Early ECMO initiation in the emergency department for refractory hypoxemic respiratory failure caused by NaDCC intoxication.

We describe a case of acute respiratory failure caused by inhalation of gas formed from a reaction of intentional dissolution of sodium dichloroisocyanurate (NaDCC) tablets in water. A patient had refractory respiratory failure despite the use of conventional therapy, including lung-protective mechanical ventilation. Early veno-venous extracorporeal membrane oxygenation (VV-ECMO) support was initiated in the emergency department (ED). The patient was weaned from ECMO on hospital day 6 and discharged from the ICU on hospital day 27. Cases of severe inhalation injury with acute respiratory failure refractory to conventional treatments and mechanical ventilator support may benefit from VV-ECMO. Literature on early initiation of ED-VV-ECMO in NaDCC-induced refractory respiratory failure is rare. This case may be used as a guide in the management of subsequent cases as it shows that early initiation of ED-VV-ECMO was beneficial to the patient.

Src-mediated phosphorylation of ßPix-b regulates dendritic spine morphogenesis.

PAK-interacting guanine nucleotide exchange factor (ßPix; also known as Arhgef7) has been implicated in many actin-based cellular processes, including spine morphogenesis in neurons. However, the molecular mechanisms by which ßPix controls spine morphology remain elusive. Previously, we have reported the expression of several alternative spliced ßPix isoforms in the brain. Here, we report a novel finding that the b isoform of ßPix (ßPix-b) mediates the regulation of spine and synapse formation. We found that ßPix-b, which is mainly expressed in neurons, enhances spine and synapse formation through preferential localization at spines. In neurons, glutamate treatment efficiently stimulates Rac1 GEF activity of ßPix-b. The glutamate stimulation also promotes Src-mediated phosphorylation of ßPix-b in both an AMPA receptor- and NMDA receptor-dependent manner. Tyrosine 598 (Y598) of ßPix-b is identified as the major Src-mediated phosphorylation site. Finally, Y598 phosphorylation of ßPix-b enhances its Rac1 GEF activity that is critical for spine and synapse formation. In conclusion, we provide a novel mechanism by which ßPix-b regulates activity-dependent spinogenesis and synaptogenesis via Src-mediated phosphorylation.

Clinical relationship between blood concentration and clinical symptoms in aconitine intoxication.

BACKGROUND: Aconitine is well-known for its potential analgesic, anti-inflammatory, and circulation promoting effects and has been widely used as a folk medicine in South Korea. Owing to its extremely toxic nature and relatively low safety margin, intoxication is sometimes fatal. The toxic compound mainly affects the central nervous system, heart, and muscle, resulting in cardiovascular complications. PURPOSE: To determine the exact relationship between blood concentration of aconitine and clinical manifestation. BASIC PROCEDURES: The National Forensic Service (NFS) was commissioned to assist in a quantitative analysis of highly toxic aconitine and corresponding blood concentrations by analyzing the body fluids of three patients who were suspected of aconitine poisoning. MAIN FINDINGS: Aconitine blood values tested by the NFS showed that patients with a blood concentration below a certain level developed symptoms slowly and showed a high severity of clinical manifestation. There was no correlation between blood concentration and symptoms or ECG results. CONCLUSIONS: In case of suspected aconitine poisoning, an emergency care department should be visited, even with symptomatic improvement, and the patient should be monitored for at least 24 h, depending on the level of recovery and changes in ECG results.

Intramembrane proteolysis of an extracellular serine protease, epithin/PRSS14, enables its intracellular nuclear function.

BACKGROUND: Epithin/PRSS14, a type II transmembrane serine protease, is an emerging target of cancer therapy because of its critical roles in tumor progression and metastasis. In many circumstances, the protease, through its ectodomain shedding, exists as a soluble form and performs its proteolytic functions in extracellular environments increasing cellular invasiveness. The seemingly functional integrity of the soluble form raises the question of why the protease is initially made as a membrane-associated protein. RESULTS: In this report, we show that the epithin/PRSS14 intracellular domain (EICD) can be released from the membrane by the action of signal peptide peptidase-like 2b (SPPL2b) after ectodomain shedding. The EICD preferentially localizes in the nucleus and can enhance migration, invasion, and metastasis of epithelial cancer when heterologously expressed. Unbiased RNA-seq analysis and subsequent antibody arrays showed that EICD could control the gene expression of chemokines involved in cell motility, by increasing their promoter activities. Finally, bioinformatics analysis provided evidence for the clinical significance of the intramembrane proteolysis of epithin/PRSS14 by revealing that the poor survival of estrogen receptor (ER)-negative breast cancer patients with high epithin/PRSS14 expression is further worsened by high levels of SPPL2b. CONCLUSIONS: These results show that ectodomain shedding of epithin/PRSS14 can initiate a unique and synchronized bidirectional signal for cancer metastasis: extracellularly broadening proteolytic modification of the surrounding environment and intracellularly reprogramming the transcriptome for metastatic conversion. Clinically, this study also suggests that the intracellular function of epithin/PRSS14 should be considered for targeting this protease for anti-cancer treatment.

ßPix heterozygous mice have defects in neuronal morphology and social interaction.

ßPix activates Rho family small GTPases, Rac1 and Cdc42 as a guanine nucleotide exchange factor. Although overexpression of ßPix in cultured neurons indicates that ßPix is involved in spine morphogenesis and synapse formation in vitro, the in vivo role of ßPix in the neuron is not well understood. Recently, we generated ßPix knockout mice that showed lethality at embryonic day 9.5. Here, we investigate the neuronal role of ßPix using ßPix heterozygous mice that are viable and fertile. ßPix heterozygous mice show decreased expression levels of ßPix proteins in various tissues including the brain. Cultured hippocampal neurons from ßPix heterozygous mice show a decrease in neurite length and complexity as well as synaptic density. Both excitatory and inhibitory synapse densities are decreased in these neurons. Golgi-staining of hippocampal tissues from the brain of these mice show reduced dendritic complexity and spine density in the hippocampal neurons. Expression levels of NMDA- and AMPA-receptor subunits and Git1 protein in hippocampal tissues are also decreased in these mice. Behaviorally, ßPix heterozygous mice exhibit impaired social interaction. Altogether, these results indicate that ßPix is required for neurite morphogenesis and synapse formation, and the reduced expression of ßPix proteins results in a defect in social behavior.

Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains.

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-ß TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.

Vimentin filaments regulate integrin-ligand interactions by binding to the cytoplasmic tail of integrin ß3.

Vimentin, an intermediate filament protein induced during epithelial-to-mesenchymal transition, is known to regulate cell migration and invasion. However, it is still unclear how vimentin controls such behaviors. In this study, we aimed to find a new integrin regulator by investigating the H-Ras-mediated integrin suppression mechanism. Through a proteomic screen using the integrin ß3 cytoplasmic tail protein, we found that vimentin might work as an effector of H-Ras signaling. H-Ras converted filamentous vimentin into aggregates near the nucleus, where no integrin binding can occur. In addition, an increase in the amount of vimentin filaments accessible to the integrin ß3 tail enhanced talin-induced integrin binding to its ligands by inducing integrin clustering. In contrast, the vimentin head domain, which was found to bind directly to the integrin ß3 tail and compete with endogenous vimentin filaments for integrin binding, induced nuclear accumulation of vimentin filaments and reduced the amount of integrin-ligand binding. Finally, we found that expression of the vimentin head domain can reduce cell migration and metastasis. From these data, we suggest that filamentous vimentin underneath the plasma membrane is involved in increasing integrin adhesiveness, and thus regulation of the vimentin-integrin interaction might control cell adhesion.

Disruption of TACE-filamin interaction can inhibit TACE-mediated ectodomain shedding.

Ectodomain shedding regulates functions of many membrane proteins through the cleavage of their juxtamembrane region mainly by a disintegrin and metalloproteinase family proteinases. Tumor necrosis factor-alpha converting enzyme (TACE) is known to be responsible for phorbol myristate acetate (PMA)-induced shedding of various membrane proteins. How PMA regulates TACE-dependent shedding and how TACE exhibits substrate specificity without proteolysis of other membrane proteins are questionable. Here, we show that TACE can interact with an actin-binding protein, filamin, through 20th filamin repeat. We found that the interaction between TACE and filamin was increased by PMA treatment. In addition, loss of filamin or specific disruption of TACE-filamin interaction inhibited ectodomain shedding of representative TACE substrates, CD44 and amyloid protein precursor. From these data, we suggest that filamin may work as a scaffold that can recruit TACE and its substrates in a PMA-dependent manner to achieve substrate specificity for TACE.

Calmodulin Mediates Ca2+-Dependent Inhibition of Tie2 Signaling and Acts as a Developmental Brake During Embryonic Angiogenesis.

OBJECTIVE: Angiogenesis, the process of building complex vascular structures, begins with sprout formation on preexisting blood vessels, followed by extension of the vessels through proliferation and migration of endothelial cells. Based on the potential therapeutic benefits of preventing angiogenesis in pathological conditions, many studies have focused on the mechanisms of its initiation as well as control. However, how the extension of vessels is terminated remains obscure. Thus, we investigated the negative regulation mechanism. APPROACH AND RESULTS: We report that increased intracellular calcium can induce dephosphorylation of the endothelial receptor tyrosine kinase Tie2. The calcium-mediated dephosphorylation was found to be dependent on Tie2-calmodulin interaction. The Tyr1113 residue in the C-terminal end loop of the Tie2 kinase domain was mapped and found to be required for this interaction. Moreover, mutation of this residue into Phe impaired both the Tie2-calmodulin interaction and calcium-mediated Tie2 dephosphorylation. Furthermore, expressing a mutant Tie2 incapable of binding to calmodulin or inhibiting calmodulin function in vivo causes unchecked growth of the vasculature in Xenopus. Specifically, knockdown of Tie2 in Xenopus embryo retarded the sprouting and extension of intersomitic veins. Although human Tie2 expression in the Tie2-deficient animals almost completely rescued the retardation, the Tie2(Y1113F) mutant caused overgrowth of intersomitic veins with strikingly complex and excessive branching patterns. CONCLUSIONS: We propose that the calcium/calmodulin-dependent negative regulation of Tie2 can be used as an inhibitory signal for vessel growth and branching to build proper vessel architecture during embryonic development.

Filamin A is required in injured axons for HDAC5 activity and axon regeneration.

Microtubule dynamics are important for axon growth during development as well as axon regeneration after injury. We have previously identified HDAC5 as an injury-regulated tubulin deacetylase that functions at the injury site to promote axon regeneration. However, the mechanisms involved in the spatial control of HDAC5 activity remain poorly understood. Here we reveal that HDAC5 interacts with the actin binding protein filamin A via its C-terminal domain. Filamin A plays critical roles in HDAC5-dependent tubulin deacetylation because, in cells lacking filamin A, the levels of acetylated tubulin are elevated markedly. We found that nerve injury increases filamin A axonal expression in a protein synthesis-dependent manner. Reducing filamin A levels or interfering with the interaction between HDAC5 and filamin A prevents injury-induced tubulin deacetylation as well as HDAC5 localization at the injured axon tips. In addition, neurons lacking filamin A display reduced axon regeneration. Our findings suggest a model in which filamin A local translation following axon injury controls localized HDAC5 activity to promote axon regeneration.

DW1029M, a novel botanical drug candidate, inhibits advanced glycation end-product formation, rat lens aldose reductase activity, and TGF-ß1 signaling.

DW1029M is a botanical extract consisting of Morus bark and Puerariae radix, produced by Dong-Wha Pharmaceutical, for nephroprotective drug development; it has been in phase II clinical trials in Korea. In our mechanistic investigations, we found that DW1029M inhibits advanced glycation end products (AGEs), rat lens aldose reductase (RLAR), and transforming growth factor (TGF)-ß1 signaling, all of which are implicated in diabetic complications such as diabetic nephropathy and diabetic retinopathy. DW1029M inhibits AGE formation via Fe(2+) chelation. The extract contains 13 active constituents that inhibit AGE formation, 8 active constituents that inhibit RLAR activity, and 1 inhibitor of TGF-ß1 signaling. Our results suggest DW1029M protects against diabetic nephropathy via blockade of AGE formation, RLAR activity, and TGF-ß1 signaling.

Shedding of epithin/PRSS14 is induced by TGF-ß and mediated by tumor necrosis factor-α converting enzyme.

Epithin/PRSS14, a type II transmembrane serine protease, plays critical roles in cancer metastasis. Previously, we have reported that epithin/PRSS14 undergoes ectodomain shedding in response to phorbol myristate acetate (PMA) stimulation. In this study, we show that transforming growth factor-ß (TGF-ß) induces rapid epithin/PRSS14 shedding through receptor mediated pathway in 427.1.86 thymoma cells. Tumor necrosis factor-α converting enzyme (TACE) is responsible for this shedding. Amino acid sequence encompassing the putative shedding cleavage site of epithin/PRSS14 exhibit strong homology to the cleavage site of l-selectin, a known TACE substrate. TACE inhibitor, TAPI-0 and TACE siRNA greatly reduced TGF-ß-induced epithin/PRSS14 shedding. TGF-ß treatment induces translocation of intracellular pool of TACE to the membrane where epithin/PRSS14 resides. These findings suggest that TGF-ß induces epithin/PRSS14 shedding by mediating translocation of epithin/PRSS14 sheddase, TACE, to the membrane.

Activity-dependent modulation of the interaction between CaMKIIα and Abi1 and its involvement in spine maturation.

Remodeling of dendritic spines through regulation of actin dynamics is a key event in activity-dependent structural plasticity. However, the molecular mechanism underlying this process is poorly understood. Here, we show that activity-dependent modulation of Abl interactor 1-Ca(2+)/calmodulin-dependent kinase IIα (Abi1-CaMKIIα) interaction, and thereby their activity, is important for regulation of spine morphology in cultured rat hippocampal neurons. Abi1 interacts with CaMKIIα at resting conditions through Abi1's tSNARE (target membrane-associated SNARE), which harbors striking homology with CaMKIIα regulatory domain. The interaction of the two proteins, Abi1 and CaMKIIα, results in their simultaneous inhibition, inhibition of CaMKIIα activity, and also inhibition of Abi1-dependent Rac activation. Their functional impediment is released when they dissociate from each other by calmodulin binding through glutamate receptor activation. Before dissociation, Abi1 is phosphorylated by CaMKIIα at serine 88, which may involve in regulation of Rac activation and spine maturation. Our results suggest that modulation of the interaction between Abi1 and CaMKIIα, through the glutamate receptor pathway, may be a molecular mechanism underlying activity-regulated structural plasticity in rat hippocamapal neurons.

Epithin/PRSS14 proteolytically regulates angiopoietin receptor Tie2 during transendothelial migration.

Epithin/PRSS14, a type II transmembrane serine protease, is involved in normal epithelial development and tumor progression. Here we report, as an interacting substrate of epithin, a receptor tyrosine kinase Tie2 that is well known for important roles in the vessel stability. Epithin interacts with and degrades the Tie2 extracellular portion that contains the ligand-binding domain. Epithin is located in the neighbor of Tie2-expressing vessels in normal tissue. Furthermore, epithin can cleave and degrade Tie2 not only in the same cell but also from neighboring cells nearby, resulting in the degradation of the Tie2 ectodomain. The remaining Tie2 fragment was highly phosphorylated and was able to recruit a downstream effector, phosphatidylinositol 3-kinase. Knocking down epithin expression using short hairpin RNA in thymoma cell severely impaired the migration through endothelial cells that show the actin rearrangement during the process. The diminution of epithin protein expression in 4T1 breast cancer cells caused the significant decrease in the number of transendothelial migrating cells in vitro as well as in those of metastasizing tumor nodules in vivo, Therefore, we propose that epithin, which regulates endothelial Tie2 functions, plays a critical role in the fine tuning of transendothelial migration for normal and cancer cells.

Retropancreatic fascia is absent along the pancreas facing the superior mesenteric artery: a histological study using elderly donated cadavers.

To determine the fascial configuration between the superior mesenteric artery and vein and the posterior aspect of the pancreas, we examined histological sections of 10 elderly donated cadavers without pathology in the abdomen. The retropancreatic fascia was absent along the pancreatic parenchyma facing the artery and vein. Abundant nerves along the artery were separated from the pancreas by loose tissue almost 10 mm in thickness. In addition, anterior renal fasciae facing the pancreatic body were not evident in these specimens, possibly due to the degeneration of the left adrenal gland with age. Thus, a definite renal fascia was restricted on the lateral and posterior sides of the left kidney. These findings suggest that interactions between a pancreatic tumor and nerves would require migration of cancer cells over a long distance. Conversely, attachment of the enlarged tumor mass to the nerves may be necessary for the invasion. The anterior renal fascia may fuse with the retropancreatic fascia.

Neuronal specific ßPix-b stimulates actin-dependent processes via the interaction between its PRD and WH1 domain of N-WASP.

ßPix, a Pak-interacting nucleotide exchange factor (Cool-1/p85SPR), is a Cdc42/Rac1-specific guanine nucleotide exchange factor (GEF) involved in various actin-related processes. Many previous studies have focused on ubiquitously expressed ßPix-a, while the role of the neuronal-specific isoform ßPix-b is still unknown, especially whether its role is distinct from or similar to ßPix-a. Here we show that unlike ßPix-a, overexpression of ßPix-b stimulates actin-dependent comet formation in BHK21 cells. This effect is attributed to the interaction between its proline-rich domain (PRD) and the WH1 domain of N-WASP. In addition, we show that overexpression of ßPix-b stimulates actin-dependent dendritic spine formation in rat hippocampal neurons in culture, a formation that is blocked by co-expression of the WH1 domain of N-WASP or the PRD of ßPix-b. Knocking-down endogenous expression of ßPix-b by shRNA reduced the number of dendritic spines, which were rescued only by PRD-containing ßPix-b mutants. GEF activity of ßPix-b is also required for these effects. The results show that neuronal-specific ßPix-b stimulates actin-dependent processes in cells via the interaction between its PRD and the WH1 domain of N-WASP. Our results identify N-WASP as the first protein shown to interact with the PRD of ßPix-b, raising the possibility that, as an N-WASP WH1-binding protein, ßPix-b may regulate N-WASP's activity in cells.

2',4',6'-Tris(methoxymethoxy) chalcone (TMMC) attenuates the severity of cerulein-induced acute pancreatitis and associated lung injury.

Acute pancreatitis (AP) is an inflammatory disease involving acinar cell injury and rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. 2',4',6'-Tris (methoxymethoxy) chalcone (TMMC), a synthetic chalcone derivative, displays potent anti-inflammatory effects. Therefore, we aimed to investigate whether TMMC might affect the severity of AP and pancreatitis-associated lung injury in mice. We used the cerulein hyperstimulation model of AP. Severity of pancreatitis was determined in cerulein-injected mice by histological analysis and neutrophil sequestration. The pretreatment of mice with TMMC reduced the severity of AP and pancreatitis-associated lung injury and inhibited several biochemical parameters (activity of amylase, lipase, trypsin, trypsinogen, and myeloperoxidase and production of proinflammatory cytokines). In addition, TMMC inhibited pancreatic acinar cell death and production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 by inhibiting NF-κB and extracellular signal-regulated protein kinase 1/2 (ERK1/2) activation. Neutralizing antibodies for TNF-α, IL-1ß, and IL-6 inhibited cerulein-induced cell death in isolated pancreatic acinar cells. Moreover, pharmacological blockade of NF-κB/ERK1/2 reduced acinar cell death and production of TNF-α, IL-1ß, and IL-6 in isolated pancreatic acinar cells. In addition, posttreatment of mice with TMMC showed reduced severity of AP and lung injury. Our results suggest that TMMC may reduce the complications associated with pancreatitis.

PRSS14/Epithin is induced in macrophages by the IFN-γ/JAK/STAT pathway and mediates transendothelial migration.

PRSS14/Epithin (also known as matriptase and ST14), a member of the type II transmembrane serine proteases, is primarily found in a subpopulation of normal epithelial cells and in epithelial cancers. Its known functions include maintaining the epithelial barrier, thymic development, and cancer progression. In this study, we show that several macrophage cell lines and activated bone marrow-derived macrophages also express PRSS14/Epithin. Surface expression, as well as cytoplasmic expression, was detectable upon activation by IFN-γ, but not TNF-α or TGF-ß. Induction of the protein appeared to be restricted to macrophages. IFN-γ showed a biphasic regulation in RAW264.7 cells, and upregulated expression was sustained for several days. This induction by IFN-γ was partially through the increase of PRSS14/Epithin mRNA production, which is downstream of the JAK pathway, shown by the inhibition by tyrphostin AG490. Using chromatin immunoprecipitation, we verified that two sites among six putative STAT1 binding sites in the PRSS14/Epithin promoter were occupied by STAT1 upon activation. Treatment with IFN-γ enhanced the serum-triggered transendothelial migration of RAW264.7 cells, but not that of PRSS14/Epithin knock-down RAW264.7 cells, although they express multiple markers such as ICAM1, CD80, and CD40 at normal levels. These data strongly suggest that PRSS14/Epithin plays an important role in the transendothelial migration of activated macrophages in the inflammatory microenvironment, and the mode of action is similar to the events in cancer metastasis.

Interaction of microtubules and actin with the N-terminus of ßPix-b(L) directs cellular pinocytosis.

ßPix is a Rac/Cdc42 guanine nucleotide exchange factor (GEF) that is known to be a regulator of actin cytoskeleton remodeling. Recently, a novel splicing isoform, ßPix-b(L), was identified as an alternative translational product of the ßPix-b mRNA with an extended N-terminus comprising a partial calponin homology (CH) domain and a serine-rich (SR) domain. However, the cellular function of ßPix-b(L) is largely unknown. In the current study, we analyzed the genomic DNA structure and cellular functions of ßPix-b(L). The results of this study demonstrate that ßPix is composed of 24 exons and 21 introns spanning around 100 kb. RT-PCR experiments revealed that there are two forms of ßPix mRNA with distinct 5' UTRs that are the result of alternative splicing of exon 1 and 2 from ßPix genomic DNA. In addition, affinity chromatography analysis and a pull-down assay with the N-terminal region of ßPix-b(L) revealed that ßPix-b(L) interacts with tubulin and actin via its N-terminal CH and SR domains, respectively. Interaction with tubulin enabled ßPix-b(L) to bundle the microtubule and form membrane protrusions. Furthermore, the N-terminus of ßPix-b(L) was also critical for its localization to cellular vesicles. Functionally, ßPix-b(L) induced pinocytosis through cooperative action of the CH and Dbl homology (DH) domains, demonstrating the role of ßPix-b(L) in the regulation of membrane dynamics.

Beta-Pix-dynamin 2 complex promotes colorectal cancer progression by facilitating membrane dynamics.

PURPOSE: Spatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (ßPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying ßPix-mediated cancer cell invasion remain unclear. METHODS: The clinical significance of ßPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for ßPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of ßPix on CRC progression. A ßPix-SH3 antibody delivery system was used to determine the effects of the ßPix-Dyn2 complex in CRC cells. RESULTS: We found that the Src homology 3 (SH3) domain of ßPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The ßPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of ßPix enhanced ßPix-Dyn2 complex formation. Disruption of the ßPix-Dyn2 complex by ßPix-SH3 antibodies targeting intracellular ßPix inhibited CRC cell invasion. CONCLUSIONS: Our data indicate that spatiotemporal regulation of the Src-ßPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the ßPix-Dyn2 complex may be a useful therapeutic strategy for CRC.