Cell Biology

Myotonic dystrophy type 1 (DM1) is a genetic disorder that causes muscle weakness and myotonia. In DM1 patients, cardiac electrical manifestations include conduction defects and atrial fibrillation. DM1 results in the expansion of a CTG transcribed into CUG-containing transcripts that accumulate in the nucleus as RNA foci and alter the activity of several splicing regulators. The underlying pathological mechanism involves two key RNA-binding proteins (MBNL and CELF) with expanded CUG repeats that sequester MBNL and alter the activity of CELF resulting in spliceopathy and abnormal electrical activity. In the present study, we identified two DM1 patients with heart conduction abnormalities and characterized their hiPSC lines. Two differentiation protocols were used to investigate both the ventricular and the atrial electrophysiological aspects of DM1 and unveil the impact of the mutation on voltage-gated ion channels, electrical activity, and calcium homeostasis in DM1 cardiomyocytes derived from hiPSCs. Our analysis revealed the presence of molecular hallmarks of DM1, including the accumulation of RNA foci and sequestration of MBNL1 in DM1 hiPSC-CMs. We also observed mis-splicing of SCN5A and haploinsufficiency of DMPK. Furthermore, we conducted separate characterizations of atrial and ventricular electrical activity, conduction properties, and calcium homeostasis. Both DM1 cell lines exhibited reduced density of sodium and calcium currents, prolonged action potential duration, slower conduction velocity, and impaired calcium transient propagation in both ventricular and atrial cardiomyocytes. Notably, arrhythmogenic events were recorded, including both ventricular and atrial arrhythmias were observed in the two DM1 cell lines. These findings enhance our comprehension of the molecular mechanisms underlying DM1 and provide valuable insights into the pathophysiology of ventricular and atrial involvement.

Generating atrial-like cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial for modeling and treating atrial-related diseases, such as atrial arrythmias including atrial fibrillations. However, it is essential to obtain a comprehensive understanding of the electrophysiological properties of these cells. The objective of the present study was to investigate the molecular, electrical, and biophysical properties of several ion channels, especially NaV1.5 channels, in atrial hiPSC cardiomyocytes. Atrial cardiomyocytes were obtained by the differentiation of hiPSCs treated with retinoic acid (RA). The quality of the atrial specification was assessed by qPCR, immunocytofluorescence, and western blotting. The electrophysiological properties of action potentials (APs), Ca2+ dynamics, K+ and Na+ currents were investigated using patch-clamp and optical mapping approaches. We evaluated mRNA transcript and protein expressions to show that atrial cardiomyocytes ex...

Background: The ability to differentiate patient-specific human induced pluripotent stem cells (hiPSC) into cardiac myocytes (hiPSC-CM) offers novel perspectives for cardiovascular research. A number of studies, which reported mainly on current-voltage curves used hiPSC-CM to model voltage-gated Na þ channel (Na v ) dysfunction. However, the expression patterns and precise biophysical and pharmacological properties of Na v channels from hiPSC-CM remain unknown. Our objective was to study the characteristics of Na v channels from hiPSC-CM and assess the appropriateness of this novel cell model. Methods: We generated hiPSC-CM using the recently described monolayer-based differentiation protocol. Results: hiPSC-CM expressed cardiac-specific markers, exhibited spontaneous electrical and contractile activities, and expressed distinct Na v channels subtypes. Electrophysiological, pharmacological, and molecular characterizations revealed that, in addition to the main Na v 1.5 channel, the neuronal TTX-sensitive Na v 1.7 channel was also significantly expressed in hiPSC-CM. Most of the Na þ currents were resistant to tetrodotoxin block. Therapeutic concentrations of lidocaine, a class I antiarrhythmic drug, also inhibited Na þ currents in a use-dependent manner. Na v 1.5 and Na v 1.7 expression and maturation patterns of hiPSC-CM and native Human cardiac tissues appeared to be similar. The four Na v b regulatory subunits were expressed in hiPSC-CM, with b3 being the preponderant subtype. Conclusions: The findings indicated that hiPSC-CM robustly express Na v 1.5 channels which exhibited molecular and pharmacological properties similar to those in native cardiac tissues. Interestingly, neuronal Na v 1.7 channels were also expressed in hiPSC-CM and are expected to be responsible for the TTX-sensitive Na v current.

P2X receptors are ligand-gated ion channels which are activated by excitatory neurotransmitter ATP. Despite considerable evidence of signaling by extracellular nucleotides in other sensory systems, P2X receptors in the visual system have only rarely been studied, and almost nothing is known about their functional significance in the retina. To determine whether ATP plays a role in the modulation of vertical retinal signal pathways, we examined the expression of P2X receptor mRNA in freshly isolated bipolar cells of the rat retina Ž . Brown Norway, P25 using the single-cell RT-PCR technique. Positive amplification signals were found in about 33% of the bipolar cells for P2X , P2X and P2X but not for P2X mRNA. We conclude that at least a subpopulation of bipolar cells in the rat retina 3 4 5 7 expresses ionotropic P2 receptors of the P2X type and that these possibly exert a neuromodulatory influence on information processing in the retina.

P2X receptors are ligand-gated ion channels activated by adenosine triphosphate and expressed in a broad variety of tissues. The present study demonstrates the expression of various types of purinergic P2X receptors in identified retinal ganglion cells (RGCs) of the adult rat retina. Single-cell reverse transcription polymerase chain reaction (SC-RT-PCR) resulted in a positive amplification signal for all P2X receptor subunit mRNAs examined (P2X , P2X ). Immunohistochemistry with P2X receptor subunit-specific antibodies showed 3 -5 7 3,4 a labelling of neurons in the ganglion cell layer and inner nuclear layer. Our data suggest that extracellular ATP acts directly on RGCs via several types of P2X receptors and may provide neuromodulatory influences on information processing in the retina.

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Current approaches to monitor and quantify cell division in live cells, and reliably distinguish between acytokinesis and endoreduplication, are limited and complicate determination of stem cell pool identities. Here we overcome these limitations by generating an in vivo reporter system using the scaffolding protein anillin fused to enhanced green fluorescent protein, to provide high spatiotemporal resolution of mitotic phase. This approach visualizes cytokinesis and midbody formation as hallmarks of expansion of stem and somatic cells, and enables distinction from cell cycle variations. High-resolution microscopy in embryonic heart and brain tissues of enhanced green fluorescent protein-anillin transgenic mice allows live monitoring of cell division and quantitation of cell cycle kinetics. Analysis of cell division in hearts post injury shows that border zone cardiomyocytes in the infarct respond with increasing ploidy, but not cell division. Thus, the enhanced green fluorescent protein-anillin system enables monitoring and measurement of cell division in vivo and markedly simplifies in vitro analysis in fixed cells.

To resolve the metabolite redox cycling associated with our earlier clinical compound 2, we carried out lead optimization of lead molecule 1. Compound 4 showed improved lipophilic ligand efficiency and demonstrated robust glucose lowering in diet-induced obese mice without a liability in predictive preclinical drug safety studies. Thus, it was selected as a clinical candidate and further studied in type 2 diabetic patients. Clinical data suggests no evidence of metabolite cycling, which is consistent with the preclinical profiling of metabolism.

Inflammation and renal tubular injury are major features of acute kidney injury (AKI). Many cytokines and chemokines are released from injured tubular cells and acts as proinflammatory mediators. However, the role of IL-19 in the pathogenesis of AKI is not defined yet. In bilateral renal ischemia/reperfusion injury (IRI)-induced and HgCl 2 -induced AKI animal models, realtime quantitative (RTQ)-PCR showed that the kidneys, livers, and lungs of AKI mice expressed significantly higher IL-19 and its receptors than did sham control mice. Immunohistochemical staining showed that IL-19 and its receptors were strongly stained in the kidney, liver, and lung tissue of AKI mice. In vitro, IL-19 upregulated MCP-1, TGF-b1, and IL-19, and induced mitochondria-dependent apoptosis in murine renal tubular epithelial M-1 cells. IL-19 upregulated TNF-a and IL-10 in cultured HepG2 cells, and it increased IL-1b and TNF-a expression in cultured A549 cells. In vivo, after renal IRI or a nephrotoxic dose of HgCl 2 treatment, IL-20R1-deficient mice (the deficiency blocks IL-19 signaling) showed lower levels of blood urea nitrogen (BUN) in serum and less tubular damage than did wild-type mice. Therefore, we conclude that IL-19 mediates kidney, liver, and lung tissue damage in murine AKI and that blocking IL-19 signaling may provide a potent therapeutic strategy for treating AKI.

In this article, we have briefly reviewed the molecular mechanism involved in DNA damage and repair at various checkpoints of the cell cycle in eukaryotic cells. Eukaryotic cells have an ability to develop a response to DNA damage that can be caused by environmental factors such as chemicals, xenobiotics, free radicals, ionizing radiation (IR) or products of intracellular metabolism and also due to products of medical therapy. In response to these insults, the following reactions can take place in the cellular environment: (a) In the case of DNA damage, the protein machinery is activated and attaches itself to the site of the lesion which results in cell cycle arrest at the G1 to S phase (the G1/S checkpoint), DNA replication phase (the intra-S checkpoint), or G2 to mitosis phase (the G2/M checkpoint) until the lesion undergoes repair; (b) As there are many different lesions possible, the following DNA repair mechanisms are activated which include direct repair, base excision repair, nucleotide excision repair, mismatch repair, and double strand breaks (DSBs) such as, homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. In HR, the most important emerging proteins are the tumor suppressor proteins BRCA1, BRCA2, and Rad51 which play an important role in maintaining the genomic integrity by protecting cells from double strand breaks. Furthermore, interaction of Rad51 with BRCA2 protein complexes are essential for HR, which can be visualized by microscope as a foci and are thought to be representative sites where repair mechanism can take place. In addition, we have given special focus to the recent finding in the interaction of Rad51 with BRCA2 protein in double strand breaks by HR.

Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn can initiate neoplastic transformation of porcine ovarian putative stem cells (poPSCs). Immunomagnetically isolated poPSCs were expanded ex vivo in the presence of Ndn or Bdn, for 7 and 14 days. Results show that pharmacological doses of both Ndn and Bdn, already after 7 days of poPSCs culture, caused a significant increase of selected, stemness-related markers of cancer cells: CD44 and CD133. Notably, Ndn also negatively affected poPSCs growth not only by suppressing their proliferation and mitochondrial respiration but also by inducing apoptosis. This observation shows, for the first time, that chronic exposure to Ndn or Bdn represents a precondition that might enha...

We have recently shown that activating stimuli induce platelet microRNA modulation and proteome reorganization. However, how platelets modulate the resident mRNAs has to be elucidated. Among the possible mechanisms, mRNA splicing may be potentially relevant. Methods: Purified platelets from healthy volunteers were activated in vitro with collagen or Thrombin Receptor Activating Peptide. Transcriptome analysis by RNA-Seq and in silico intron retention analysis were applied to search for splicing events caused by activation. High-resolution isoelectric focusing liquid chromatography-mass spectrometry (HiRIEF LC-MS) methodology was employed to investigate proteome changes at deep coverage that were related to mRNA splicing events. Results: Extensive computational analysis following RNA-Seq revealed several splicing events occurring in activated platelets (Fig. and). By applying unbiased proteogenomics, we correlated intron retention events in resting platelets to exon-exon junctions frequency after activation (Fig. ), identifying a set of transcripts with reduced intron retention, indicating mRNA maturation, and high peptide representation at exon-exon junctions in activated vs resting platelets. These results indicate that pre-mRNA maturation of platelet-specific transcripts occurs during activation. This phenomenon may result in changes of platelet proteomics, ultimately leading to modulation of platelet function.

Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results

The ability of the zebrafish heart to regenerate following injury makes it a valuable model to deduce why this capability in mammals is limited to early neonatal stages. Although metabolic reprogramming and glycosylation remodeling have emerged as key aspects in many biological processes, how they may trigger a cardiac regenerative response in zebrafish is still a crucial question. Here, by using an up-to-date panel of transcriptomic, proteomic and glycomic approaches, we identify a metabolic switch from mitochondrial oxidative phosphorylation to glycolysis associated with membrane glycosylation remodeling during heart regeneration. Importantly, we establish the N- and O-linked glycan structural repertoire of the regenerating zebrafish heart, and link alterations in both sialylation and high mannose structures across the phases of regeneration. Our results show that metabolic reprogramming and glycan structural remodeling are potential drivers of tissue regeneration after cardiac in...

Aedes aegypti is the main vector for yellow fever, dengue, chikungunya and Zika viruses. Recent outbreaks of dengue and chikungunya have been reported in Kenya. Presence and abundance of this vector is associated with the risk for the occurrence and transmission of these diseases. This study aimed to characterize the presence and abundance of Ae. aegypti adult mosquitoes from rural and urban sites in western and coastal regions of Kenya. Presence and abundance of Ae. aegypti adult mosquitoes were determined indoors and outdoors in two western (urban Kisumu and rural Chulaimbo) and two coastal (urban Ukunda and rural Msambweni) sites in Kenya. Sampling was performed using quarterly human landing catches, monthly Prokopack automated aspirators and monthly Biogents-sentinel traps. A total of 2,229 adult Ae. aegypti mosquitoes were collected: 785 (35.2%) by human landing catches, 459 (20.6%) by Prokopack aspiration and 985 (44.2%) by Biogents-sentinel traps. About three times as many Ae. aegypti mosquitoes were collected in urban than rural sites (1,650 versus 579). Comparable numbers were collected in western (1,196) and coastal (1,033) sites. Over 80% were collected outdoors through human landing catches and Prokopack aspiration. The probability of collecting Ae. aegypti mosquitoes by human landing catches was significantly higher in the afternoon than morning hours (P<0.001), outdoors than indoors (P<0.001) and in urban than rural sites (P = 0.008). Significantly more Ae. aegypti mosquitoes were collected using Prokopack aspiration outdoors than indoors (P<0.001) and in urban than rural areas (P<0.001). Significantly more mosquitoes were collected using Biogents-sentinel traps in urban than rural areas (P = 0.008) and in western than coastal sites (P = 0.006). The probability of exposure to Ae. aegypti bites was highest in urban areas, outdoors and in the afternoon hours. These characteristics have major implications for the possible transmission of arboviral diseases and for the planning of surveillance and control programs.

We aimed to evaluate a computer-aided diagnosis (CADx) system for lung nodule classification focussing on (i) usefulness of the conventional CADx system (hand-crafted imaging feature + machine learning algorithm), (ii) comparison between support vector machine (SVM) and gradient tree boosting (XGBoost) as machine learning algorithms, and (iii) effectiveness of parameter optimization using Bayesian optimization and random search. Data on 99 lung nodules (62 lung cancers and 37 benign lung nodules) were included from public databases of CT images. A variant of the local binary pattern was used for calculating a feature vector. SVM or XGBoost was trained using the feature vector and its corresponding label. Tree Parzen Estimator (TPE) was used as Bayesian optimization for parameters of SVM and XGBoost. Random search was done for comparison with TPE. Leave-one-out cross-validation was used for optimizing and evaluating the performance of our CADx system. Performance was evaluated using area under the curve (AUC) of receiver operating characteristic analysis. AUC was calculated 10 times, and its average was obtained. The best averaged AUC of SVM and XGBoost was 0.850 and 0.896, respectively; both were obtained using TPE. XGBoost was generally superior to SVM. Optimal parameters for achieving high AUC were obtained with fewer numbers of trials when using TPE, compared with random search. Bayesian optimization of SVM and XGBoost parameters was more efficient than random search. Based on observer study, AUC values of two board-certified radiologists were 0.898 and 0.822. The results show that diagnostic accuracy of our CADx system was comparable to that of radiologists with respect to classifying lung nodules.

Pulmonary epithelial injury is central to the pathogenesis of many lung diseases, such as asthma, pulmonary fibrosis, and the acute respiratory distress syndrome. Regulated epithelial repair is crucial for lung homeostasis and prevents scar formation and inflammation that accompany dysregulated healing. The extracellular matrix (ECM) plays an important role in epithelial repair after injury. Vitronectin is a major ECM component that promotes epithelial repair. However, the factors that modify cell-vitronectin interactions after injury and help promote epithelial repair are not well studied. Inter-␣-trypsin inhibitor (IaI) is an abundant serum protein. IaI heavy chains contain von Willebrand A domains that can bind the arginine-glycine-aspartate domain of vitronectin. We therefore hypothesized that IaI can bind vitronectin and promote vitronectin-induced epithelial repair after injury. We show that IaI binds vitronectin at the arginine-glycine-aspartate site, thereby promoting epithelial adhesion and migration in vitro. Furthermore, we show that IaIdeficient mice have a dysregulated response to epithelial injury in vivo, consisting of decreased proliferation and epithelial metaplasia. We conclude that IaI interacts not only with hyaluronan, as previously reported, but also other ECM components like vitronectin and is an important regulator of cellular repair after injury. Epithelial injury is a crucial component in the pathogenesis of many lung diseases. Bronchial epithelial injury occurs chronically in asthmatic patients (1, 2). Furthermore, alveolar and bronchiolar epithelial injury are early triggers in idiopathic pulmonary fibrosis (3) and in lung transplant rejection (4), respectively. In acute respiratory distress syndrome, diffuse alveolar epithelial injury initiates the inflammatory and fibrotic response that leads to lung dysfunction (5). It is now believed that a dysregulated response to epithelial injury ultimately causes fibroproliferation, scar formation, and respiratory failure in acute as well as chronic lung injury (3). It is therefore important to understand the epithelial repair process after pulmonary epithelial injury, if we are to develop causal treatments for these diseases. The mechanisms governing epithelial repair are incompletely understood. Epithelial repair encompasses cell prolifer-ation, migration, and differentiation. All of these processes require cell interactions with the extracellular matrix (ECM). 2 ECM components like tenascin C, fibronectin, and vitronectin promote epithelial regeneration through integrin binding. Vitronectin (Vn) is a pluripotent 75-kDa plasma and ECM glycoprotein that regulates a number of biological processes such as coagulation, complement activation, and wound healing. Vn promotes cell adhesion and migration via binding primarily to integrins ␣ v ␤ 1 , ␣ v ␤ 3 , ␣ v ␤ 5 , and ␣ v ␤ 6 . After cell binding, Vn can protect bronchial epithelial cells from apoptosis (6) by inducing Akt phosphorylation and preventing caspase and Fas-associated with death domain (FADD) activation ( ). Vn also binds to non-integrin cell receptors such as urokinase-type plasminogen activator receptor to promote changes in cell morphology, migration, and signal transduction (8). Consequently, Vn deficiency impairs bronchial (6) and alveolar (9) epithelial repair. Cell-Vn interactions are modulated by other extracellular factors. For example, plasminogen activator inhibitor 1 (PAI-1) is bound to circulating Vn and forms multimers with Vn upon extravasation to the extracellular space (10), thus possibly activating Vn into an adhesive form (11). Furthermore, Vn possesses several domains that can function as possible ligands, such as a somatomedin B domain, an arginine-glycine-aspartate (RGD) domain, and a heparin-binding domain. However, the extent to which other serum or ECM factors may interact with Vn and influence cell-Vn interactions is unclear. In this report, we investigated possible interactions between Vn and the serum and ECM protein inter-␣-trypsin inhibitor. Inter-␣-trypsin inhibitor (IaI) is a complex protein found in relatively high concentrations in mammalian plasma. It is made up of a light chain (called bikunin for its two Kunitz domains), which confers the protease inhibitory activity, as well as two heavy chains (12). The precise functions of the heavy chains are unknown. Heavy chains contain a von Willebrand Type A (vWA) domain, and they have been shown to bind to hyaluronan and thereby stabilize the extracellular matrix. However, vWA domains are fairly promiscuous and can bind to a large array of proteins, including RGD domains. Furthermore, IaI is expressed by epithelial cells under stress conditions and is incorporated into de novo ECM structures produced by stressed epithelia (13). We therefore hypothesized that IaI may

After traumatic injury, healing of mammalian ligaments is typically associated with fibrotic scarring as opposed to scar-free regeneration. In contrast, here we show that the ligament supporting the jaw joint of adult zebrafish is capable of rapid and complete scar-free healing. Following surgical transection of the jaw joint ligament, we observe breakdown of ligament tissue adjacent to the cut sites, expansion of mesenchymal tissue within the wound site, and then remodeling of extracellular matrix (ECM) to a normal ligament morphology. Lineage tracing of mature ligamentocytes following transection shows that they dedifferentiate, undergo cell cycle re-entry, and contribute to the regenerated ligament. Single-cell RNA sequencing of the regenerating ligament reveals dynamic expression of ECM genes in neural-crest-derived mesenchymal cells, as well as diverse immune cells expressing the endopeptidase-encoding gene legumain. Analysis of legumain mutant zebrafish shows a requirement for...

Alteration to endoplasmic reticulum (ER) proteostasis is observed on a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR-target genes. Here, we designed an ATF6f-XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has stronger an effect in reducing the abnormal aggregation of mutant huntingtin and alpha-synuclein when compared to XBP1s or ATF6 alone. We developed a gene ...

Parkinson&#39;s disease (PD) compromises motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta. At the histopathological level, PD is characterized by the accumulation of Lewy bodies, large protein inclusions containing aggregated αSynuclein (αSyn). The progression of PD involves the spreading of αSyn misfolding through the brain mediated by a prion-like mechanism, where the protein is transferred between cells. Here we report that αSyn internalization is a dynamic process, where the protein transits through different sub-cellular compartments. Importantly, cells incorporating αSyn develop larger protein-like inclusions when compared to αSyn producing cells. We developed a new tool to monitor cell-to-cell transfer of αSyn in vivo using an adeno-associated viral (AAV) vector expressing αSyn fused to a red fluorescent protein in addition to soluble EGFP to label donor cells. Intra-nigral delivery of this reporter AAV construct allowed the visualiz...

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1 G93A (ACM-hSOD1 G93A ) quickly enhances Na v channelmediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1 G93A , but not ACM-hSOD1 WT , increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1 G93A -mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1 G86R or TDP43 A315T . We further find that co-application of ACM-SOD1 G93A with blockers of Na v channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1 G93A induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyperexcitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death.

In the cochlea, Reissner&#39;s membrane separates the scala media endolymphatic compartment that sustains the positive endocochlear potential and ion composition necessary for sound transduction, from the scala vestibuli perilymphatic compartment. It is known that with sustained elevated sound levels, adenosine 5&#39;-triphosphate (ATP) is released into the endolymph and ATP-gated ion channels on the epithelial cells lining the endolymphatic compartment shunt the electrochemical driving force, contributing to protective purinergic hearing adaptation. This study characterises the properties of epithelial cell P2X2-type ATP-activated membrane conductance in the mouse Reissner&#39;s membrane, which forms a substantial fraction of the scale media surface. The cells were found to express two isoforms (a and b) of the P2X2 subunit arising from alternative splicing of the messenger RNA (mRNA) transcript that could contribute to the trimeric subunit assembly. The ATP-activated conductance d...

Peripherin, a type III intermediate filament protein, forms part of the cytoskeleton in a subset of neurons, most of which have peripheral fibre projections. Studies suggest a role for peripherin in axon outgrowth and regeneration, but evidence for this in sensory and brain tissues is limited. The exclusive expression of peripherin in a sub-population of primary auditory neurons, the type II spiral ganglion neurons (SGN) prompted our investigation of the effect of peripherin gene deletion (pphKO) on these neurons. We used confocal immunofluorescence to examine the establishment of the innervation of the cochlear outer hair cells by the type II SGN neurites in vivo and in vitro, in wildtype (WT) and pphKO mice, in the first postnatal week. The distribution of the type II SGN nerve fibres was normal in pphKO cochleae. However, using P1 spiral ganglion explants under culture conditions where the majority of neurites were derived from type II SGN, pphKO resulted in increased numbers of neurites/explant compared WT controls. Type II SGN neurites from pphKO explants extended ~ double the distance of WT neurites, and had reduced complexity based on greater distance between turning points. Addition of brain-derived neurotrophic factor (BDNF) to the culture media increased neurite number in WT and KO explants ~30-fold, but did not affect neurite length or distance between turning. These results indicate that peripherin may interact with other cytoskeletal elements to regulate outgrowth of the peripheral neurites of type II SGN, distinguishing these neurons from the type I SGN innervating the inner hair cells.

L1, a neural cell adhesion molecule of the immunoglobulin superfamily, is widely expressed in the nervous system and important in axonal outgrowth, guidance, synapse formation, and signaling. Gene deletion studies emphasize the significance of L1 during development of the central nervous system and L1 is crucial for the topographic targeting of retinal axons. In contrast to the brain and retina, the role of L1 in the inner ear is largely unknown. While previous studies have localized L1 in the developing inner ear of the chicken and mouse, its function during the innervation of the cochlea still remains largely unclear. We therefore investigated the functional role of L1 in the mammalian inner ear. Our aim was to determine whether or not L1 can modulate type I and/or type II spiral ganglion neuron outgrowth using an in vitro alternate choice assay. We found that L1, presented in stripe micropatterns, provide directional cues to neonatal rodent type I but not type II inner ear spiral ganglion neurites. The results suggest that L1 may play a role in axonal pathfinding of type I spiral ganglion dendrites toward their inner hair cell targets, but not of type II toward the outer hair cells.

Introduction-Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na + /H + exchange and H 2 O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously. Methods-We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport. Results-piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport. Conclusions-Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

The definitive treatment for end-stage renal disease is kidney transplantation, which remains limited by organ availability and post-transplant complications. Alternatively, an implantable bioartificial kidney could address both problems while enhancing the quality and length of patient life. An implantable bioartificial kidney requires a bioreactor containing renal cells to replicate key native cell functions, such as water and solute reabsorption, and metabolic and endocrinologic functions. Here, we report a proof-of-concept implantable bioreactor containing silicon nanopore membranes to offer a level of immunoprotection to human renal epithelial cells. After implantation into pigs without systemic anticoagulation or immunosuppression therapy for 7 days, we show that cells maintain &gt;90% viability and functionality, with normal or elevated transporter gene expression and vitamin D activation. Despite implantation into a xenograft model, we find that cells exhibit minimal damage,...

Hemodialysis using hollow-fiber membranes provides life-sustaining treatment for nearly 2 million patients worldwide with end stage renal disease (ESRD). However, patients on hemodialysis have worse long-term outcomes compared to kidney transplant or other chronic illnesses. Additionally, the underlying membrane technology of polymer hollow-fiber membranes has not fundamentally changed in over four decades. Therefore, we have proposed a fundamentally different approach using microelectromechanical systems (MEMS) fabrication techniques to create thin-flat sheets of silicon-based membranes for implantable or portable hemodialysis applications. The silicon nanopore membranes (SNM) have biomimetic slit-pore geometry and uniform pores size distribution that allow for exceptional permeability and selectivity. A quantitative diffusion model identified structural limits to diffusive solute transport and motivated a new microfabrication technique to create SNM with enhanced diffusive transpo...

Critical failures associated with current engineered bone grafts involve insufficient induction of osteogenesis of the implanted cells and lack of vascular integration between graft scaffold and host tissue. This study investigated the combined effects of surface microtextures and biochemical supplements to achieve osteogenic differentiation of human mesenchymal stem cells (hMSCs) and revascularization of the implants in vivo. Cells were cultured on 10μm micropost-textured polydimethylsiloxane (PDMS) substrates in either proliferative basal medium (BM) or osteogenic medium (OM). In vitro data revealed that cells on microtextured substrates in OM had dense coverage of extracellular matrix, whereas cells in BM displayed more cell spreading and branching. Cells on microtextured substrates in OM demonstrated a higher gene expression of osteoblast-specific markers, namely collagen I, alkaline phosphatase, bone sialoprotein, and osteocalcin, accompanied by substantial amount of bone matri...

The role electrical charge plays in determining glomerular permeability to macromolecules remains unclear. If the glomerular basement membrane (GBM) has any significant role in permselectivity, physical principles would suggest a negatively charged GBM would reject similarly charged more than neutral species. However, recent in vivo studies with negative and neutral glomerular probes showed the opposite. Whether this observation is due to unique characteristics of the probes used or is a general physiological phenomenon remains to be seen. The goal of this study was to use the basement membrane deposited by Madin-Darby canine kidney epithelial cells as a simple model of a biologically derived, negatively charged filter to evaluate size- and charge-based sieving properties. Fluorescein isothiocyanate-labeled carboxymethylated Ficoll 400 (FITC-CM Ficoll 400) and amino-4-methyl-coumarin-labeled Ficoll 400 (AMC Ficoll 400) were used as negatively charged and neutral tracer molecules, re...

Growth factor binding to transmembrane protein receptors is generally understood to initiate cell signaling. Receptor binding of heparin-binding growth factors (HB-GFs), such as fibroblast growth factor-2 (FGF-2), is regulated by interactions with heparan sulfate proteoglycans. While there is some specificity for binding to heparan sulfate, overlap in sites for different growth factors may allow for cross regulation. Here we demonstrate, using experiments and computer simulations, that the HB-GFs FGF-2 and heparin-binding EGF-like growth factor (HB-EGF) can cross regulate receptor binding of the other despite having unique receptors. The ability of HSPG to stabilize HB-GF receptor binding is critical for competing growth factors to modulate receptor binding with both enhanced and reduced binding possible depending on this stabilization process. HSPG density and affinity for HB-GF are also critical factors for HB-GF cross regulation. Simulations further reveal that HB-GF can regulate receptor binding of non-HB-GFs such as EGF even when the two proteins share no binding sites when other HB-GF are present within the network. Proliferation studies demonstrate potentiation of HB-EGF-induced growth by FGF-2 indicating that competition networks can alter biological response. Exogenous manipulation of cellular responses to growth factors in complex living systems will require understanding the HSPG-controlled network.

Although it is well recognized that cell-matrix interactions are based on both molecular and geometrical characteristics, the relationship between specific cell types and the three-dimensional morphology of the surface to which they are attached is poorly understood. This is particularly true for glomerular podocytes -the gatekeepers of glomerular filtrationwhich completely enwrap the glomerular basement membrane with their primary and secondary ramifications. Nanotechnologies produce biocompatible materials which offer the possibility to build substrates which differ only by topology in order to mimic the spatial organization of diverse basement membranes. With this in mind, we produced and utilized rough and porous surfaces obtained from silicon to analyze the behavior of two diverse ramified cells: glomerular podocytes and a neuronal cell line used as a control. Proper differentiation and development of ramifications of both cell types was largely influenced by topographical characteristics. Confirming previous data, the neuronal cell line acquired features of maturation on rough nanosurfaces. In contrast, podocytes developed and matured preferentially on nanoporous surfaces provided with grooves, as shown by the organization of the actin cytoskeleton stress fibers and the proper development of vinculin-positive focal adhesions. On the basis of these findings, we suggest that in vitro studies regarding podocyte attachment to the glomerular basement membrane should take into account the geometrical properties of the surface on which the tests are conducted because physiological cellular activity depends on the three-dimensional microenvironment.

Overexpression of PMP22 is responsible for the most common form of inherited neuropathy, Charcot-Marie-Tooth disease (CMT) type 1A. The PMP22-transgenic rat (CMT rat) is an animal model of CMT1A, and its peripheral nerves show the characteristic features of ongoing demyelination and remyelination that is also seen in CMT1A patients. Since Schwann cell proliferation is a prominent feature of peripheral nerves in inherited peripheral neuropathies, we examined proliferation and the expression of cyclin D1 in CMT rats. D-type cyclins are required for the initial steps in cell division and nuclear import is crucial for the function of cyclin D1 in promoting cell proliferation. Like normal myelinating Schwann cells in wild-type rats, remyelinating Schwann cells in CMT rats show perinuclear cyclin D1 expression. Schwann cells with nuclear cyclin D1 expression, as well as proliferating Schwann cells, were both associated with demyelinated axonal segments. Supernumerary onion bulb Schwann cells, however, do not express cyclin D1 and were not proliferating. Thus, cyclin D1 expression and its subcellular localization correlate directly with distinct physiological states of Schwann cells in this animal model of CMT1A.

Neurons regulate Schwann cell proliferation, but little is known about the molecular basis of this interaction. We have examined the possibility that cyclin D1 is a key regulator of the cell cycle in Schwann cells. Myelinating Schwann cells express cyclin D1 in the perinuclear region, but after axons are severed, cyclin D1 is strongly upregulated in parallel with Schwann cell proliferation and translocates into Schwann cell nuclei. During development, cyclin D1 expression is confined to the perinuclear region of proliferating Schwann cells and the analysis of cyclin D1-null mice indicates that cyclin D1 is not required for this type of Schwann cell proliferation. As in the adult, injury to immature peripheral nerves leads to translocation of cyclin D1 to Schwann cell nuclei and injury-induced proliferation is impaired in both immature and mature cyclin D1-deficient Schwann cells. Thus, our data indicate that the molecular mechanisms regulating proliferation of Schwann cells during development or activated by axonal damage are fundamentally different.

Inducible transgenesis provides a valuable technique for the analysis of gene function in vivo. We report the generation and characterization of mouse lines carrying glia lineage-specific transgenes expressing an improved variant of the tamoxifen-inducible Cre recombinase, CreERT2, where the recombinase is fused to a mutated ligand binding domain of the human estrogen receptor. Using a PLP-CreERT2 transgene, we have generated mice that show specific inducible Cre function, as analyzed by cross-breeding experiments into the Rosa26 Cre-LacZ reporter line, in developing and adult Schwann cells, in mature myelinating oligodendrocytes, and in undifferentiated NG2-positive oligodendrocyte precursors in the adult. Using a P0Cx-CreERT2 transgene, we have also established mouse lines with inducible Cre function specifically in the Schwann cell lineage. These tamoxifen-inducible CreERT2 lines will allow detailed spatiotemporally controlled analysis of gene functions in loxP-based conditional mutant mice in both developing and adult Schwann cells and in the oligodendrocyte lineage.

Recently, we demonstrated that the microRNA 424(322)/503 [miR-424(322)/503] cluster is transcriptionally controlled by transforming growth factor β (TGF-β) in the mammary epithelium. Induction of this microRNA cluster impacts mammary epithelium fate by regulating apoptosis and insulin-like growth factor 1 (IGF1) signaling. Here, we expanded our finding to demonstrate that miR-424(322)/503 is an integral component of the cell cycle arrest mediated by TGF-β. Mechanistically, we showed that after TGF-β exposure, increased levels of miR-424(322)/503 reduce the expression of the cell cycle regulator CDC25A. miR-424(322)/503-dependent posttranscriptional downregulation of CDC25A cooperates with previously described transcriptional repression of the CDC25A promoter and proteasome-mediated degradation to reduce the levels of CDC25A expression and to induce cell cycle arrest. We also provide evidence that the TGF-β/miR-424(322)/503 axis is part of the mechanism that regulates the proliferati...

The mammary gland is a very dynamic organ that undergoes continuous remodeling. The critical regulators of this process are not fully understood. Here we identify the microRNA cluster miR-424(322)/503 as an important regulator of epithelial involution after pregnancy. Through the generation of a knockout mouse model, we found that regression of the secretory acini of the mammary gland was compromised in the absence of miR-424(322)/503. Mechanistically, we show that miR-424(322)/503 orchestrates cell life and death decisions by targeting BCL-2 and IGF1R (insulin growth factor-1 receptor). Furthermore, we demonstrate that the expression of this microRNA cluster is regulated by TGF-β, a well-characterized regulator of mammary involution. Overall, our data suggest a model in which activation of the TGF-β pathway after weaning induces the transcription of miR-424(322)/503, which in turn down-regulates the expression of key genes. Here, we unveil a previously unknown, multilayered regulat...

The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. Here we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments, reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, DacyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress (H 2 O 2 ), but no change in heavy metal resistance or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group.

Cellular adhesion plays an important role in numerous fundamental physiological and pathological processes. Its measurement is relatively complex, requires sophisticated equipment, and, in most cases, cannot be carried out without breaking the links between the studied cell and its target. In this contribution, we propose a novel, nanomotion-based, technique that overcomes these drawbacks. The applied force is generated by the studied cell itself (nanomotion), whereas cellular movements are detected by traditional optical microscopy and dedicated software. The measurement is non-destructive, single-cell sensitive, and permits following the evolution of the adhesion as a function of time. We applied the technique on different strains of the fungal pathogen Candida albicans on a fibronectin-coated surface. We demonstrated that this novel approach can significantly simplify, accelerate, and make more affordable living cells–substrate adhesion measurements.

Wnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica TEM to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D-SIM imaging of isolated egg cortices demonstrated the concentration gradient-like distribution of Dsh in the VCD, whereas higher resolution STED imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Ds...

SUMMARYThe organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes [1]. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled is a cytoplasmic Wnt pathway effector required for axis development in diverse species [2-4], but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dishevelled localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes, but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-condensation mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome asso...

The activation of Wnt/β-catenin (cWnt) signaling at the future posterior end of early embryos is a highly conserved mechanism for initiating pattern formation along the anterior-posterior (AP) axis in bilaterians. Moreover, in many bilaterian taxa, in addition, to activation of cWnt signaling at the posterior end, inhibition of cWnt signaling at the anterior end is required for normal development of anterior structures. In most cases, inhibition of cWnt signaling at the anterior end occurs around gastrulation and it is typically mediated by secreted factors that block signal transduction through the cWnt cell surface receptor-ligand complex. This phenomenon has been fairly well characterized, but the emerging role for intracellular inhibition of cWnt signaling in future anterior blastomeres—in cleavage stage embryos—to regulate correct AP patterning is less well understood. To investigate this process in an invertebrate deuterostome embryo we studied the function of Axin, a critical...

The ß-catenin protein has two major known functions in animal cells. It keeps epithelial tissue homeostasis by its connection with Adherens Junctions (AJ), and it serves as a transcriptional cofactor along with Lef/Tcf to enter the nucleus and regulate target genes of the Wnt/ß-catenin (cWnt) signaling pathway. To assess the ancestral role of ß-catenin during development we examined its distribution and function in the ctenophoreMnemiopsis leidyi(one of the earliest branching animal phyla) by using ctenophore-specific antibodies and mRNA injection. We found that ß-catenin protein never localizes to cell-cell contacts during embryogenesis as it does in other metazoans, most likely because ctenophore-cadherins do not have the cytoplasmic domain required for interaction with the catenin proteins. Downregulation of zygoticMlß-catenin signaling led to the loss of endodermal and mesodermal tissues indicating that nuclear ß-catenin may have a deep role in germ-layer evolution. Our results ...

Pattern formation along the animal-vegetal (AV) axis in sea urchin embryos is initiated when canonical Wnt (cWnt) signaling is activated in vegetal blastomeres. The mechanisms that restrict cWnt signaling to vegetal blastomeres are not well understood, but there is increasing evidence that the egg's vegetal cortex plays a critical role in this process by mediating localized "activation" of Disheveled (Dsh). To investigate how Dsh activity is regulated along the AV axis, sea urchin-specific Dsh antibodies were used to examine expression, subcellular localization, and posttranslational modification of Dsh during development. Dsh is broadly expressed during early sea urchin development, but immunolocalization studies revealed that this protein is enriched in a punctate pattern in a novel vegetal cortical domain (VCD) in the egg. Vegetal blastomeres inherit this VCD during embryogenesis, and at the 60-cell stage Dsh puncta are seen in all cells that display nuclear β-catenin. Analysis of Dsh post-translational modification using twodimensional Western blot analysis revealed that compared to Dsh pools in the bulk cytoplasm, this protein is differentially modified in the VCD and in the 16-cell stage micromeres that partially inherit this domain. Dsh localization to the VCD is not directly affected by disruption of microfilaments and microtubules, but unexpectedly, microfilament disruption led to degradation of all the Dsh pools in unfertilized eggs over a period of incubation suggesting that microfilament integrity is required for maintaining Dsh stability. These results demonstrate that a pool of differentially modified Dsh in the VCD is selectively inherited by the vegetal blastomeres that activate cWnt signaling in early embryos, and suggests that this domain functions as a scaffold for localized Dsh activation. Localized cWnt activation regulates AV axis patterning in many metazoan embryos. Hence, it is possible that the VCD is an evolutionarily conserved cytoarchitectural domain that specifies the AV axis in metazoan ova.

All endodermal and mesenchymal cells of the sea urchin embryo descend from the vegetal plate, a thickened epithelium of approximately 50 cells arising at the early blastula stage. Cell types that derive from the vegetal plate are specified conditionally by inductive interactions with underlying micromeres, but the molecular details of vegetal-plate specification remain unresolved. In a search for regulatory proteins that have roles in vegetal-plate specification, a screen was performed to clone Kriippel/Krox-related genes from a Strongylocentrotus purpuratus embryo eDNA library. One newly identified clone, named SpKroxl, contained four zinc fingers and a leucine zipper domain. SpKroxl expression was low in unfertilized eggs, increased severalfold to the early blastula stage and decreased between the early gastmla and pluteus stages. SpKroxl mRNA was first seen in maeromeres of 16-cell stage embryos and was restricted to cells of the developing vegetal plate thereafter. Vegetal-plate expression corresponded to a ring of cells around the blastopore and overlapped the expression patterns of other genes with potential roles in vegetal plate-specification. As the vegetal-plate ceils invaginated into the blastopore, SpKroxl expression was lost, suggesting that its role was not in endoderm differentiation per se but rather in the initial establishment of the vegetal plate.

At the 16-cell stage, the sea urchin embryo is partitioned along the animal-veg-eta1 axis into eight mesomeres, four macromeres, and four micromeres. The micromeres, unlike the other blastomeres, are autonomously specified to produce skeletogenic mesenchymal cells and are also required to induce the vegetal-plate territory. A long-held belief is that micromeres inherit localized maternal determinants that endow them with their cell autonomous behavior and inducing capabilities, Here, we present evidence that an orthodenticle-related protein, SpOtx appears transiently in nuclei of micromeres but not in nuclei of mesomeres and macromeres. At later stages of development, SpOtx was translocated into nuclei of all cells. To address the possibility that SpOtx was retained in the cytoplasm at early developmental stages, we searched for cytoplasmic proteins that interact with SpOtx. A proline-rich region of SpOtx resembling an SH3-binding domain was used to screen an embryo cDNA expression library, and a cDNA clone was isolated and shown to be a-actinin. A yeast two-hybrid analysis showed a specific interaction between the prolinerich region of SpOtx and a putative SH3 domain of the sea urchin a-actinin. Because micromeres lack an actin-based cytoskeleton, the results suggested that, at the vegetal pole of the 16-cell stage embryo, SpOtx was translocated into micromere nuclei, whereas in other blastomeres SpOtx was actively retained in the cytoplasm by binding to a-actinin. The transient appearance of SpOtx in micromere nuclei may be associated with the specification of micromere cell fate.