The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.

Phosphatidylinositol phosphate kinases (PIPKs) are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate) is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα) and type IIα (PIPKIIα) in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3- cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5)P2.

Extracellular pH is usually maintained around 7.4 in multicellular organisms, and cells are optimized to proliferate under this condition. Here, we find cells can adapt to a more acidic pH of 6.5 and become addicted to this acidic microenvironment by expressing phosphatase of regenerating liver (PRL), a driver of cancer malignancy. Genome-scale CRISPR-Cas9 knockout screening and subsequent analyses revealed that PRL promotes H+ extrusion and acid addiction by stimulating lysosomal exocytosis. Further experiments using cultured cells and Caenorhabditis elegans clarified the molecular link between PRL and lysosomal exocytosis across species, involving activation of lysosomal Ca2+ channel TRPML by ROS. Indeed, disruption of TRPML in cancer cells abolished PRL-stimulated lysosomal exocytosis, acid addiction, and metastasis. Thus, PRL is the molecular switch turning cells addicted to an acidic condition, which should benefit cancer cells to thrive in an acidic tumor microenvironment.

Adenomatous polyposis coli (APC) is a tumor-suppressing protein whose inactivation triggers the formation of colorectal polyps. Numerous studies using cell lines or genetically engineered mice have revealed its role in suppressing Wnt/β-catenin signaling pathway and regulating cell proliferation and differentiation. Here, we performed genetic analyses of APC using a three-dimensional organoid culture of mouse colon epithelia, which enables the detailed examination of epithelial properties. Analyses of Apc-knockout colon organoids not only confirmed the importance of APC in suppressing Wnt/β-catenin signaling and regulating cell differentiation, but also revealed several novel features: a significant decrease in proliferating speed and an increase in cross-sectional area of cells. Moreover, we found a significant number of lysozyme-positive Paneth-like cells, which were never observed in wild-type colon tissues or organoids, but have been reported to emerge in colon cancers. Therefore, APC autonomously suppresses ectopic differentiation into lysozyme-positive cells, specifically in the colon epithelia. Colon organoids would be an ideal material to investigate the molecular mechanism and biological importance of the ectopic differentiation associated with cancer development.

To determine whether the Arp2/3 complex activated by N-WASP (VCA) branches actin filaments at the side (side branching), or at the barbed (B-)end (end branching) of the mother filaments, we have directly observed the branching process of actin filaments and examined single-molecule unbinding under optical microscope. We found that side branching was predominant, though not exclusive. At the initial stage of polymerization, the branching at the B-end occurred and subsequently the side branching started to occur. In either type of branching, the mother and daughter filaments elongated at nearly the same rate (growing type). Independently of the stage of polymerization, branching due to the direct coupling of filaments with an acute angle to the mother filaments (a coupling type) occurred. Phalloidin suppressed the growing type of branching but not the coupling type, implying that actin monomers are required for the former but not the latter. We found, by single molecule measurements using optical tweezers, that the Arp2/3 complex attaches to the side of actin filaments and the N-WASP appears to detach from the actin-Arp2/3 complex at 6-7 pN.

Abnormalities in insulin-induced glucose incorporation in skeletal muscle were observed in Type 2 diabetes. Our previous studies revealed that the binding between skeletal muscle and kidney-enriched inositol polyphosphate phosphatase (SKIP) and p21-activated protein kinase (Pak1) at the plasma membrane is induced insulin-dependently and that this binding mediated a rapid and efficient termination of insulin signaling and a subsequent glucose uptake into skeletal muscle cells. Here, we identified 11-amino-acids peptide within kinase domain of Pak1, necessary and sufficient for SKIP binding. Expression of this region in C2C12 cells resulted in an increase in insulin signaling. Supplementation of a synthetic peptide of this sequence increased insulin signaling and insulin-induced glucose uptake into skeletal muscle cell lines. These findings suggest the physiological role of Pak1-SKIP binding in the regulation of insulin signaling in skeletal muscle.

Ca(2+) influx triggers sperm capacitation; however, the underlying regulatory mechanisms remain incompletely understood. Here, we show that CNNM4, a Mg(2+) transporter, is required for Ca(2+) influx during capacitation. We find that Cnnm4-deficient male mice are almost infertile because of sperm dysfunction. Motion analyses show that hyperactivation, a qualitative change in the mode of sperm motility during capacitation, is abrogated in Cnnm4-deficient sperm. In contrast, tyrosine phosphorylation of flagellar proteins, a hallmark of capacitation, is excessively augmented. These seemingly paradoxical phenotypes of Cnnm4-deficient sperm are very similar to those of sperm lacking a functional cation channel of sperm (CatSper) channel, which plays an essential role in Ca(2+) influx during sperm capacitation. Ca(2+) imaging analyses demonstrate that Ca(2+) influx is perturbed in Cnnm4-deficient sperm, and forced Ca(2+) entry into these sperm normalizes the level of tyrosine phosphorylation. Furthermore, we confirm the importance of CNNM4 in sperm by generating germ-cell-specific Cnnm4-deficient mice. These results suggest a new role of CNNM4 in sperm Ca(2+) homeostasis.

CD44, a transmembrane receptor, is expressed in the standard or variant form and plays a critical role in tumor progression and metastasis. This protein regulates cell adhesion and migration in breast cancer cells. We previously reported that phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi regulates cell migration and invasion in breast cancer cell lines. In this study, we showed that an increase in PI(4)P levels at the Golgi by knockdown of PI(4)P phosphatase SAC1 increased the expression of standard CD44, variant CD44, and ezrin/radixin phosphorylation and enhanced the formation of focal adhesions mediated by CD44 and ezrin/radixin in MCF7 and SK-BR-3 cells. In contrast, knockdown of PI 4-kinase IIIβ in highly invasive MDA-MB-231 cells decreased these factors. These results suggest that SAC1 expression and PI(4)P at the Golgi are important in tumor progression and metastasis and are potential prognostic markers of breast cancers.

Intracellular Mg(2+) levels are strictly regulated; however, the biological importance of intracellular Mg(2+) levels and the pathways that regulate them remain poorly understood. Here, we determined that intracellular Mg(2+) is important in regulating both energy metabolism and tumor progression. We determined that CNNM4, a membrane protein that stimulates Mg(2+) efflux, binds phosphatase of regenerating liver (PRL), which is frequently overexpressed in malignant human cancers. Biochemical analyses of cultured cells revealed that PRL prevents CNNM4-dependent Mg(2+) efflux and that regulation of intracellular Mg(2+) levels by PRL and CNNM4 is linked to energy metabolism and AMPK/mTOR signaling. Indeed, treatment with the clinically available mTOR inhibitor rapamycin suppressed the growth of cancer cells in which PRL was overexpressed. In ApcΔ(14/+) mice, which spontaneously form benign polyps in the intestine, deletion of Cnnm4 promoted malignant progression of intestinal polyps to adenocarcinomas. IHC analyses of tissues from patients with colon cancer demonstrated an inverse relationship between CNNM4 expression and colon cancer malignancy. Together, these results indicate that CNNM4-dependent Mg(2+) efflux suppresses tumor progression by regulating energy metabolism.

Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the strict control of GTPase-activating proteins (GAPs). ARAP1 (Arf GAP with Rho GAP domain, ankyrin repeat, and PH domain 1) is an Arf GAP molecule with multiple PH domains that recognize phosphatidylinositol 3,4,5-trisphosphate. We found that growth factor stimulation induced localization of ARAP1 to an area of the plasma membrane inside the ring structure of circular dorsal ruffles (CDRs). Moreover, expression of ARAP1 increased the size of the CDR filamentous-actin ring in an Arf GAP activity-dependent manner, whereas smaller CDRs were formed by ARAP1 knockdown. In addition, expression of a dominant-negative mutant of Arf1 and Arf5, the substrates of ARAP1, expanded the size of CDRs, suggesting that the two Arf isoforms regulate ring structure downstream of ARAP1. Therefore our results reveal a novel molecular mechanism of CDR ring size control through the ARAP1-Arf1/5 pathway.

The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPases in C. elegans.

Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of approximately 220 A in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an approximately 600 A diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role.

Blood pressure has a daily pattern, with higher values in the active period. Its elevation at the onset of the active period substantially increases the risk of fatal cardiovascular events. Renin secretion stimulated by renal sympathetic neurons is considered essential to this process; however, its regulatory mechanism remains largely unknown. Here, we show the importance of transient receptor potential melastatin-related 6 (TRPM6), a Mg2+-permeable cation channel, in augmenting renin secretion in the active period. TRPM6 expression is significantly reduced in the distal convoluted tubule of hypotensive Cnnm2-deficient mice. We generate kidney-specific Trpm6-deficient mice and observe a decrease in blood pressure and a disappearance of its circadian variation. Consistently, renin secretion is not augmented in the active period. Furthermore, renin secretion after pharmacological activation of β-adrenoreceptor, the target of neuronal stimulation, is abrogated, and the receptor expression is decreased in renin-secreting cells. These results indicate crucial roles of TRPM6 in the circadian regulation of blood pressure.

To date, good experimental animal models of renal anemia are not available. Therefore, the purpose of this study was to establish a novel approach to induce chronic kidney disease (CKD) with severe anemia by oral administration of adenine in rodents. Adenine was administered to 6-week-old male C57BL/6 mice (25 and 50 mg/kg body weight) by oral gavage daily for 28 days. Serum creatinine and BUN as well as hematocrit, hemoglobin (Hb) and plasma erythropoietin (EPO) levels were monitored to assess renal function and anemia, respectively. Adenine at 25 mg/kg for 28 days slightly increased plasma creatinine levels, but did not induce anemia. In contrast, 50 mg/kg of adenine daily for 28 days showed severe renal dysfunction (plasma creatinine 1.9 ± 0.10 mg/dL) and anemia (hematocrit 36.5 ± 1.0% and EPO 28 ± 2.4 pg/mL) as compared with vehicle-treated mice (0.4 ± 0.02 mg/dL, 49.6 ± 1.6% and 61 ± 4.0 pg/mL, respectively). At the end of experiment, level of Hb also significantly reduced in 50 mg/kg adenine administration group. Remarkable histological changes of kidney tissues characterized by interstitial fibrosis and cystic appearance in tubules were observed in 50 mg/kg of adenine treatment group. These results have demonstrated that oral dosing with adenine at 50 mg/kg for 28 days is suitable to induce a stable anemia associated with CKD in mice.

α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion.

Several aging phenotypes, including age-related memory impairment (AMI), are thought to be caused by cumulative oxidative damage. In Drosophila, age-related impairments in 1 hr memory can be suppressed by reducing activity of protein kinase A (PKA). However, the mechanism for this effect has been unclear. Here we show that decreasing PKA suppresses AMI by reducing activity of pyruvate carboxylase (PC), a glial metabolic enzyme whose amounts increase upon aging. Increased PC activity causes AMI through a mechanism independent of oxidative damage. Instead, increased PC activity is associated with decreases in D-serine, a glia-derived neuromodulator that regulates NMDA receptor activity. D-serine feeding suppresses both AMI and memory impairment caused by glial overexpression of dPC, indicating that an oxidative stress-independent dysregulation of glial modulation of neuronal activity contributes to AMI in Drosophila.

Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP(3)-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP(3).

Axonal branching is one of the key processes within the enormous complexity of the nervous system to enable a single neuron to send information to multiple targets. However, the molecular mechanisms that control branch formation are poorly understood. In particular, previous studies have rarely addressed the mechanisms underlying axonal bifurcation, in which axons form new branches via splitting of the growth cone. We demonstrate that DISCO Interacting Protein 2 (DIP2) is required for precise axonal bifurcation in Drosophila mushroom body (MB) neurons by suppressing ectopic bifurcation and regulating the guidance of sister axons. We also found that DIP2 localize to the plasma membrane. Domain function analysis revealed that the AMP-synthetase domains of DIP2 are essential for its function, which may involve exerting a catalytic activity that modifies fatty acids. Genetic analysis and subsequent biochemical analysis suggested that DIP2 is involved in the fatty acid metabolization of acyl-CoA. Taken together, our results reveal a function of DIP2 in the developing nervous system and provide a potential functional relationship between fatty acid metabolism and axon morphogenesis.

Mechanistic target of rapamycin complex 1 (mTORC1) plays a pivotal role in controlling cell growth and metabolism in response to nutrients and growth factors. The activity of mTORC1 is dually regulated by amino acids and growth factor signaling, and amino acid-dependent mTORC1 activity is regulated by mTORC1 interaction with the Ragulator-Rag GTPase complex, which is localized to the surface of lysosomes via a membrane-anchored protein, p18/Lamtor1. However, the physiological function of p18-Ragulator-dependent mTORC1 signaling remains elusive. The present study evaluated the function of p18-mediated mTORC1 signaling in the intestinal epithelia using p18 conditional knockout mice. In p18 knockout colonic crypts, mTORC1 was delocalized from lysosomes, and in vivo mTORC1 activity was markedly decreased. Histologically, p18 knockout crypts exhibited significantly increased proliferating cells and dramatically decreased mucin-producing goblet cells, while overall crypt architecture and enteroendocrine cell differentiation were unaffected. Furthermore, p18 knockout crypts normally expressed transcription factors implicated in crypt differentiation, such as Cdx2 and Klf4, indicating that p18 ablation did not affect the genetic program of cell differentiation. Analysis of colon crypt organoid cultures revealed that both p18 ablation and rapamycin treatment robustly suppressed development of mucin-producing goblet cells. Hence, p18-mediated mTORC1 signaling could promote the anabolic metabolism required for robust mucin production in goblet cells to protect the intestinal epithelia from various external stressors.Key words: mTORC1, p18/lamtor1, intestinal epithelium, goblet cells, mucin.