Distinct roles of the two VPS33 proteins in the endolysosomal system in Caenorhabditis elegans.

Sec1/Munc-18 (SM) family proteins are essential regulators in intracellular transport in eukaryotic cells. The SM protein Vps33 functions as a core subunit of two tethering complexes, class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) in the endocytic pathway in yeast. Metazoan cells possess two Vps33 proteins, VPS33A and VPS33B, but their precise roles remain unknown. Here, we present a comparative analysis of Caenorhabditis elegans null mutants for these proteins. We found that the vps-33.1 (VPS33A) mutants exhibited severe defects in both endocytic function and endolysosomal biogenesis in scavenger cells. Furthermore, vps-33.1 mutations caused endocytosis defects in other tissues, and the loss of maternal and zygotic VPS-33.1 resulted in embryonic lethality. By contrast, vps-33.2 mutants were viable but sterile, with terminally arrested spermatocytes. The spermatogenesis phenotype suggests that VPS33.2 is involved in the formation of a sperm-specific organelle. The endocytosis defect in the vps-33.1 mutant was not restored by the expression of VPS-33.2, which indicates that these proteins have nonredundant functions. Together, our data suggest that VPS-33.1 shares most of the general functions of yeast Vps33 in terms of tethering complexes in the endolysosomal system, whereas VPS-33.2 has tissue/organelle specific functions in C. elegans.

Aberrant alternative splicing of RHOA is associated with loss of its expression and activity in diffuse-type gastric carcinoma cells.

RhoA is a member of Rho family small GTPases that regulates diverse cellular functions. Recent large-scale sequencing studies have identified recurrent somatic mutations of RHOA in diffuse-type gastric carcinoma (DGC), indicating that RHOA is a driver of DGC. In this study, we investigated the possible abnormalities of RHOA in a panel of gastric carcinoma (GC) cell lines. Pulldown assay and immunoblot analysis showed that the activity and expression of RhoA were detectable in all GC cell lines tested, except for two DGC cell lines, HSC-59 and GSU. RHOA coding region sequencing revealed that aberrant alternative splicing of RHOA occurred in these cell lines. Quantitative real-time PCR analysis showed that the expression of wild-type RHOA was nearly undetectable, whereas splicing variants were almost exclusively expressed in HSC-59 and GSU cell lines. However, the expression levels of RHOA splicing variants were very low and the corresponding proteins were not detected by immunoblotting. Moreover, the splicing isoforms of RhoA protein were neither efficiently expressed nor activated even if ectopically expressed in cells. These results indicate that aberrant alternative splicing of RHOA results in the loss of its activity and expression in DGC cells.

Enhancement of catecholamine release from PC12 cells by the traditional Japanese medicine, rikkunshito.

BACKGROUND: Rikkunshito is a traditional Japanese herbal medicine that is used to treat appetite loss associated with cancer and other disorders. The formulation contains various constituents that influence cell signaling, and rikkunshito may accordingly affect human homeostasis through multiple regulatory pathways, including those governed by the endocrine system. We investigated the actions of rikkunshito on catecholamine release from PC12 cells, an adrenal chromaffin cell line. METHODS: The actions of rikkunshito on PC12 cells were evaluated by measuring intracellular cAMP levels, tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP) mRNA expression levels, and catecholamine levels in the culture medium. The transcriptional activation of VIP gene by rikkunshito was assessed by using a VIP promoter-driven reporter gene assay. RESULTS: Rikkunshito dose-dependently enhanced forskolin-induced elevations in cAMP in PC12 cells, and also increased the gene expression of TH and VIP. The transcriptional activation of VIP gene by rikkunshito was confirmed. Norepinephrine and dopamine secretion into the culture medium of PC12 cells were also dose-dependently augmented by rikkunshito and/or forskolin, but experiments with a protein kinase C (PKC) activator and a phosphodiesterase inhibitor revealed that the effects of rikkunshito were not simply due to the modulation of PKC or phosphodiesterase activity. CONCLUSIONS: These findings suggest that rikkunshito enhances the release of catecholamines by a novel mechanism involving cAMP.

Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1.

Naturally arising CD25+CD4+ regulatory T cells (T(R) cells) are engaged in the maintenance of immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses, such as autoimmune disease and allergy. T(R) cells specifically express the transcription factor Foxp3, a key regulator of T(R)-cell development and function. Ectopic expression of Foxp3 in conventional T cells is indeed sufficient to confer suppressive activity, repress the production of cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and upregulate T(R)-cell-associated molecules such as CD25, cytotoxic T-lymphocyte-associated antigen-4, and glucocorticoid-induced TNF-receptor-family-related protein. However, the method by which Foxp3 controls these molecular events has yet to be explained. Here we show that the transcription factor AML1 (acute myeloid leukaemia 1)/Runx1 (Runt-related transcription factor 1), which is crucially required for normal haematopoiesis including thymic T-cell development, activates IL-2 and IFN-gamma gene expression in conventional CD4+ T cells through binding to their respective promoters. In natural T(R) cells, Foxp3 interacts physically with AML1. Several lines of evidence support a model in which the interaction suppresses IL-2 and IFN-gamma production, upregulates T(R)-cell-associated molecules, and exerts suppressive activity. This transcriptional control of T(R)-cell function by an interaction between Foxp3 and AML1 can be exploited to control physiological and pathological T-cell-mediated immune responses.

Tissue factor-induced fibrinogenesis mediates cancer cell clustering and multiclonal peritoneal metastasis.

Peritoneal metastasis is one of the most frequent causes of death in several types of advanced cancers; however, the underlying molecular mechanisms remain largely unknown. In this study, we exploited multicolor fluorescent lineage tracking to investigate the clonality of peritoneal metastasis in mouse xenograft models. When peritoneal metastasis was induced by intraperitoneal or orthotopic injection of multicolored cancer cells, each peritoneally metastasized tumor displayed multicolor fluorescence regardless of metastasis sites, indicating that it consists of multiclonal cancer cell populations. Multicolored cancer cell clusters form within the peritoneal cavity and collectively attach to the peritoneum. In vitro, peritoneal lavage fluid or cleared ascitic fluid derived from cancer patients induces cancer cell clustering, which is inhibited by anticoagulants. Cancer cell clusters formed in vitro and in vivo are associated with fibrin formation. Furthermore, tissue factor knockout in cancer cells abrogates cell clustering, peritoneal attachment, and peritoneal metastasis. Thus, we propose that cancer cells activate the coagulation cascade via tissue factor to form fibrin-mediated cell clusters and promote peritoneal attachment; these factors lead to the development of multiclonal peritoneal metastasis and may be therapeutic targets.

Application of an intracellular stability test of a novel missense menin mutant to the diagnosis of multiple endocrine neoplasia type 1.

Germline MEN1 mutation analysis is a powerful tool for an early diagnosis of multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant familial cancer syndrome characterized by the parathyroid, pituitary and gastroenteropancreatic endocrine tumors. However, the clinical significance of MEN1 gene variants, especially missense and in-frame mutations as well as some splicing mutations, is not always obvious. We have previously shown that mutant menin proteins associated with MEN1 are rapidly degraded by the ubiquitin-proteasome pathway. We also demonstrated by a fluorescent immunocytochemical stability test that the stability of missense and in-frame deletion mutants varies widely but that unstable mutants were found only in MEN1 and related disorders and not in normal polymorphisms. In the present study, we evaluated by this stability test the pathogenicity of a novel MEN1 missense mutation, c.1118C>T, encoding a P373L mutant menin, identified in a suspected MEN1 patient. The results demonstrated that the mutant menin is highly unstable, indicating that this mutation is causative for MEN1. These findings encouraged us to proceed with presymptomatic genetic screening for this mutation among the family members, which resulted in the identification of asymptomatic mutation carriers. Thus, the information from the menin stability test was useful for genetic diagnosis and counseling of MEN1 in the case with a previously unreported MEN1 missense mutation.

A novel splice site mutation of the MEN1 gene identified in a patient with primary hyperparathyroidism.

Heterozygous germline mutation of the tumor suppressor gene MEN1 is responsible for multiple endocrine neoplasia type 1 (MEN1), a familial cancer syndrome characterized by pituitary, parathyroid and enteropancreatic tumors. Various mutations have been identified throughout the entire gene region in patients with MEN1 and its incomplete forms often manifested as familial isolated hyperparathyroidism and apparently sporadic parathyroid tumor. Mutation analysis of the MEN1 gene is a powerful tool for the early diagnosis of MEN1; however, the clinical significance of the identified mutations is not always obvious. In this study, a previously unreported missense MEN1 mutation, c.824G>T was identified in a patient with primary hyperparathyroidism and evaluated for its pathogenicity. This mutation was predicted to generate a putative missense menin protein, R275M. A stability test of the menin protein demonstrated that the stability of R275M mutant was reduced only slightly as compared with wild type menin, and therefore could not preclude the possibility that it was a rare benign polymorphism. However, further analysis of leukocyte mRNA and minigene experiments indicated that the mutant c.824G>T allele gives rise to abnormally spliced menin mRNA, and thereby confirmed that c.824G>T mutation is causative for MEN1. Thus, leukocyte mRNA analysis has been demonstrated useful to identify a splicing mutation of the MEN1 gene.

Receptor Tyrosine Kinases Amplified in Diffuse-Type Gastric Carcinoma: Potential Targeted Therapies and Novel Downstream Effectors.

Gastric cancer (GC) is a major cause of cancer-related death worldwide. Patients with an aggressive subtype of GC, known as diffuse-type gastric carcinoma (DGC), have extremely poor prognoses. DGC is characterized by rapid infiltrative growth, massive desmoplastic stroma, frequent peritoneal metastasis, and high probability of recurrence. These clinical features and progression patterns of DGC substantially differ from those of other GC subtypes, suggesting the existence of specific oncogenic signals. The importance of gene amplification and the resulting aberrant activation of receptor tyrosine kinase (RTK) signaling in the malignant progression of DGC is becoming apparent. Here, we review the characteristics of RTK gene amplification in DGC and its importance in peritoneal metastasis. These insights may potentially lead to new targeted therapeutics.

Integrin α5 mediates cancer cell-fibroblast adhesion and peritoneal dissemination of diffuse-type gastric carcinoma.

Diffuse-type gastric carcinoma (DGC) has a poor prognosis due to its rapid diffusive infiltration and frequent peritoneal dissemination. DGC is associated with massive fibrosis caused by aberrant proliferation of cancer-associated fibroblasts (CAFs). Previously, we reported that direct heterocellular interaction between cancer cells and CAFs is important for the peritoneal dissemination of DGC. In this study, we aimed to identify and target the molecules that mediate such heterocellular interactions. Monoclonal antibodies (mAbs) against intact DGC cells were generated and subjected to high-throughput screening to obtain several mAbs that inhibit the adhesion of DGC cells to CAFs. Immunoprecipitation and mass spectrometry revealed that all mAbs recognized integrin α5 complexed with integrin ß1. Blocking integrin α5 in DGC cells or fibronectin, a ligand of integrin α5ß1, deposited on CAFs abrogated the heterocellular interaction. Administration of mAbs or knockout of integrin α5 in DGC cells suppressed their invasion led by CAFs in vitro and peritoneal dissemination in a mouse xenograft model. Altogether, these findings demonstrate that integrin α5 mediates the heterotypic cancer cell-fibroblast interaction during peritoneal dissemination of DGC and may thus be a therapeutic target.

Correlation of mutant menin stability with clinical expression of multiple endocrine neoplasia type 1 and its incomplete forms.

Germline mutations of the tumor suppressor gene MEN1 are found not only in typical multiple endocrine neoplasia type 1 (MEN1) but also in its incomplete forms such as familial isolated hyperparathyroidism (FIHP) and apparently sporadic parathyroid tumor (ASPT). No definitive genotype-phenotype correlation has been established between these clinical forms and MEN1 gene mutations. We previously demonstrated that mutant menin proteins associated with MEN1 are rapidly degraded by the ubiquitin-proteasome pathway. To examine whether the intracellular stability of mutant menin is correlated with clinical phenotypes, we developed a method of evaluating menin stability and examined 20 mutants associated with typical MEN1 (17 missense, two in-frame deletion, one nonsense) and 21 mutants associated with FIHP or ASPT (19 missense, two in-frame deletion). All tested mutants associated with typical MEN1 showed reduced stability. Some missense and in-frame deletion mutants (G28A, R171W, T197I, E255K, E274A, Y353del and E366D) associated with FIHP or ASPT were almost as stable as or only slightly less stable than wild-type menin, while others were as unstable as those associated with typical MEN1. Some stable mutants exhibited substantial biological activities when tested by JunD-dependent transactivation assay. These findings suggest that certain missense and in-frame mutations are fairly stable and retain intrinsic biological activity, and might be specifically associated with incomplete clinical phenotypes. The menin stability test will provide useful information for the management of patients carrying germline MEN1 mutations especially when they have missense or in-frame variants of ambiguous clinical significance.

PLEKHA5 regulates the survival and peritoneal dissemination of diffuse-type gastric carcinoma cells with Met gene amplification.

Met gene amplification has been found in a subset of malignant carcinomas, including diffuse-type gastric carcinoma (DGC), which has a poor prognosis owing to rapid infiltrative invasion and frequent peritoneal dissemination. Met is considered a promising therapeutic target for DGC. However, DGC cells with Met gene amplification eventually acquire resistance to Met inhibitors. Therefore, identification of alternate targets that mediate Met signaling and confer malignant phenotypes is critical. In this study, we conducted a phosphoproteomic analysis of DGC cells possessing Met gene amplification and identified Pleckstrin Homology Domain Containing A5 (PLEKHA5) as a protein that is tyrosine-phosphorylated downstream of Met. Knockdown of PLEKHA5 selectively suppressed the growth of DGC cells with Met gene amplification by inducing apoptosis, even though they had acquired resistance to Met inhibitors. Moreover, PLEKHA5 silencing abrogated the malignant phenotypes of Met-addicted DGC cells, including peritoneal dissemination in vivo. Mechanistically, PLEKHA5 knockdown dysregulates glycolytic metabolism, leading to activation of the JNK pathway that promotes apoptosis. These results indicate that PLEKHA5 is a novel downstream effector of amplified Met and is required for the malignant progression of Met-addicted DGC.

SHP2 as a Potential Therapeutic Target in Diffuse-Type Gastric Carcinoma Addicted to Receptor Tyrosine Kinase Signaling.

Diffuse-type gastric carcinoma (DGC) exhibits aggressive progression associated with rapid infiltrative growth, massive fibrosis, and peritoneal dissemination. Gene amplification of Met and fibroblast growth factor receptor 2 (FGFR2) receptor tyrosine kinases (RTKs) has been observed in DGC. However, the signaling pathways that promote DGC progression downstream of these RTKs remain to be fully elucidated. We previously identified an oncogenic tyrosine phosphatase, SHP2, using phospho-proteomic analysis of DGC cells with Met gene amplification. In this study, we characterized SHP2 in the progression of DGC and assessed the therapeutic potential of targeting SHP2. Although SHP2 was expressed in all gastric carcinoma cell lines examined, its tyrosine phosphorylation preferentially occurred in several DGC cell lines with Met or FGFR2 gene amplification. Met or FGFR inhibitor treatment or knockdown markedly reduced SHP2 tyrosine phosphorylation. Knockdown or pharmacological inhibition of SHP2 selectively suppressed the growth of DGC cells addicted to Met or FGFR2, even when they acquired resistance to Met inhibitors. Moreover, SHP2 knockdown or pharmacological inhibition blocked the migration and invasion of Met-addicted DGC cells in vitro and their peritoneal dissemination in a mouse xenograft model. These results indicate that SHP2 is a critical regulator of the malignant progression of RTK-addicted DGC and may be a therapeutic target.

Caspase 8 and menin expressions are not correlated in human parathyroid tumors.

Menin is lost by the sequential inactivation of both MEN1 alleles in subsets of non-hereditary endocrine tumors as well as those associated with multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant hereditary cancer syndrome characterized by multiple tumors including parathyroid, pituitary and enteropancreatic endocrine tumors. Loss of menin has been reported to be associated with lowered caspase 8 expression and resistance to apoptosis in murine fibroblasts and in pancreatic islet tumors arising in heterozygous MEN1 gene knockout mice, the animal model of the human MEN1 syndrome. We confirmed by menin-knockdown experiments with specific siRNA that menin is crucial for caspase 8 expression in human culture cells while overexpression of menin did not increase caspase 8 protein over basal levels. We then examined expression of menin, caspase 8 and cyclin-dependent kinase inhibitors p27(Kip1) and p15(Ink4b) by Western blotting in human parathyroid tumors surgically resected from patients with MEN1 and those with non-hereditary primary hyperparathyroidism. The menin and p27(Kip1) expression levels were correlated with MEN1 mutation status that was confirmed by DNA analysis. The caspase 8 and p15(Ink4b) protein levels were variable among tumors, and were not correlated with menin protein levels. These findings suggest that human endocrine tumors lacking menin may not always exhibit lowered caspase 8 expression and hence may not be resistant to apoptosis-inducing therapy.

MEN1 gene and its mutations: basic and clinical implications.

Heterozygous germline mutations of the tumor-suppressor gene MEN1 are responsible for multiple endocrine neoplasia type 1 (MEN1), a dominantly inherited familial cancer syndrome characterized by pituitary, parathyroid, and enteropancreatic tumors. Various mutations have been identified throughout the entire gene region in patients with MEN1 and related disorders. Neither mutation hot spot nor phenotype­genotype correlation has been established in MEN1 although some missense mutations may be specifically associated with a phenotype of familial isolated hyperparathyroidism. The gene product menin has been implicated in multiple roles, including gene transcription, maintenance of genomic integrity, and control of cell division and differentiation. These multiple functions are likely to be conferred by association with multiple protein factors. Occurrence of MEN1-causing missense mutations throughout menin also suggests the requirement of multiple binding factors for its full tumor-suppressive activity. The effect of menin depletion is highly tissue specific, but its underlying mechanism remains to be elucidated. A DNA test for MEN1 germline mutations is a useful tool for diagnosis of MEN1 although it needs further improvements

Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins.

Role of tyramine in calcium dynamics of GABAergic neurons and escape behavior in Caenorhabditis elegans.

BACKGROUND: Tyramine, known as a "trace amine" in mammals, modulates a wide range of behavior in invertebrates; however, the underlying cellular and circuit mechanisms are not well understood. In the nematode Caenorhabditis elegans (C. elegans), tyramine affects key behaviors, including foraging, feeding, and escape responses. The touch-evoked backward escape response is often coupled with a sharp omega turn that allows the animal to navigate away in the opposite direction. Previous studies have showed that a metabotropic tyramine receptor, SER-2, in GABAergic body motor neurons controls deep body bending in omega turns. In this study, we focused on the role of tyramine in GABAergic head motor neurons. Our goal is to understand the mechanism by which tyraminergic signaling alters neural circuit activity to control escape behavior. RESULTS: Using calcium imaging in freely moving C. elegans, we found that GABAergic RME motor neurons in the head had high calcium levels during forward locomotion but low calcium levels during spontaneous and evoked backward locomotion. This calcium decrease was also observed during the omega turn. Mutant analyses showed that tbh-1 mutants lacking only octopamine had normal calcium responses, whereas tdc-1 mutants lacking both tyramine and octopamine did not exhibit the calcium decrease in RME. This neuromodulation was mediated by SER-2. Moreover, tyraminergic RIM neuron activity was negatively correlated with RME activity in the directional switch from forward to backward locomotion. These results indicate that tyramine released from RIM inhibits RME via SER-2 signaling. The omega turn is initiated by a sharp head bend when the animal reinitiates forward movement. Interestingly, ser-2 mutants exhibited shallow head bends and often failed to execute deep-angle omega turns. The behavioral defect and the abnormal calcium response in ser-2 mutants could be rescued by SER-2 expression in RME. These results suggest that tyraminergic inhibition of RME is involved in the control of omega turns. CONCLUSION: We demonstrate that endogenous tyramine downregulates calcium levels in GABAergic RME motor neurons in the head via the tyramine receptor SER-2 during backward locomotion and omega turns. Our data suggest that this neuromodulation allows deep head bending during omega turns and plays a role in the escape behavior in C. elegans.

Menin missense mutants associated with multiple endocrine neoplasia type 1 are rapidly degraded via the ubiquitin-proteasome pathway.

MEN1 is a tumor suppressor gene that is responsible for multiple endocrine neoplasia type 1 (MEN1) and that encodes a 610-amino-acid protein, called menin. While the majority of germ line mutations identified in MEN1 patients are frameshift and nonsense mutations resulting in truncation of the menin protein, various missense mutations have been identified whose effects on menin activity are unclear. For this study, we analyzed a series of menin proteins with single amino acid alterations and found that all of the MEN1-causing missense mutations tested led to greatly diminished levels of the affected proteins in comparison with wild-type and benign polymorphic menin protein levels. We demonstrate here that the reduced levels of the mutant proteins are due to rapid degradation via the ubiquitin-proteasome pathway. Furthermore, the mutants, but not wild-type menin, interact both with the molecular chaperone Hsp70 and with the Hsp70-associated ubiquitin ligase CHIP, and the overexpression of CHIP promotes the ubiquitination of the menin mutants in vivo. These findings reveal that MEN1-causing missense mutations lead to a loss of function of menin due to enhanced proteolytic degradation, which may be a common mechanism for inactivating tumor suppressor gene products in familial cancer.

Actinin-1 and actinin-4 play essential but distinct roles in invadopodia formation by carcinoma cells.

Invadopodia are ventral membrane protrusions formed by cancer cells that degrade the extracellular matrix (ECM) during tumor invasion and metastasis. Formation of invadopodia is initiated by the assembly of actin filaments (F-actin) that results from the coordinated activation of several actin regulatory proteins. Actinin-1 and actinin-4 are actin bundling proteins expressed in non-muscle cells and actinin-4 is preferentially associated with malignant phenotypes of carcinoma cells. In this study, we investigated the role of actinin-1 and -4 in invadopodia formation. Expression of both actinin-1 and -4 tended to be higher in invasive and metastatic breast carcinoma cell lines than in non-invasive ones. Immunofluorescence analysis revealed that actinin-1 and -4 colocalized at core actin structures of invadopodia. Time-lapse imaging showed that appearance of both actinins at invadopodia is concomitant with the assembly of F-actin. Knockdown of either actinin-1 or actinin-4 suppressed the formation of invadopodia and degradation of the ECM by carcinoma cells. Interestingly, overexpression of actinin-4, but not actinin-1, significantly promoted the formation of invadopodia and this activity required the actin binding domains and the unique N-terminal motif that exists only in actinin-4. These results demonstrate that both actinin-1 and actinin-4 participate in the assembly of F-actin at invadopodia. Additionally, actinin-4 may have a selective advantage in accelerating invadopodia-mediated invasion of carcinoma cells.

A new platform for long-term tracking and recording of neural activity and simultaneous optogenetic control in freely behaving Caenorhabditis elegans.

BACKGROUND: Real-time recording and manipulation of neural activity in freely behaving animals can greatly advance our understanding of how neural circuits regulate behavior. Ca2+ imaging and optogenetic manipulation with optical probes are key technologies for this purpose. However, integrating the two optical approaches with behavioral analysis has been technically challenging. NEW METHOD: Here, we developed a new imaging system, ICaST (Integrated platform for Ca2+ imaging, Stimulation, and Tracking), which combines an automatic worm tracking system and a fast-scanning laser confocal microscope, to image neurons of interest in freely behaving C. elegans. We optimized different excitation wavelengths for the concurrent use of channelrhodopsin-2 and G-CaMP, a green fluorescent protein (GFP)-based, genetically encoded Ca2+ indicator. RESULTS: Using ICaST in conjunction with an improved G-CaMP7, we successfully achieved long-term tracking and Ca2+ imaging of the AVA backward command interneurons while tracking the head of a moving animal. We also performed all-optical manipulation and simultaneous recording of Ca2+ dynamics from GABAergic motor neurons in conjunction with behavior monitoring. COMPARISON WITH EXISTING METHOD(S): Our system differs from conventional systems in that it does not require fluorescent markers for tracking and can track any part of the worm's body via bright-field imaging at high magnification. Consequently, this approach enables the long-term imaging of activity from neurons or nerve processes of interest with high spatiotemporal resolution. CONCLUSION: Our imaging system is a powerful tool for studying the neural circuit mechanisms of C. elegans behavior and has potential for use in other small animals.

Detection of APC gene deletion by double competitive polymerase chain reaction in patients with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant familial cancer syndrome caused by germline mutations of the tumor suppressor adenomatous polyposis coli (APC) gene. Heterozygous apc mutations have been identified in the majority of classical FAP patients who develop more than 100 colorectal adenomas. However, classical FAP patients often fail to display germline APC mutations detectable by routine mutation analysis. These apparently mutation-negative cases may be caused by heterozygous large genomic deletions. In the present study, FAP patients who showed no APC germline mutation detectable by the protein truncation assay and direct sequencing of protein coding exons were screened for APC gene deletion by a gene dose assay based on double competitive polymerase chain reaction. Gene dosage measurements within exon 15 of the APC gene identified two patients with gene deletion and one with possible gene duplication among 41 apparently mutation-negative patients. The deleted sequences in the two patients were determined by fine gene dose mapping around the APC gene and nucleotide sequencing of the deletion breakpoints. They were approximately 435-kilobase pair (kb) and 737-kb regions including the whole APC gene and flanked by a 4-base pair repeat and LINE-1 repetitive sequences, respectively. The chimeric LINE-1 element created at the breakpoint in the latter case also contained a short sequence derived from another LINE-1 element, suggesting a complex unequal homologous recombination event. These findings indicate that this gene dose assay is a useful technique to detect large gene deletions of the APC gene and to determine their genomic breakpoints.