiac tissues by immunofluorescence assays. Taken together, NRIP is a novel ACTN2-interacting protein. To investigate insights into in vivo function of NRIP, we generated conventional NRIP-null mice. The H&E staining results are shown in the hearts of NRIP KO mice are enlarged and dilated and the cell width of NRIP KO cardiomyocyte is increased. The EM of NRIP KO heart muscles reveal the reduction of I-band width and extension length of Z-disc in sarcomere structure; and the echocardiography shows the diminished fractional shortening in heart functions. Additionally, the calcium transient and sarcomere contraction length in cardiomyocytes of NRIP KO is weaker and shorter than wt; respectively. In conclusion, NRIP is a novel Z-disc protein and has function for maintenance of sarcomere integrity structure and function for calcium transient and muscle contraction.

Structure and Regulation of the Movement of Human Myosin VIIA

Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail). Deletion of the C-terminal 40 amino acids and mutation of the basic residues in this region (R2176A or K2179A) abolished the inhibition. Electron microscopy revealed that HM7AFull is a monomer in which the tail domain bends back toward the head-neck domain to form a compact structure. This compact structure is extended at high ionic strength or in the presence of Ca(2+). Although myosin VIIA has five isoleucine-glutamine (IQ) motifs, the neck length seems to be shorter than the expected length of five bound calmodulins. Supporting this observation, the IQ domain bound only three calmodulins in Ca(2+), and the first IQ motif failed to bind calmodulin in EGTA. These results suggest that the unique IQ domain of HM7A is important for the tail-neck interaction and, therefore, regulation. Cellular studies revealed that dimer formation of HM7A is critical for its translocation to filopodial tips and that the tail domain (HM7ATail) markedly reduced the filopodial tip localization of the HM7AΔTail dimer, suggesting that the tail-inhibition mechanism is operating in vivo. The translocation of the HM7AFull dimer was significantly less than that of the HM7AΔTail dimer, and R2176A/R2179A mutation rescued the filopodial tip translocation. These results suggest that HM7A can transport its cargo molecules, such as USH1 proteins, upon release of the tail-dependent inhibition.

Network Analyses Reveal Shifts in Transcript Profiles and Metabolites That Accompany the Expression of SUN and an Elongated Tomato Fruit.

SUN controls elongated tomato (Solanum lycopersicum) shape early in fruit development through changes in cell number along the different axes of growth. The gene encodes a member of the IQ domain family characterized by a calmodulin binding motif. To gain insights into the role of SUN in regulating organ shape, we characterized genome-wide transcriptional changes and metabolite and hormone accumulation after pollination and fertilization in wild-type and SUN fruit tissues. Pericarp, seed/placenta, and columella tissues were collected at 4, 7, and 10 d post anthesis. Pairwise comparisons between SUN and the wild type identified 3,154 significant differentially expressed genes that cluster in distinct gene regulatory networks. Gene regulatory networks that were enriched for cell division, calcium/transport, lipid/hormone, cell wall, secondary metabolism, and patterning processes contributed to profound shifts in gene expression in the different fruit tissues as a consequence of high expression of SUN. Promoter motif searches identified putative cis-elements recognized by known transcription factors and motifs related to mitotic-specific activator sequences. Hormone levels did not change dramatically, but some metabolite levels were significantly altered, namely participants in glycolysis and the tricarboxylic acid cycle. Also, hormone and primary metabolite networks shifted in SUN compared with wild-type fruit. Our findings imply that SUN indirectly leads to changes in gene expression, most strongly those involved in cell division, cell wall, and patterning-related processes. When evaluating global coregulation in SUN fruit, the main node represented genes involved in calcium-regulated processes, suggesting that SUN and its calmodulin binding domain impact fruit shape through calcium signaling.

Retraction: Cytoskeletal disrupting agents prevent calmodulin kinase, IQ domain and voltage-dependent facilitation of L-type Ca2+ channels.

Retraction: Cytoskeletal disrupting agents prevent calmodulin kinase, IQ domain and voltage-dependent facilitation of L-type Ca2+ channels.

IQGAP1 Is Involved in Enhanced Aggressive Behavior of Epithelial Ovarian Cancer Stem Cell-Like Cells During Differentiation.

BackgroundWide metastasis is one of characteristics of ovarian cancer. Cancer stem cells, as a source in cancer invasion and metastasis, possess powerful potential of differentiation. Scaffolding IQ domain GTPase-activating protein 1 (IQGAP1) plays a key role in the invasion and metastasis of cancer cells, but IQGAP1’s role in cancer stem cells including ovarian cancer was unclear.MethodsSpheroid culture with serum-free medium was used for enriching ovarian cancer stem cell-like cells (CSC-LCs) from 3AO cell line, and a medium with 10% fetal bovine serum was used to induce the differentiation of CSC-LCs. Immunofluorescence was for detecting the stem markers OCT4 and SOX2. The quantitative real-time-polymerase chain reaction and Western blotting were performed to determine the messenger RNA and protein expression of IQGAP1, respectively. The capacity of cell invasion was evaluated by transwell chamber assay.ResultsOvarian CSC-LCs obtained through spheroid culture showed irregularly elongated appearance, CD24 negative, and OCT4 and SOX2 positive. IQGAP1 expression was decreased in ovarian CSC-LCs compared with parental 3AO cells, but increased de novo during the differentiation of CSC-LCs. Knockdown of IQGAP1 by specific small interfering RNA remarkably weakened invasion capacity of 2-day differentiated ovarian CSC-LCs.ConclusionsIncreased IQGAP1 expression during the differentiation of CSC-LCs is involved in an aggressive cell behavior, which may contribute to metastasis of ovarian cancer.