Kif9 is an active kinesin motor required for ciliary beating and proximodistal patterning of motile axonemes.

Most motile cilia have a stereotyped structure of nine microtubule outer doublets and a single central pair of microtubules. The central pair of microtubules are surrounded by a set of proteins, termed the central pair apparatus. A specific kinesin, Klp1 projects from the central pair and contributes to ciliary motility in Chlamydomonas. The vertebrate ortholog, Kif9, is required for beating in mouse sperm flagella, but the mechanism of Kif9/Klp1 function remains poorly defined. Here, using Xenopus epidermal multiciliated cells, we show that Kif9 is necessary for ciliary motility and the proper distal localization of not only central pair proteins, but also radial spokes and dynein arms. In addition, single-molecule assays in vitro reveal that Xenopus Kif9 is a long-range processive motor, although it does not mediate long-range movement in ciliary axonemes in vivo. Together, our data suggest that Kif9 is integral for ciliary beating and is necessary for proper axonemal distal end integrity.

Neural tube closure requires the endocytic receptor Lrp2 and its functional interaction with intracellular scaffolds.

Pathogenic mutations in the endocytic receptor LRP2 in humans are associated with severe neural tube closure defects (NTDs) such as anencephaly and spina bifida. Here, we have combined analysis of neural tube closure in mouse and in the African Clawed Frog Xenopus laevis to elucidate the etiology of Lrp2-related NTDs. Lrp2 loss of function impaired neuroepithelial morphogenesis, culminating in NTDs that impeded anterior neural plate folding and neural tube closure in both model organisms. Loss of Lrp2 severely affected apical constriction as well as proper localization of the core planar cell polarity (PCP) protein Vangl2, demonstrating a highly conserved role of the receptor in these processes, which are essential for neural tube formation. In addition, we identified a novel functional interaction of Lrp2 with the intracellular adaptor proteins Shroom3 and Gipc1 in the developing forebrain. Our data suggest that, during neurulation, motifs within the intracellular domain of Lrp2 function as a hub that orchestrates endocytic membrane removal for efficient apical constriction, as well as PCP component trafficking in a temporospatial manner.

Ordered deployment of distinct ciliary beating machines in growing axonemes of vertebrate multiciliated cells.

The beating of motile cilia requires the coordinated action of diverse machineries that include not only the axonemal dynein arms, but also the central apparatus, the radial spokes, and the microtubule inner proteins. These machines exhibit complex radial and proximodistal patterns in mature axonemes, but little is known about the interplay between them during motile ciliogenesis. Here, we describe and quantify the relative rates of axonemal deployment for these diverse cilia beating machineries during the final stages of differentiation of Xenopus epidermal multiciliated cells.

Twinfilin1 controls lamellipodial protrusive activity and actin turnover during vertebrate gastrulation.

The dynamic control of the actin cytoskeleton is a key aspect of essentially all animal cell movements. Experiments in single migrating cells and in vitro systems have provided an exceptionally deep understanding of actin dynamics. However, we still know relatively little of how these systems are tuned in cell-type-specific ways, for example in the context of collective cell movements that sculpt the early embryo. Here, we provide an analysis of the actin-severing and depolymerization machinery during vertebrate gastrulation, with a focus on Twinfilin1 (Twf1) in Xenopus. We find that Twf1 is essential for convergent extension, and loss of Twf1 results in a disruption of lamellipodial dynamics and polarity. Moreover, Twf1 loss results in a failure to assemble polarized cytoplasmic actin cables, which are essential for convergent extension. These data provide an in vivo complement to our more-extensive understanding of Twf1 action in vitro and provide new links between the core machinery of actin regulation and the specialized cell behaviors of embryonic morphogenesis.

Acquisition of Streptomycin Resistance by Oxidative Stress Induced by Hydrogen Peroxide in Radiation-Resistant Bacterium Deinococcus geothermalis.

Streptomycin is used primarily to treat bacterial infections, including brucellosis, plague, and tuberculosis. Streptomycin resistance easily develops in numerous bacteria through the inhibition of antibiotic transfer, the production of aminoglycoside-modifying enzymes, or mutations in ribosomal components with clinical doses of streptomycin treatment. (1) Background: A transposable insertion sequence is one of the mutation agents in bacterial genomes under oxidative stress. (2) Methods: In the radiation-resistant bacterium Deinococcus geothermalis subjected to chronic oxidative stress induced by 20 mM hydrogen peroxide, active transposition of an insertion sequence element and several point mutations in three streptomycin resistance (SmR)-related genes (rsmG, rpsL, and mthA) were identified. (3) Results: ISDge6 of the IS5 family integrated into the rsmG gene (dgeo_2335), called SrsmG, encodes a ribosomal guanosine methyltransferase resulting in streptomycin resistance. In the case of dgeo_2840-disrupted mutant strains (S1 and S2), growth inhibition under antibiotic-free conditions was recovered with increased growth yields in the presence of 50 µg/mL streptomycin due to a streptomycin-dependent (SmD) mutation. These mutants have a predicted proline-to-leucine substitution at the 91st residue of ribosomal protein S12 in the decoding center. (4) Conclusions: Our findings show that the active transposition of a unique IS element under oxidative stress conditions conferred antibiotic resistance through the disruption of rsmG. Furthermore, chronic oxidative stress induced by hydrogen peroxide also induced streptomycin resistance caused by point and frameshift mutations of streptomycin-interacting residues such as K43, K88, and P91 in RpsL and four genes for streptomycin resistance.

A systematic, label-free method for identifying RNA-associated proteins in vivo provides insights into vertebrate ciliary beating machinery.

Cell-type specific RNA-associated proteins are essential for development and homeostasis in animals. Despite a massive recent effort to systematically identify RNA-associated proteins, we currently have few comprehensive rosters of cell-type specific RNA-associated proteins in vertebrate tissues. Here, we demonstrate the feasibility of determining the RNA-associated proteome of a defined vertebrate embryonic tissue using DIF-FRAC, a systematic and universal (i.e., label-free) method. Application of DIF-FRAC to cultured tissue explants of Xenopus mucociliary epithelium identified dozens of known RNA-associated proteins as expected, but also several novel RNA-associated proteins, including proteins related to assembly of the mitotic spindle and regulation of ciliary beating. In particular, we show that the inner dynein arm tether Cfap44 is an RNA-associated protein that localizes not only to axonemes, but also to liquid-like organelles in the cytoplasm called DynAPs. This result led us to discover that DynAPs are generally enriched for RNA. Together, these data provide a useful resource for a deeper understanding of mucociliary epithelia and demonstrate that DIF-FRAC will be broadly applicable for systematic identification of RNA-associated proteins from embryonic tissues.

Mutations in Kinesin family member 6 reveal specific role in ependymal cell ciliogenesis and human neurological development.

Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular system of the brain, with localized flow being established by the polarized beating of the ependymal cell (EC) cilia. Here, we report a homozygous one base-pair deletion, c.1193delT (p.Leu398Glnfs*2), in the Kinesin Family Member 6 (KIF6) gene in a child displaying neurodevelopmental defects and intellectual disability. To test the pathogenicity of this novel human KIF6 mutation we engineered an analogous C-terminal truncating mutation in mouse. These mutant mice display severe, postnatal-onset hydrocephalus. We generated a Kif6-LacZ transgenic mouse strain and report expression specifically and uniquely within the ependymal cells (ECs) of the brain, without labeling other multiciliated mouse tissues. Analysis of Kif6 mutant mice with scanning electron microscopy (SEM) and immunofluorescence (IF) revealed specific defects in the formation of EC cilia, without obvious effect of cilia of other multiciliated tissues. Dilation of the ventricular system and defects in the formation of EC cilia were also observed in adult kif6 mutant zebrafish. Finally, we report Kif6-GFP localization at the axoneme and basal bodies of multi-ciliated cells (MCCs) of the mucociliary Xenopus epidermis. Overall, this work describes the first clinically-defined KIF6 homozygous null mutation in human and defines KIF6 as a conserved mediator of neurological development with a specific role for EC ciliogenesis in vertebrates.

Redox potential change by the cystine importer affected on enzymatic antioxidant protection in Deinococcus geothermalis.

Radiation resistant bacteria genus Deinococcus species were well studied on DNA repair and anti-oxidative stress response mechanisms. There are many protection factors as enzymatic and nonenzymatic involved. One of them is intracellular redox potential as like thiol compounds including cysteine acts as primary protectant against oxidation stress. A gene cluster consisting of the genes Dgeo_1986 and Dgeo_1987 of Deinococcus geothermalis was identified as a cystine importer. The expression levels of dgeo_1986 and dgeo_1987 were up-regulated by over 60-fold and 4-fold during the late exponential (L) growth phase, respectively. The double-knockout mutant of dgeo_1986 and dgeo_1987 was reduced in cystine and thiol concentrations and leading to enhanced sensitivity against H2O2 stress. The expression of catalase (Dgeo_2728) as an enzymatic anti-oxidant is more induced in the wild-type strain than the Δdgeo_1986-87 strain at the late growth phase. The expression level of the oxidative stress response regulator OxyR (Dgeo_1888) is dependent on the intracellular redox balance. That is, when the intracellular thiol content was reduced in the wild-type strain during the L phase, OxyR was clearly induced. Interestingly, the expression level of OxyR was higher in the Δdgeo_1986-87 strain than in the wild-type strain upon H2O2 treatment. Although OxyR was induced by H2O2 treatment in Δdgeo_1986-87 strain, where intracellular redox potential of cystine was reduced as a thiol compound due to reduced cystine import, the relative level of expression of catalase was unexpectedly down-regulated. Therefore, the catalase induction system as an enzymatic antioxidant protection should be affected via the cystine importer but not rely on the OxyR controlled manner.

TTC25 Deficiency Results in Defects of the Outer Dynein Arm Docking Machinery and Primary Ciliary Dyskinesia with Left-Right Body Asymmetry Randomization.

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

Integration of over 9,000 mass spectrometry experiments builds a global map of human protein complexes.

Macromolecular protein complexes carry out many of the essential functions of cells, and many genetic diseases arise from disrupting the functions of such complexes. Currently, there is great interest in defining the complete set of human protein complexes, but recent published maps lack comprehensive coverage. Here, through the synthesis of over 9,000 published mass spectrometry experiments, we present hu.MAP, the most comprehensive and accurate human protein complex map to date, containing > 4,600 total complexes, > 7,700 proteins, and > 56,000 unique interactions, including thousands of confident protein interactions not identified by the original publications. hu.MAP accurately recapitulates known complexes withheld from the learning procedure, which was optimized with the aid of a new quantitative metric (k-cliques) for comparing sets of sets. The vast majority of complexes in our map are significantly enriched with literature annotations, and the map overall shows improved coverage of many disease-associated proteins, as we describe in detail for ciliopathies. Using hu.MAP, we predicted and experimentally validated candidate ciliopathy disease genes in vivo in a model vertebrate, discovering CCDC138, WDR90, and KIAA1328 to be new cilia basal body/centriolar satellite proteins, and identifying ANKRD55 as a novel member of the intraflagellar transport machinery. By offering significant improvements to the accuracy and coverage of human protein complexes, hu.MAP (http://proteincomplexes.org) serves as a valuable resource for better understanding the core cellular functions of human proteins and helping to determine mechanistic foundations of human disease.

The hyaloid vasculature facilitates basement membrane breakdown during choroid fissure closure in the zebrafish eye.

A critical aspect of vertebrate eye development is closure of the choroid fissure (CF). Defects in CF closure result in colobomas, which are a significant cause of childhood blindness worldwide. Despite the growing number of mutated loci associated with colobomas, we have a limited understanding of the cell biological underpinnings of CF closure. Here, we utilize the zebrafish embryo to identify key phases of CF closure and regulators of the process. Utilizing Laminin-111 as a marker for the basement membrane (BM) lining the CF, we determine the spatial and temporal patterns of BM breakdown in the CF, a prerequisite for CF closure. Similarly, utilizing a combination of in vivo time-lapse imaging, ß-catenin immunohistochemistry and F-actin staining, we determine that tissue fusion, which serves to close the fissure, follows BM breakdown closely. Periocular mesenchyme (POM)-derived endothelial cells, which migrate through the CF to give rise to the hyaloid vasculature, possess distinct actin foci that correlate with regions of BM breakdown. Disruption of talin1, which encodes a regulator of the actin cytoskeleton, results in colobomas and these correlate with structural defects in the hyaloid vasculature and defects in BM breakdown. cloche mutants, which entirely lack a hyaloid vasculature, also possess defects in BM breakdown in the CF. Taken together, these data support a model in which the hyaloid vasculature and/or the POM-derived endothelial cells that give rise to the hyaloid vasculature contribute to BM breakdown during CF closure.

Bio-Inspired Distributed Transmission Power Control Considering QoS Fairness in Wireless Body Area Sensor Networks.

Recently, the development of wireless body area sensor network (WBASN) has accelerated due to the rapid development of wireless technology. In the WBASN environment, many WBASNs coexist where communication ranges overlap with each other, resulting in the possibility of interference. Although nodes in a WBASN typically operate at a low power level, to avoid adversely affecting the human body, high transmission rates may be required to support some applications. In addition to this, since many varieties of applications exist in the WBASN environment, each prospective user may have different quality of service (QoS) requirements. Hence, the following issues should be considered in the WBASN environment: (1) interference between adjacent WBASNs, which influences the performance of a specific system, and (2) the degree of satisfaction on the QoS of each user, i.e., the required QoS such as user throughput should be considered to ensure that all users in the network are provided with a fair QoS satisfaction. Thus, in this paper, we propose a transmission power adjustment algorithm that addresses interference problems and guarantees QoS fairness between users. First, we use a new utility function to measure the degree of the satisfaction on the QoS for each user. Then, the transmission power of each sensor node is calculated using the Cucker-Smale model, and the QoS satisfaction of each user is synchronized dispersively. The results of simulations show that the proposed algorithm performs better than existing algorithms, with respect to QoS fairness and energy efficiency.

In vivo analysis of hyaloid vasculature morphogenesis in zebrafish: A role for the lens in maturation and maintenance of the hyaloid.

Two vascular networks nourish the embryonic eye as it develops - the hyaloid vasculature, located at the anterior of the eye between the retina and lens, and the choroidal vasculature, located at the posterior of the eye, surrounding the optic cup. Little is known about hyaloid development and morphogenesis, however. To begin to identify the morphogenetic underpinnings of hyaloid formation, we utilized in vivo time-lapse confocal imaging to characterize morphogenesis of the zebrafish hyaloid through 5 days post fertilization (dpf). Our data segregate hyaloid formation into three distinct morphogenetic stages: Stage I: arrival of hyaloid cells at the lens and formation of the hyaloid loop; Stage II: formation of a branched hyaloid network; Stage III: refinement of the hyaloid network. Utilizing fixed and dissected tissues, distinct Stage II and Stage III aspects of hyaloid formation were quantified over time. Combining in vivo imaging with microangiography, we demonstrate that the hyaloid system becomes fully enclosed by 5dpf. To begin to identify the molecular and cellular mechanisms underlying hyaloid morphogenesis, we identified a recessive mutation in the mab21l2 gene, and in a subset of mab21l2 mutants the lens does not form. Utilizing these "lens-less" mutants, we determined whether the lens was required for hyaloid morphogenesis. Our data demonstrate that the lens is not required for Stage I of hyaloid formation; however, Stages II and III of hyaloid formation are disrupted in the absence of a lens, supporting a role for the lens in hyaloid maturation and maintenance. Taken together, this study provides a foundation on which the cellular, molecular and embryologic mechanisms underlying hyaloid morphogenesis can be elucidated.

Label-free proteomic comparison reveals ciliary and nonciliary phenotypes of IFT-A mutants.

DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila. A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.

Ancient eukaryotic protein interactions illuminate modern genetic traits and disorders.

All eukaryotes share a common ancestor from roughly 1.5 - 1.8 billion years ago, a single-celled, swimming microbe known as LECA, the Last Eukaryotic Common Ancestor. Nearly half of the genes in modern eukaryotes were present in LECA, and many current genetic diseases and traits stem from these ancient molecular systems. To better understand these systems, we compared genes across modern organisms and identified a core set of 10,092 shared protein-coding gene families likely present in LECA, a quarter of which are uncharacterized. We then integrated >26,000 mass spectrometry proteomics analyses from 31 species to infer how these proteins interact in higher-order complexes. The resulting interactome describes the biochemical organization of LECA, revealing both known and new assemblies. We analyzed these ancient protein interactions to find new human gene-disease relationships for bone density and congenital birth defects, demonstrating the value of ancestral protein interactions for guiding functional genetics today.

Label-free proteomic comparison reveals ciliary and non-ciliary phenotypes of IFT-A mutants.

DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila . A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes.

Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in Tetrahymena thermophila. From over 19,000 XLs, we identified 4,757 unique amino acid interactions among 1,143 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the Intraflagellar Transport system, axonemal dynein arms, radial spokes, the 96 nm ruler, and microtubule inner proteins, among others. Guided by this dataset, we used vertebrate multiciliated cells to reveal novel functional interactions among several poorly-defined human ciliopathy proteins. The dataset therefore provides a powerful resource for studying the basic biology of an ancient organelle and the molecular etiology of human genetic disease.

Transposition of insertion sequences by dielectric barrier discharge plasma and gamma irradiation in the radiation-resistant bacterium Deinococcus geothermalis.

Insertion sequences (ISs) of the radiation-resistant bacterium Deinococcus geothermalis are transposed into other loci by oxidative stress through hydrogen peroxide treatment. Gamma irradiation and dielectric barrier discharge (DBD) plasma radiation are known to produce a variety of oxidative stress agents such as reactive oxygen species and reactive nitrogen species. Therefore, to determine whether the transposition of ISs was induced in D. geothermalis by both gamma irradiation and DBD plasma radiation, we selected non-pigmented mutants with disrupted target genes encoding carotenoid biosynthesis enzymes such as a phytoene synthase (dgeo_0523) and a phytoene desaturase (dgeo_0524). Different DNA-binding protein-deficient mutants exhibited novel transposition of ISs. Dps (dgeo_0257), OxyR (dgeo_1888), and the LysR (dgeo_2840) family regulator, in addition to cystine importer-disrupted and -overexpressed mutants (dgeo_1986-87 and dgeo_1985R) and wild-type D. geothermalis were tested in this study. Active IS transposition was not detected in two wild-type control species (Deinococcus radiodurans and Deinococcus radiopugnans) after phenotypic selection in gamma irradiation. Our finding demonstrated that gamma irradiation triggers the transposition of particular IS elements, especially ISDge2 and ISDge3 of the IS1 family, ISDge5 of the IS701 family, and ISDge6 of the IS5 family in wild-type strain and the Δdgeo_0257, Δdgeo_1986-87, Δdgeo_1985R, and Δdgeo_2840 mutants. Furthermore, DBD plasma radiation triggered the transposition of ISDge11 of the IS4 family in the wild-type strain; ISDge6 of the IS5 family on Δdgeo_0257, Δdgeo_1888 and Δdgeo_2840; ISDge5 of the IS701 family on Δdgeo_0257 strain.

ARVCF catenin controls force production during vertebrate convergent extension.

The design of an animal's body plan is encoded in the genome, and the execution of this program is a mechanical progression involving coordinated movement of proteins, cells, and whole tissues. Thus, a challenge to understanding morphogenesis is connecting events that occur across various length scales. Here, we describe how a poorly characterized adhesion effector, Arvcf catenin, controls Xenopus head-to-tail axis extension. We find that Arvcf is required for axis extension within the intact organism but not within isolated tissues. We show that the organism-scale phenotype results from a defect in tissue-scale force production. Finally, we determine that the force defect results from the dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to membranes. These results provide a comprehensive understanding of Arvcf function during axis extension and produce an insight into how a cellular-scale defect in adhesion results in an organism-scale failure of development.

Influence of Redox Imbalances on the Transposition of Insertion Sequences in Deinococcus geothermalis.

The transposition of insertion sequence elements was evaluated among different Deinococcus geothermalis lineages, including the wild-type, a cystine importer-disrupted mutant, a complemented strain, and a cystine importer-overexpressed strain. Cellular growth reached early exponential growth at OD600 2.0 and late exponential growth at OD600 4.0. Exposing the cells to hydrogen peroxide (80-100 mM) resulted in the transposition of insertion sequences (ISs) in genes associated with the carotenoid biosynthesis pathway. Particularly, ISDge7 (an IS5 family member) and ISDge5 (an IS701 family member) from the cystine importer-disrupted mutant were transposed into phytoene desaturase (dgeo_0524) via replicative transposition. Further, the cystine importer-overexpressed strain Δdgeo_1985R showed transposition of both ISDge2 and ISDge5 elements. In contrast, IS transposition was not detected in the complementary strain. Interestingly, a cystine importer-overexpressing strain exhibited streptomycin resistance, indicating that point mutation occurred in the rpsL (dgeo_1873) gene encoding ribosomal protein S12. qRT-PCR analyses were then conducted to evaluate the expression of oxidative stress response genes, IS elements, and low-molecular-weight thiol compounds such as mycothiol and bacillithiol. Nevertheless, the mechanisms that trigger IS transposition in redox imbalance conditions remain unclear. Here, we report that the active transposition of different IS elements was affected by intracellular redox imbalances caused by cystine importer deficiencies or overexpression.