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Cancer stem‐like cells (CSCs) induce drug resistance and recurrence of tumors when they experience DNA replication stress. However, the mechanisms underlying DNA replication stress in CSCs and its compensation remain unclear. Here, we demonstrate that upregulated c‐Myc expression induces stronger DNA replication stress in patient‐derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini‐chromosome maintenance protein 10 (MCM10), a firing (activating) factor of DNA replication origins, to compensate for DNA replication stress in CSCs. MCM10 expression is upregulated in CSCs and is maintained by c‐Myc. c‐Myc‐dependent collisions between RNA transcription and DNA replication machinery may occur in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to these collisions to ensure the progression of replication. Moreover, patient‐derived breast CSCs were found to be dependent on MCM10 for their maintenance, even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. Further, MCM10 depletion decreased the growth of cancer cells, but not of normal cells. Therefore, MCM10 may robustly compensate for DNA replication stress and facilitate genome duplication in cancer cells in the S‐phase, which is more pronounced in CSCs. Overall, we provide a preclinical rationale to target the c‐Myc‐MCM10 axis for preventing drug resistance and recurrence of tumors., We provide evidence that upregulated c‐Myc expression induces stronger DNA replication stress in patient‐derived breast cancer stem‐like cells than in differentiated cancer cells. Our results suggest critical roles for mini‐chromosome maintenance protein 10 (MCM10), which is a firing (activating) factor of the DNA replication origins, to compensate for the DNA replication stress.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) are effective in patients with non–small‐cell lung cancer (NSCLC) harboring EGFR mutations. However, due to acquired resistance to EGFR‐TKIs, even patients on third‐generation osimertinib have a poor prognosis. Resistance mechanisms are still not fully understood. Here, we demonstrate that the increased expression of MUSASHI‐2 (MSI2), an RNA‐binding protein, is a novel mechanism for resistance to EGFR‐TKIs. We found that after a long‐term exposure to gefitinib, the first‐generation EGFR‐TKI lung cancer cells harboring the EGFR‐TKI‐sensitive mutations became resistant to both gefitinib and osimertinib. Although other mutations in EGFR were not found, expression levels of Nanog, a stemness core protein, and activities of aldehyde dehydrogenase (ALDH) were increased, suggesting that cancer stem‐like properties were increased. Transcriptome analysis revealed that MSI2 was among the stemness‐related genes highly upregulated in EGFR‐TKI‐resistant cells. Knockdown of MSI2 reduced cancer stem‐like properties, including the expression levels of Nanog, a core stemness factor. We demonstrated that knockdown of MSI2 restored sensitivity to osimertinib or gefitinib in EGFR‐TKI‐resistant cells to levels similar to those of parental cells in vitro. An RNA immunoprecipitation (RIP) assay revealed that antibodies against MSI2 were bound to Nanog mRNA, suggesting that MSI2 increases Nanog expression by binding to Nanog mRNA. Moreover, overexpression of MSI2 or Nanog conferred resistance to osimertinib or gefitinib in parental cells. Finally, MSI2 knockdown greatly increased the sensitivity to osimertinib in vivo. Collectively, our findings provide proof of principle that targeting the MSI2‐Nanog axis in combination with EGFR‐TKIs would effectively prevent the emergence of acquired resistance., Osimertinib (AZD9291) is a third‐generation irreversible, oral EGFR‐TKI that is effective without serious adverse effects, however, similar to earlier generations of EGFR‐TKIs, acquired resistance to osimertinib remains a major challenge. We discovered MSI2 as a novel target to overcome resistance to EGFR‐TKIs including osimertinib. Mechanistically, we demonstrate that MSI2 confers cancer stem‐like properties in lung adenocarcinoma cells, leading to resistance to the EGFR‐TKIs.

Rodent models mimic the heterogeneity of head and neck cancer (HNC) malignancies and are used to investigate HNC-associated biomarkers and evaluate drug responses. To assess the utility of patient-derived xenografts (PDXs) as an HNC model, 18 tumour samples were obtained from surgical specimens of patients with HNC and implanted into non-obese diabetic severe combined immunodeficient mice. The histological features of PDXs and corresponding patient samples were compared. Furthermore, the present study investigated how PDX responses to anticancer drugs mimic patient clinical responses, as well as the expression of adenosine triphosphate-binding cassette transporters through chemotherapy in an HNC-PDX model. A total of five PDXs from patients with HNC exhibiting high correspondence with histopathological features of the original patient samples were established (establishment rate, 28%). The responses of three PDXs to cisplatin were associated with clinical responses of the patients. ABC transporter expression was augmented in one PDX model after anticancer drug treatment, but not in PBS-treated passaged PDXs. PDX models exhibited similar biological and chemosensitive characteristics to those of the primary tumours. PDXs could be a useful preclinical tool to test novel therapeutic agents and identify novel targets and biomarkers in HNC.

Cancer stem-like cells (CSCs) are responsible for the drug resistance of tumors and recurrence while they experience DNA replication stress. However, the underlying mechanisms that cause DNA replication stress in CSCs and how they compensate for this stress remain unclear. Here we provide evidence that upregulated c-Myc expression induces stronger DNA replication stress in patient-derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini-chromosome maintenance protein 10 (MCM10), which is a firing (activating) factor of the DNA replication origins, to compensate for the DNA replication stress. Expression levels of MCM10 are upregulated in CSCs and maintained by c-Myc. c-Myc-dependent collisions may take place between RNA transcription and DNA replication machinery in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to the collisions to ensure replication progression. Moreover, patient-derived breast CSCs were dependent on MCM10 for their maintenance even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. In addition, MCM10 depletion decreased the growth of cancer cells but not normal cells. Therefore, MCM10 is likely to robustly compensate for DNA replication stress and facilitate genome duplication in the S-phase in cancer cells, which is more pronounced in CSCs. We provide a preclinical rationale to target the c-Myc-MCM10 axis to prevent drug resistance and recurrence.

Triple negative breast cancer (TNBC) is currently the only major breast tumor subtype without effective targeted therapy and, as a consequence, usually presents a poor outcome. Due to its more aggressive phenotype, there is an urgent clinical need to identify novel biomarkers that discriminate individuals with poor prognosis. We hypothesize that miRNAs can be used to this end because they are involved in the initiation and progression of tumors by altering the expression of their target genes. To identify a prognostic biomarker in TNBC, we analyzed the miRNA expression of a cohort composed of 185 patients diagnosed with TNBC using penalized Cox regression models. We identified a four-biomarker signature based on miR-221, miR-1305, miR-4708, and RMDN2 expression levels that allowed for the subdivision of TNBC into high- or low-risk groups (Hazard Ratio – HR = 0.32; 95% Confidence Interval - CI = 0.11–0.91; p = 0.03) and are also statistically associated with survival outcome in subgroups of postmenopausal status (HR = 0.19; 95% CI = 0.04–0.90; p= 0.016), node negative status (HR = 0.12; 95% CI = 0.01–1.04; p = 0.026), and tumors larger than 2cm (HR = 0.21; 95% CI = 0.05–0.81; p = 0.021). This four-biomarker signature was significantly associated with TNBC as an independent prognostic factor for survival.

The CD74-Neuregulin1 (NRG1) fusion gene was recently identified as novel driver of invasive mucinous adenocarcinoma, a malignant form of lung cancer. However, the function of the CD74-NRG1 fusion gene in adenocarcinoma pathogenesis and the mechanisms by which it may impart protumorigenic characteristics to cancer stem cells (CSC) is still unclear. In this study, we found that the expression of the CD74-NRG1 fusion gene increased the population of lung cancer cells with CSC-like properties. CD74-NRG1 expression facilitated sphere formation not only of cancer cells, but also of nonmalignant lung epithelial cells. Using a limiting dilution assay in a xenograft model, we further show that the CD74-NRG1 fusion gene enhanced tumor initiation. Mechanistically, we found that CD74-NRG1 expression promoted the phosphorylation of ErbB2/3 and activated the PI3K/Akt/NF-κB signaling pathway. Furthermore, the expression of the secreted insulin-like growth factor 2 (IGF2) and phosphorylation of its receptor, IGF1R, were enhanced in an NF-κB–dependent manner in cells expressing CD74-NRG1. These findings suggest that CD74-NRG1–induced NF-κB activity promotes the IGF2 autocrine/paracrine circuit. Moreover, inhibition of ErbB2, PI3K, NF-κB, or IGF2 suppressed CD74-NRG1–induced tumor sphere formation. Therefore, our study provides a preclinical rationale for developing treatment approaches based on these identified pathways to suppress CSC properties that promote tumor progression and recurrence. Cancer Res; 76(4); 974–83. ©2016 AACR.

Axon paths in developing spinal cords Sensory neurons entering the spinal cord take different paths as inputs for pain and proprioception diverge. Working with chick and mouse embryos, Guy et al. found that glycerophospholipids produced by radial glial cells guide these neural fibers, or axons, in the developing spinal cord. A soluble glycerophospholipid released by the cells provided an inhibitory signal to the pain-sensitive axons, keeping them on their own unique pathway. Science , this issue p. 974

The epidermal growth factor receptor (EGFR) and its family members are involved in many aspects of tumor biological processes. Aberrant activation of the EGFR tyrosine kinase by mutations or protein overexpression is observed in various types of human cancer, including lung cancer. EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, are highly effective in lung cancer patients who harbor active mutations in the EGFR gene. However, patients who are initially sensitive to EGFR-TKIs eventually relapse within few years.Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and is associated with a high frequency of EGFR mutations. This review describes the EGFR mutations that determine the sensitivity to EGFR-TKIs and the current understanding of the molecular mechanisms of acquired resistance to EGFR-TKIs in NSCLC. Furthermore, the authors describe recent strategies developed to overcome acquired resistance using second-generation EGFR-TKIs and combination therapies with several molecular-targeting drugs.Although recent findings have contributed to our understanding of the mechanism of acquired resistance and helped the development of novel strategies to overcome such resistance, the underlying mechanisms are complex and additional research is necessary to develop effective therapeutic strategies for individual patients with lung cancer.

Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)—the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in gefitinib-resistant cancer cells. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence. Citation Format: Noriko Gotoh, Tatsunori Nishimura, Asuka Nakata, Shin-ichi Horike, Susumu Kohno, Chiaki Takahashi, Tomoyoshi Soga, Arinobu Tojo. Cancer stem-like properties and drug resistance are dependent on purine synthetic metabolism mediated by the mitochondrial enzyme MTHFD2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 168.

PtdIns(3, 4, 5)P(3)-dependent Rac exchanger (P-Rex) 1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac. P-Rex1 is activated by G protein betagamma subunits (Gbetagamma), and the Gbetagamma-induced activation is inhibited by cAMP-dependent protein kinase A (PKA). However, the details of regulatory mechanism of P-Rex1 remain to be clarified. In the present study, we investigated the mechanism of activation and inhibition of P-Rex1 using various truncated and alanine-substituted mutants and found that the domain-domain interaction of P-Rex1 is important for Gbetagamma-induced activation and PKA-induced inhibition. Immunoprecipitation analysis showed that the second Disheveled/EGL-10/Pleckstrin (DEP) and first PSD-95/Dlg/ZO-1 (PDZ) domains of P-Rex1 associate with the inositol polyphosphate-4-phosphatase (IP4P) domain. Carboxyl-terminal truncation on the IP4P domain or mutations in the protein-binding pocket of the first PDZ domain abolished the association. Analysis of in vitro guanine nucleotide exchange assay, PAK1/2 phosphorylation, and Rac-specific actin reorganization revealed that Gbetagamma could activate a complex of the P-Rex1 mutant lacking the IP4P domain and the isolated IP4P domain as well as full-length P-Rex1. Moreover, PKA phosphorylation prevented the domain-domain interaction and Gbetagamma-binding. These results provide a new insight into the regulation of other Rho-family GEFs and cell responses induced by the heterotrimeric G protein.

Glycerophospholipids, the structural components of cell membranes, have not been considered to be spatial cues for intercellular signaling because of their ubiquitous distribution. We identified lyso-phosphatidyl-β-D-glucoside (LysoPtdGlc), a hydrophilic glycerophospholipid, and demonstrated its role in modality-specific repulsive guidance of spinal cord sensory axons. LysoPtdGlc is locally synthesized and released by radial glia in a patterned spatial distribution to regulate the targeting of nociceptive but not proprioceptive central axon projections. Library screening identified the G protein-coupled receptor GPR55 as a high-affinity receptor for LysoPtdGlc, and GPR55 deletion or LysoPtdGlc loss of function in vivo caused the misallocation of nociceptive axons into proprioceptive zones. These findings show that LysoPtdGlc/GPR55 is a lipid-based signaling system in glia-neuron communication for neural development.

There is a high death rate of lung cancer patients. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in some lung adenocarcinoma patients with EGFR mutations. However, a significant number of patients show primary and acquire resistance to EGFR-TKIs. Although the Akt kinase is commonly activated due to various resistance mechanisms, the key targets of Akt remain unclear. Here, we show that the Akt-β-catenin pathway may be a common resistance mechanism. We analyzed gene expression profiles of gefitinib-resistant PC9M2 cells that were derived from gefitinib-sensitive lung cancer PC9 cells and do not have known resistance mechanisms including EGFR mutation T790M. We found increased expression of Axin, a β-catenin target gene, increased phosphorylation of Akt and GSK3, accumulation of β-catenin in the cytoplasm/nucleus in PC9M2 cells. Both knockdown of β-catenin and treatment with a β-catenin inhibitor at least partially restored gefitinib sensitivity to PC9M2 cells. Lung adenocarcinoma tissues derived from gefitinib-resistant patients displayed a tendency to accumulate β-catenin in the cytoplasm. We provide a rationale for combination therapy that includes targeting of the Akt-β-catenin pathway to improve the efficacy of EGFR-TKIs.

The aim of this study was to identify differentially-expressed miRNAs in the serum of non-small cell lung cancer (NSCLC) patients that might be a clinically-useful tool for lung cancer early detection. We performed miRNA expression profile analysis using TaqMan OpenArray Human panel in a discovery set of 70 serum samples obtained at lung tumor resection and 22 non-cancer subjects (NC). Selected serum miRNAs were then validated by quantitative PCR using an independent validation set of serum samples from LC patients (n = 84) and NC (n = 23). Sixty miRNAs were significantly up-regulated and 31 were down-regulated in the serum from NSCLC patients versus NC (adjusted p p p

PURPOSE: To identify stage I lung adenocarcinoma patients with a poor prognosis who will benefit from adjuvant therapy. PATIENTS AND METHODS: Whole gene expression profiles were obtained at 19 time points over a 48-hour time course from human primary lung epithelial cells that were stimulated with epidermal growth factor (EGF) in the presence or absence of a clinically used EGF receptor tyrosine kinase (RTK)-specific inhibitor, gefitinib. The data were subjected to a mathematical simulation using the State Space Model (SSM). "Gefitinib-sensitive" genes, the expressional dynamics of which were altered by addition of gefitinib, were identified. A risk scoring model was constructed to classify high- or low-risk patients based on expression signatures of 139 gefitinib-sensitive genes in lung cancer using a training data set of 253 lung adenocarcinomas of North American cohort. The predictive ability of the risk scoring model was examined in independent cohorts of surgical specimens of lung cancer. RESULTS: The risk scoring model enabled the identification of high-risk stage IA and IB cases in another North American cohort for overall survival (OS) with a hazard ratio (HR) of 7.16 (P = 0.029) and 3.26 (P = 0.0072), respectively. It also enabled the identification of high-risk stage I cases without bronchioalveolar carcinoma (BAC) histology in a Japanese cohort for OS and recurrence-free survival (RFS) with HRs of 8.79 (P = 0.001) and 3.72 (P = 0.0049), respectively. CONCLUSION: The set of 139 gefitinib-sensitive genes includes many genes known to be involved in biological aspects of cancer phenotypes, but not known to be involved in EGF signaling. The present result strongly re-emphasizes that EGF signaling status in cancer cells underlies an aggressive phenotype of cancer cells, which is useful for the selection of early-stage lung adenocarcinoma patients with a poor prognosis. TRIAL REGISTRATION: The Gene Expression Omnibus (GEO) GSE31210.

Aryl hydrocarbon receptor (AhR) is a transcription factor that works as a dioxin receptor and is also involved in various physiological phenomena, including development and cell proliferation. Here, we show that the Galpha13 signal destabilizes AhR by promoting the ubiquitination of AhR. Galpha13 interacts directly with AhR-interacting protein (AIP) and inhibits the interaction between AhR and AIP, a crucial interacting protein of AhR. Strikingly, a reporter gene assay and a quantitative reverse transcription-PCR analysis indicate that the Galpha13 signal shows a potent inhibitory effect on the ligand-induced transcriptional activation of AhR. Galpha13 results in the nuclear translocation of AhR in a ligand-independent manner. However, in the presence of active Galpha13, AhR fails to form the active transcriptional complex. Taken together, we propose a new negative regulation of dioxin signalling by the G protein.

The cell adhesion molecule L1 plays crucial roles in axon tract development. In vitro, L1 presented as a culture substrate stimulates axon elongation by binding to L1 expressed on the growth cone. In migrating growth cones, L1 is endocytosed via the AP-2/clathrin-mediated pathway at the central domain, followed by anterograde vesicular transport and recycling to the plasma membrane of the leading front. It has previously been shown that this endocytic trafficking of L1 is critical for axon elongation (Kamiguchi and Yoshihara [2001] J. Neurosci. 21:9194–9203). Adjacent to the AP-2 recognition site, the L1 cytoplasmic domain has a cluster of acidic amino acids containing Ser1181 that can be phosphorylated by casein kinase II (CKII; Wong et al. [1996a] J. Neurochem. 66:779–786). In this paper, we demonstrate that Ser1181 phosphorylation by CKII is implicated in both normal endocytic trafficking of L1 and L1-stimulated axon growth. Whereas L1 is sorted into transferrin-positive endosomes after endocytosis, pharmacological inhibition of CKII caused some population of L1 to be internalized into transferrin-negative compartments. Single-amino-acid mutations at Ser1181, which either prevent or mimic phosphorylation by CKII, caused similar missorting of internalized L1. Furthermore, dorsal root ganglion neurons that had been treated with a CKII inhibitor or transfected with the L1 mutants showed impaired ability to extend axons on an L1 substrate but not on other control substrates. These results demonstrate the novel role of CKII in L1-mediated axon elongation and stress the importance of functional linkage between L1 phosphorylation and L1 trafficking in migrating growth cones. © 2007 Wiley-Liss, Inc.

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, we demonstrate that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function.

Non-small cell lung cancer (NSCLC) is the commonest and the most fatal histological subtype of lung cancer. About 10-30% of patients diagnosed as stage IA (the earliest stage of a lung tumor, smaller than three centimeters in diameter with no evidence of regional lymph node and/or regional metastasis) and submitted to surgery die due to recurrence. Therefore, the identification of prognostic biomarkers for stage IA lung adenocarcinoma with poor prognosis is of great importance to select patients who will be benefited by adjuvant therapy. To the best of our knowledge, there is not a set of genes that identifies these patients. It is known that epidermal growth factor (EGF) signaling pathway is closely related to aggressive phenotypes of lung and other cancers. EGFR tyrosine kinase-specific inhibitor, namely gefitinib is expected to alter the gene expression patterns caused by EGFR tyrosine kinase activity. We performed microarray assays in order to obtain the entire gene expression time course profile of human primary lung epithelial cells that were stimulated with EGF in both the presence and absence of gefitinib. The time courses are composed of 19 time points along 48 hours. The data were subjected to a mathematical analysis, namely the State Space Model (SSM) in order to select genes with gene expressions altered by gefitinib. One hundred thirty nine genes were identified. These genes were used as expression signatures to train a risk scoring model that classifies patients in high- or low-risk (risk of dying in five years). This model was trained by using a data set composed of 253 North American patients with lung adenocarcinomas. Then, the predictive ability of the risk scoring model was examined in two independent cohorts composed of North American and Japanese patients. The model enabled the statistically significant identification of high-risk stage IA lung adenocarcinoma in both cohorts, with hazard ratios (HRs) for death of 7.16 (P=0.029) for North American and 10.98 (P=0.008) for Japanese. The set of 139 genes altered by gefitinib includes several ones that are involved in biological aspects of cancer phenotypes but are yet unknown to be involved in EGF signaling. This result strongly re-emphasizes that EGF signaling status underlies aggressive phenotype of cancer cells, and also suggests the first set of genes that are useful for the identification of stage IA lung adenocarcinoma patients with poor prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-99. doi:1538-7445.AM2012-LB-99

During axon guidance, growth cones navigate toward attractive cues by inserting new membrane on the cue side. This process depends on Ca(2+) release from endoplasmic reticulum (ER) Ca(2+) channels, but the Ca(2+) sensor and effector governing this asymmetric vesicle export remain unknown. We identified a protein complex that controls asymmetric ER Ca(2+)-dependent membrane vesicle export. The Ca(2+)-dependent motor protein myosin Va (MyoVa) tethers membrane vesicles to the ER via a common binding site on the two major ER Ca(2+) channels, inositol 1,4,5-trisphosphate and ryanodine receptors. In response to attractive cues, micromolar Ca(2+) from ER channels triggers MyoVa-channel dissociation and the movement of freed vesicles to the cue side, enabling growth cone turning. MyoVa-Ca(2+) channel interactions are required for proper long-range axon growth in developing spinal cord in vivo. These findings reveal a peri-ER membrane export pathway for Ca(2+)-dependent attraction in axon guidance.