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Key Points Wiskott–Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins activate the ARP2/3 complex to promote reorganization of the actin cytoskeleton. The N-terminal domain of the WASP and WAVE proteins contributes to stable protein–protein interactions, thereby contributing to the formation of the protein complex. Intramolecular interaction suppresses the ARP2/3-complex-activating capability of WASP and neural (N-)WASP. The role of intramolecular or intermolecular interactions in the regulation of WAVE proteins is still unclear. Phosphatidylinositol phosphates bind to WASP and WAVE proteins and contribute to the localization of these proteins, and they also regulate their capability to activate the ARP2/3 complex. Many proteins with Src-homology-3 (SH3) domains bind to the proline-rich region of WASP and WAVE proteins, and enhance the ARP2/3 activation of WASP and WAVE. Most of the proteins with SH3 domains are adaptors that link WASP and WAVE proteins to other proteins or the cell membrane. The Bin, amphiphysin, Rvs167 (BAR) and extended Fer-CIP4 homology (EFC) domains are membrane-binding and membrane-deforming domains. Most BAR- or EFC-domain-containing proteins have SH3 domains that bind to N-WASP, and are involved in endocytosis. The Rac binding (RCB, also known as the IRSp53-Mim-homology domain (IMD)) domain of IRSp53 shares structural similarity to the BAR domain and binds to the membrane. IRSp53 binds to WAVE2 for lamellipodium formation. WASP and N-WASP are involved in cell-shape changes that occur inwardly (endocytosis) and outwardly (filopodia and podosomes). WAVE proteins are involved in outward (lamellipodia) cell-shape changes only. Membrane deformation might be coupled to WASP- and WAVE-mediated cytoskeletal changes. Membrane-binding and membrane-deforming proteins have emerged as binding partners of the Wiskott–Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins that regulate the actin cytoskeleton. Membrane deformation and cytoskeletal reorganization might be coupled in processes that require alteration of membrane shapes, including endocytosis and membrane protrusion. Wiskott–Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins are scaffolds that link upstream signals to the activation of the ARP2/3 complex, leading to a burst of actin polymerization. ARP2/3-complex-mediated actin polymerization is crucial for the reorganization of the actin cytoskeleton at the cell cortex for processes such as cell movement, vesicular trafficking and pathogen infection. Large families of membrane-binding proteins were recently found to interact with WASP and WAVE family proteins, therefore providing a new layer of membrane-dependent regulation of actin polymerization.

Tension applied to the plasma membrane (PM) is a global mechanical parameter involved in cell migration. However, how membrane tension regulates actin assembly is unknown. Here, we demonstrate that FBP17, a membrane-bending protein and an activator of WASP/N-WASP-dependent actin nucleation, is a PM tension sensor involved in leading edge formation. In migrating cells, FBP17 localizes to short membrane invaginations at the leading edge, while diminishing from the cell rear in response to PM tension increase. Conversely, following reduced PM tension, FBP17 dots randomly distribute throughout the cell, correlating with loss of polarized actin assembly on PM tension reduction. Actin protrusive force is required for the polarized accumulation, indicating a role for FBP17-mediated activation of WASP/N-WASP in PM tension generation. In vitro experiments show that FBP17 membrane-bending activity depends on liposomal membrane tension. Thus, FBP17 is the local activator of actin polymerization that is inhibited by PM tension in the feedback loop that regulates cell migration. Itoh and colleagues find that the membrane-bending protein FBP17 is released from membranes at the leading edge of migrating cells following increased tension involving a feedback loop, in which FBP17 also promotes tension through actin protrusion formation.

Cell migration is a critical step in tumor invasion and metastasis, and regulation of this process will lead to appropriate therapies for treating cancer. Cancer cells migrate in various ways, according to cell type and degree of differentiation. The different types of cell migration are regulated by different mechanisms. Reorganization of the actin cytoskeleton is the primary mechanism of cell motility and is essential for most types of cell migration. Actin reorganization is regulated by Rho family small GTPases such as Rho, Rac, and Cdc42. These small GTPases transmit extracellular chemotactic signals to downstream effectors. Of these downstream effectors, Wiskott–Aldrich syndrome protein (WASP) family proteins are key regulators of cell migration. Activated WASP family proteins induce the formation of protrusive membrane structures involved in cell migration and degradation of the extracellular matrix. Inhibition of Rho family small GTPase signaling suppresses the migration and invasion of cancer cells. Thus, control of cell migration via the actin cytoskeleton provides the possibility of regulating cancer cell invasion and metastasis. (Cancer Sci 2005; 96: 379 – 386)

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.

Wiskott–Aldrich syndrome protein (WASP) and WASP family verprolin‐homologous protein (WAVE) family proteins activate cells’ major actin nucleating machinery, the actin‐related protein 2/3 (Arp2/3) complex, leading to the formation and remodeling of cortical actin filament networks. Cortical actin regulation is critical in many aspects of cell physiology including cell–cell adhesion and cell motility, whose dysregulation is directly associated with cancer invasion and metastasis. In line with this association, the WASP and WAVE family proteins have been reported to be involved in cancer malignancies. What is puzzling, however, is that they can act as either enhancers or suppressors of cancer malignancies depending on the type of cancer and its pathological stage. We are still far from understanding the roles of the WASP and WAVE family proteins in cancer progression. Here, we summarize the recent advances of studies of the WASP and WAVE family proteins with respect to cancer invasion and we offer a model that can account for the diverse outcomes originating from dysregulated WASP and WAVE family proteins in cancer development. (Cancer Sci 2010.)

To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer. We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0-2 colorectal cancer (82.8%). Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer.

Recently, metabolome analysis has been increasingly applied to biomarker detection and disease diagnosis in medical studies. Metabolome analysis is a strategy for studying the characteristics and interactions of low molecular weight metabolites under a specific set of conditions and is performed using mass spectrometry and nuclear magnetic resonance spectroscopy. There is a strong possibility that changes in metabolite levels reflect the functional status of a cell because alterations in their levels occur downstream of DNA, RNA, and protein. Therefore, the metabolite profile of a cell is more likely to represent the current status of a cell than DNA, RNA, or protein. Thus, owing to the rapid development of mass spectrometry analytical techniques metabolome analysis is becoming an important experimental method in life sciences including the medical field. Here, we describe metabolome analysis using liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry (GC–MS), capillary electrophoresis–mass spectrometry, and matrix assisted laser desorption ionization–mass spectrometry. Then, the findings of studies about GC–MS-based metabolome analysis of gastroenterological diseases are summarized, and our research results are also introduced. Finally, we discuss the realization of disease diagnosis by metabolome analysis. The development of metabolome analysis using mass spectrometry will aid the discovery of novel biomarkers, hopefully leading to the early detection of various diseases.

Phosphoinositides play pivotal roles in the regulation of cancer cell phenotypes. Among them, phosphatidylinositol 3,4‐bisphosphate (PI(3,4)P2) localizes to the invadopodia, and positively regulates tumor cell invasion. In this study, we examined the effect of PI(3,4)P2 on focal adhesion dynamics in MDA‐MB‐231 basal breast cancer cells. Knockdown of SHIP2, a phosphatidylinositol 3,4,5‐trisphosphatase (PIP3) 5‐phosphatase that generates PI(3,4)P2, in MDA‐MB‐231 breast cancer cells, induced the development of focal adhesions and cell spreading, leading to the suppression of invasion. In contrast, knockdown of PTEN, a 3‐phosphatase that de‐phosphorylates PIP3 and PI(3,4)P2, induced cell shrinkage and increased cell invasion. Interestingly, additional knockdown of SHIP2 rescued these phenotypes. Overexpression of the TAPP1 PH domain, which binds to PI(3,4)P2, and knockdown of Lpd, a downstream effector of PI(3,4)P2, resulted in similar phenotypes to those induced by SHIP2 knockdown. Taken together, our results suggest that inhibition of PI(3,4)P2 generation and/or downstream signaling could be useful for inhibiting breast cancer metastasis. Phosphoinositides have distinct roles in basal breast cancer phenotypes. In MDA‐MB‐231 cells, which have high PTEN expression, PTEN predominantly regulates PIP3 levels, and is related to PI3‐kinase signaling. In contrast, SHIP2 plays a role in the generation of PI(3,4)P2, which in turn leads to Lpd signaling and lamellipodia formation.

Multiple functionally independent pools of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] have been postulated to occur in the cell membrane, but the existing techniques lack sufficient resolution to unequivocally confirm their presence. To analyze the distribution of PI(4,5)P₂ at the nanoscale, we developed an electron microscopic technique that probes the freeze-fractured membrane preparation by the pleckstrin homology domain of phospholipase C-δ1. This method does not require chemical fixation or expression of artificial probes, it is applicable to any cell in vivo and in vitro, and it can define the PI(4,5)P₂ distribution quantitatively. By using this method, we found that PI(4,5)P₂ is highly concentrated at the rim of caveolae both in cultured fibroblasts and mouse smooth muscle cells in vivo. PI(4,5)P₂ was also enriched in the coated pit, but only a low level of clustering was observed in the flat undifferentiated membrane. When cells were treated with angiotensin II, the PI(4,5)P₂ level in the undifferentiated membrane decreased to 37.9% within 10 sec and then returned to the initial level. Notably, the PI(4,5)P₂ level in caveolae showed a slower but more drastic change and decreased to 20.6% at 40 sec, whereas the PI(4,5)P₂ level in the coated pit was relatively constant and decreased only to 70.2% at 10 sec. These results show the presence of distinct PI(4,5)P₂ pools in the cell membrane and suggest a unique role for caveolae in phosphoinositide signaling.

Objective The roles that amino acids play in immunity and inflammation are well defined, and the relationship between inflammatory bowel disease (IBD) and certain amino acids has recently attracted attention. In this study, the levels of amino acids and trichloroacetic acid (TCA) cycle-related molecules in the colonic tissues and sera of patients with ulcerative colitis (UC) were profiled by gas chromatography/mass spectrometry (GC/MS), with the aim of evaluating whether the clinical state induced by UC leads to variations in the amino acid profile. Materials and methods Colonic biopsy samples from 22 UC patients were used, as well as serum samples from UC patients ( n  = 13), Crohn’s disease (CD) patients ( n  = 21), and healthy volunteers ( n  = 17). Results In the GC/MS-based profiling of amino acids and TCA cycle-related molecules, lower levels of 16 amino acids and 5 TCA cycle-related molecules were observed in the colonic lesion tissues of the UC patients, and the serum profiles of amino acids and TCA cycle-related molecules of the UC patients were different from those of the CD patients and healthy volunteers. Conclusions Our study raises the possibility that GC/MS-based profiling of amino acids and TCA cycle-related molecules is a useful early diagnostic tool for UC.