In colon cancer cells fascin1 regulates adherens junction remodeling.

Adherens junctions (AJs) are a defining feature of all epithelial cells. They regulate epithelial tissue architecture and integrity, and their dysregulation is a key step in tumor metastasis. AJ remodeling is crucial for cancer progression, and it plays a key role in tumor cell survival, growth, and dissemination. Few studies have examined AJ remodeling in cancer cells consequently, it remains poorly understood and unleveraged in the treatment of metastatic carcinomas. Fascin1 is an actin-bundling protein that is absent from the normal epithelium but its expression in colon cancer is linked to metastasis and increased mortality. Here, we provide the molecular mechanism of AJ remodeling in colon cancer cells and identify for the first time, fascin1's function in AJ remodeling. We show that in colon cancer cells fascin1 remodels junctional actin and actomyosin contractility which makes AJs less stable but more dynamic. By remodeling AJs fascin1 drives mechanoactivation of WNT/ß-catenin signaling and generates "collective plasticity" which influences the behavior of cells during cell migration. The impact of mechanical inputs on WNT/ß-catenin activation in cancer cells remains poorly understood. Our findings highlight the role of AJ remodeling and mechanosensitive WNT/ß-catenin signaling in the growth and dissemination of colorectal carcinomas.

Villin-1 and Gelsolin Regulate Changes in Actin Dynamics That Affect Cell Survival Signaling Pathways and Intestinal Inflammation.

BACKGROUND & AIMS: Cell stress signaling pathways result in phosphorylation of the eukaryotic translation initiation factor 2 subunit alpha (EIF2S1 or EIF2A), which affects regulation of protein translation. Translation reprogramming mitigates stress by activating pathways that result in autophagy and cell death, to eliminate damaged cells. Actin is modified during stress and EIF2A is dephosphorylated to restore homeostasis. It is not clear how actin affects EIF2A signaling. We studied the actin-binding proteins villin 1 (VIL1) and gelsolin (GSN) in intestinal epithelial cells (IECs) to determine whether they respond to cell stress response and affect signaling pathways. METHODS: We performed studies with mice with disruptions in Vil1 and Gsn (double-knockout mice). Wild-type (WT) mice either were or were not (controls) exposed to cell stressors such as tumor necrosis factor and adherent-invasive Escherichia coli. Distal ileum tissues were collected from mice; IECs and enteroids were cultured and analyzed by histology, immunoblots, phalloidin staining, immunohistochemistry, electron microscopy, and flow cytometry. HT-29 cells were incubated with cell stressors such as DTT, IFN, and adherent-invasive E coli or control agents; cells were analyzed by immunoblots and quantitative polymerase chain reaction. Green fluorescent protein and green fluorescent protein tagged mutant EIF2A were expressed from a lentiviral vector. The mouse immunity-related GTPase (IRGM1) was overexpressed in embryonic fibroblasts from dynamin1 like (DNM1L) protein-knockout mice or their WT littermates. IRGM1 was overexpressed in embryonic fibroblasts from receptor interacting serine/threonine kinase 1-knockout mice or their WT littermates. Human IRGM was overexpressed in human epithelial cell lines incubated with the DNM1L-specific inhibitor Mdivi-1. Mitochondria were analyzed by semi-quantitative confocal imaging. We performed immunohistochemical analyses of distal ileum tissues from 6-8 patients with Crohn's disease (CD) and 6-8 individuals without CD (controls). RESULTS: In IECs exposed to cell stressors, EIF2A signaling reduced expression of VIL1 and GSN. However, VIL1 and GSN were required for dephosphorylation of EIF2A and recovery from cell stress. In mouse and human IECs, prolonged, unresolved stress was accompanied by continued down-regulation of VIL1 and GSN, resulting in constitutive phosphorylation of EIF2A and overexpression of IRGM1 (or IRGM), which regulates autophagy. Overexpression of IRGM1 (or IRGM) induced cell death by necroptosis, accompanied by release of damage-associated molecular patterns (DAMPs). In double-knockout mice, constitutive phosphorylation of EIF2A and over-expression of IRGM1 resulted in spontaneous ileitis that resembled human CD in symptoms and histology. Distal ileum tissues from patients with CD had lower levels of VIL1 and GSN, increased phosphorylation of EIF2A, increased levels of IRGM and necroptosis, and increased release of nuclear DAMPs compared with controls. CONCLUSIONS: In studies of intestinal epithelial tissues from patients with CD and embryonic fibroblasts from mice, along with enteroids and human IEC lines, we found that induction of cell stress alters the cytoskeleton in IECs via changes in the actin-binding proteins VIL1 and GSN. Acute changes in actin dynamics increase IEC survival, whereas long-term changes in actin dynamics lead to IEC death and intestinal inflammation. IRGM regulates necroptosis and release of DAMPs to induce gastrointestinal inflammation, linking IRGM activity with CD.

Impaired Relaxation of Airway Smooth Muscle in Mice Lacking the Actin-Binding Protein Gelsolin.

Diverse classes of ligands have recently been discovered that relax airway smooth muscle (ASM) despite a transient increase in intracellular calcium concentrations ([Ca2+]i). However, the cellular mechanisms are not well understood. Gelsolin is a calcium-activated actin-severing and -capping protein found in many cell types, including ASM cells. Gelsolin also binds to phosphatidylinositol 4,5-bisphosphate, making this substrate less available for phospholipase Cß-mediated hydrolysis to inositol triphosphate and diacylglycerol. We hypothesized that gelsolin plays a critical role in ASM relaxation and mechanistically accounts for relaxation by ligands that transiently increase [Ca2+]i. Isolated tracheal rings from gelsolin knockout (KO) mice showed impaired relaxation to both a ß-agonist and chloroquine, a bitter taste receptor agonist, which relaxes ASM, despite inducing transiently increased [Ca2+]i. A single inhalation of methacholine increased lung resistance to a similar extent in wild-type and gelsolin KO mice, but the subsequent spontaneous relaxation was less in gelsolin KO mice. In ASM cells derived from gelsolin KO mice, serotonin-induced Gq-coupled activation increased both [Ca2+]i and inositol triphosphate synthesis to a greater extent compared to cells from wild-type mice, possibly due to the absence of gelsolin binding to phosphatidylinositol 4,5-bisphosphate. Single-cell analysis showed higher filamentous:globular actin ratio at baseline and slower cytoskeletal remodeling dynamics in gelsolin KO cells. Gelsolin KO ASM cells also showed an attenuated decrease in cell stiffness to chloroquine and flufenamic acid. These findings suggest that gelsolin plays a critical role in ASM relaxation and that activation of gelsolin may contribute to relaxation induced by ligands that relax ASM despite a transient increase in [Ca2+]i.

Regulation of directional cell migration by membrane-induced actin bundling.

During embryonic development and in metastatic cancers, cells detach from the epithelium and migrate with persistent directionality. Directional cell migration is also crucial for the regeneration and maintenance of the epithelium and impaired directional migration is linked to chronic inflammatory diseases. Despite its significance, the mechanisms controlling epithelial cell migration remain poorly understood. Villin is an epithelial-cell-specific actin modifying protein that regulates epithelial cell plasticity and motility. In motile cells villin is associated with the highly branched and the unbranched actin filaments of lamellipodia and filopodia, respectively. In this study we demonstrate for the first time that villin regulates directionally persistent epithelial cell migration. Functional characterization of wild-type and mutant villin proteins revealed that the ability of villin to self-associate and bundle actin as well as its direct interaction with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] regulates villin-induced filopodial assembly and directional cell migration. Our findings suggest that convergence of different signaling cascades could spatially restrict villin activity to areas of high PtdIns(4,5)P(2) and F-actin concentration to assemble filopodia. Furthermore, our data reveal the ability of villin to undergo actin- and PtdIns(4,5)P(2)-induced self-association, which may be particularly suited to coalesce and reorganize actin bundles within the filopodia.

Differential effects of lysophosphatidic acid and phosphatidylinositol 4,5-bisphosphate on actin dynamics by direct association with the actin-binding protein villin.

We have previously reported that the epithelial cell-specific actin-binding protein villin directly associates with phosphatidylinositol 4,5-bisphosphate (PIP(2)) through three binding sites that overlap with actin-binding sites in villin. As a result, association of villin with PIP(2) inhibits actin depolymerization and enhances actin cross-linking by villin. In this study, we demonstrate that these three PIP(2)-binding sites also bind the more hydrophilic phospholipid, lysophosphatidic acid (LPA) but with a higher affinity than PIP(2) (dissociation constant (K(d)) of 22 mum versus 39.5 mum for PIP(2)). More interestingly, unlike PIP(2), the association of villin with LPA inhibits all actin regulatory functions of villin. In addition, unlike PIP(2), LPA dramatically stimulates the tyrosine phosphorylation of villin by c-Src kinase. These studies suggest that in cells, selective interaction of villin with either PIP(2) or LPA could have dramatically different outcomes on actin reorganization as well as phospholipid-regulated cell signaling. These studies provide a novel regulatory mechanism for phospholipid-induced changes in the microfilament structure and cell function and suggest that LPA could be an intracellular regulator of the actin cytoskeleton.

Mouse intestinal tuft cells express advillin but not villin.

Tuft (or brush) cells are solitary chemosensory cells scattered throughout the epithelia of the respiratory and alimentary tract. The actin-binding protein villin (Vil1) is used as a marker of tuft cells and the villin promoter is frequently used to drive expression of the Cre recombinase in tuft cells. While there is widespread agreement about the expression of villin in tuft cells there are several disagreements related to tuft cell lineage commitment and function. We now show that many of these inconsistencies could be resolved by our surprising finding that intestinal tuft cells, in fact, do not express villin protein. Furthermore, we show that a related actin-binding protein, advillin which shares 75% homology with villin, has a tuft cell restricted expression in the gastrointestinal epithelium. Our study identifies advillin as a marker of tuft cells and provides a mechanism for driving gene expression in tuft cells but not in other epithelial cells of the gastrointestinal tract. Our findings fundamentally change the way we identify and study intestinal tuft cells.

F-actin-bundling sites are conserved in proteins with villin-type headpiece domains.

Villin is a major actin-bundling protein that assembles the brush border of intestinal and renal epithelial cells. The villin "headpiece" domain and the actin-binding residues within it regulate its actin-bundling function. Substantial experimental and theoretical information about the three-dimensional structure of the isolated villin headpiece, including a description of the actin-binding residues within the headpiece, is available. Despite that, the actin-bundling site in the full-length (FL) villin protein remains unidentified. We used this existing villin headpiece nuclear magnetic resonance data and performed mutational analysis and functional assays to identify the actin-bundling site in FL human villin protein. By careful evaluation of these conserved actin-binding residues in human advillin protein, we demonstrate their functional significance in the over 30 proteins that contain a villin-type headpiece domain. Our study is the first that combines the available structural data on villin headpiece with functional assays to identify the actin-binding residues in FL villin that regulate its filament-bundling activity. Our findings could have wider implications for other actin-bundling proteins that contain a villin-type headpiece domain.

Pediatric intrathecal baclofen management during the COVID-19 pandemic in the US and Canada.

The COVID-19 pandemic has been a challenge to healthcare systems around the world. Within pediatric rehabilitation medicine, management of intrathecal baclofen has been particularly challenging. This editorial reviews how programs in the US and Canada coped with the quickly changing healthcare environment and how we can learn from this pandemic to be prepared for future crises.

Regulation of cell structure and function by actin-binding proteins: villin's perspective.

Villin is a tissue-specific actin modifying protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells. It belongs to a large family of actin-binding proteins which includes actin-capping, -nucleating and/or -severing proteins such as gelsolin, severin, fragmin, adseverin/scinderin and actin crosslinking proteins such as dematin and supervillin. Studies done in epithelial cell lines and villin knock-out mice have demonstrated the function of villin in regulating actin dynamics, cell morphology, epithelial-to-mesenchymal transition, cell migration and cell survival. In addition, the ligand-binding properties of villin (F-actin, G-actin, calcium, phospholipids and phospholipase C-gamma1) are mechanistically important for the crosstalk between signaling pathways and actin reorganization in epithelial cells.

Regulation of cell motility by tyrosine phosphorylated villin.

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.

Perceived Exertion Is Lower When Using a Functional Electrical Stimulation Neuroprosthesis Compared With an Ankle-Foot Orthosis in Persons With Multiple Sclerosis: A Preliminary Study.

OBJECTIVE: This study investigates the direct comparison of energy cost, efficiency, and effort between an ankle-foot orthosis (AFO) and a functional electrical stimulation (FES) device for foot drop in ambulatory patients with multiple sclerosis. DESIGN: Twenty adults (32-74 years old; 55% female) with a diagnosis of multiple sclerosis resulting in foot drop participated in a crossover, counterbalanced preliminary study. Each participant was tested on different days, with each session consisting of a separate walk trial per prosthetic device. Heart rate, oxygen consumption, speed, distance, and self-reported exertion were measured independently for each device walk trial. Repeated-measures analysis of variance with device and visit number as within-participants independent variables were run for the primary outcome variables of perceived exertion, energy, and metabolic efficiency. RESULTS: A significant main effect of device was found for perceived exertion (P = 0.01), with participants reporting decreased exertion levels (Borg Scale) when using the FES compared with the AFO (mean difference, 1.63; 95% confidence interval, 0.49-2.76). Energy and efficiency did not significantly differ by device. CONCLUSION: Results suggest that further investigation of the potential advantages of FES devices over traditional AFO is warranted. TO CLAIM CME CREDITS: Complete the self-assessment activity and evaluation online at http://www.physiatry.org/JournalCME CME OBJECTIVES: Upon completion of this article, the reader should be able to: (1) describe the potential differences between an AFO and FES for the treatment of foot drop in patients with multiple sclerosis, (2) understand the mechanisms and prevalence of foot drop in patients with multiple sclerosis, and (3) recognize the potential benefit of improved perceived exertion found when using FES for the treatment of foot drop in patients with multiple sclerosis. LEVEL: Advanced ACCREDITATION: The Association of Academic Physiatrists is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.The Association of Academic Physiatrists designates this activity for a maximum of 1.5 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.

Intractable Seizures and Rehabilitation in Ciguatera Poisoning.

Ciguatera fish poisoning is the most frequently reported seafood toxin illness associated with the ingestion of contaminated tropical fish. Diagnosis relies on a history of recent tropical fish ingestion and subsequent development of gastrointestinal, cardiovascular, and neurological symptoms. Ciguatera poisoning usually has a self-limited time course, and its management involves symptomatic control and supportive care. This case report presents an uncommon case of ciguatera poisoning with prolonged intractable seizures refractory to standard antiseizure medications. The patient also had significant functional decline that responded to rigorous inpatient rehabilitation not previously described in literature.

Effect of Cognitive Demand on Functional Mobility in Ambulatory Individuals with Multiple Sclerosis.

BACKGROUND: As disease progresses, cognitive demands may affect functional mobility in individuals with multiple sclerosis (MS). The Timed Up and Go (TUG) test assesses functional mobilityin populationssuch as MS. A cognitive-demanding task can be added to the TUG test to assess its effect on functional mobility. METHODS: People with MS (n = 52) and controls (n = 57) performed three versions of the TUG test: TUG alone (TUG-alone), TUG plus reciting the alphabet (TUG-alpha), and TUG plus subtracting numbers by 3s (TUG-3s). Times to complete the TUG tests were compared among controls and three groups of participants with MS created using Expanded Disability Status Scale (EDSS) scores 0 to 3.5, 4.0 to 5.5, and 6. Differences among groups were analyzed using split-plot analysis of variance. RESULTS: Group and TUG type were significant (P < .001 for both), with no interaction effect of group × TUG type (P = .21). Mean times were 8.7, 9.4, and 11.1 seconds to perform the TUG-alone, TUG-alpha, and TUG-3s, respectively. Mean times for groups were 8.0, 8.2, 11.1, and 11.6 seconds for controls and individuals with MS and EDSS 0 to 3.5, 4.0 to 5.5, and 6, respectively. CONCLUSIONS: People with MS with an EDSS score greater than 3.5 had a statistically significant reduction in performance of the TUG test even with the addition of a simple cognitive task, which might have implications for a person's more complex everyday activities.

Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome.

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of chronic kidney disease. Here, we identified recessive mutations in the gene encoding the actin-binding protein advillin (AVIL) in 3 unrelated families with SRNS. While all AVIL mutations resulted in a marked loss of its actin-bundling ability, truncation of AVIL also disrupted colocalization with F-actin, thereby leading to impaired actin binding and severing. Additionally, AVIL colocalized and interacted with the phospholipase enzyme PLCE1 and with the ARP2/3 actin-modulating complex. Knockdown of AVIL in human podocytes reduced actin stress fibers at the cell periphery, prevented recruitment of PLCE1 to the ARP3-rich lamellipodia, blocked EGF-induced generation of diacylglycerol (DAG) by PLCE1, and attenuated the podocyte migration rate (PMR). These effects were reversed by overexpression of WT AVIL but not by overexpression of any of the 3 patient-derived AVIL mutants. The PMR was increased by overexpression of WT Avil or PLCE1, or by EGF stimulation; however, this increased PMR was ameliorated by inhibition of the ARP2/3 complex, indicating that ARP-dependent lamellipodia formation occurs downstream of AVIL and PLCE1 function. Together, these results delineate a comprehensive pathogenic axis of SRNS that integrates loss of AVIL function with alterations in the action of PLCE1, an established SRNS protein.

By moonlighting in the nucleus, villin regulates epithelial plasticity.

Villin is a tissue-specific, actin-binding protein involved in the assembly and maintenance of microvilli in polarized epithelial cells. Conversely, villin is also linked with the loss of epithelial polarity and gain of the mesenchymal phenotype in migrating, invasive cells. In this study, we describe for the first time how villin can switch between these disparate functions to change tissue architecture by moonlighting in the nucleus. Our study reveals that the moonlighting function of villin in the nucleus may play an important role in tissue homeostasis and disease. Villin accumulates in the nucleus during wound repair, and altering the cellular microenvironment by inducing hypoxia increases the nuclear accumulation of villin. Nuclear villin is also associated with mouse models of tumorigenesis, and a systematic analysis of a large cohort of colorectal cancer specimens confirmed the nuclear distribution of villin in a subset of tumors. Our study demonstrates that nuclear villin regulates epithelial-mesenchymal transition (EMT). Altering the nuclear localization of villin affects the expression and activity of Slug, a key transcriptional regulator of EMT. In addition, we find that villin directly interacts with a transcriptional corepressor and ligand of the Slug promoter, ZBRK1. The outcome of this study underscores the role of nuclear villin and its binding partner ZBRK1 in the regulation of EMT and as potential new therapeutic targets to inhibit tumorigenesis.

Both the anti- and pro-apoptotic functions of villin regulate cell turnover and intestinal homeostasis.

In the small intestine, epithelial cells are derived from stem cells in the crypts, migrate up the villus as they differentiate and are ultimately shed from the villus tips. This process of proliferation and shedding is tightly regulated to maintain the intestinal architecture and tissue homeostasis. Apoptosis regulates both the number of stem cells in the crypts as well as the sloughing of cells from the villus tips. Previously, we have shown that villin, an epithelial cell-specific actin-binding protein functions as an anti-apoptotic protein in the gastrointestinal epithelium. The expression of villin is highest in the apoptosis-resistant villus cells and lowest in the apoptosis-sensitive crypts. In this study we report that villin is cleaved in the intestinal mucosa to generate a pro-apoptotic fragment that is spatially restricted to the villus tips. This cleaved villin fragment severs actin in an unregulated fashion to initiate the extrusion and subsequent apoptosis of effete cells from the villus tips. Using villin knockout mice, we validate the physiological role of villin in apoptosis and cell extrusion from the gastrointestinal epithelium. Our study also highlights the potential role of villin's pro-apoptotic function in the pathogenesis of inflammatory bowel disease, ischemia-reperfusion injury, enteroinvasive bacterial and parasitic infections.

Spasticity and the use of intrathecal baclofen in patients with spinal cord injury.

Muscle spasms and spasticity constitute a significant problem in patients with spinal cord injury, interfering with rehabilitation and leading to impairments in quality of life in addition to medical complications. Administration of intrathecal baclofen (ITB) is indicated when spasticity continues to produce a clinical disability despite trials of oral treatments and other alternatives in patients who have functional goals and/or pain without contractures. Severe spasticity of spinal origin has been shown to respond dramatically to long-term ITB when used in appropriate patients with spasticity.

Tetraspanin CD82 inhibits protrusion and retraction in cell movement by attenuating the plasma membrane-dependent actin organization.

To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.

The role of actin bundling proteins in the assembly of filopodia in epithelial cells.

The goal of this review is to highlight how emerging new models of filopodia assembly, which include tissue specific actin-bundling proteins, could provide more comprehensive representations of filopodia assembly that would describe more adequately and effectively the complexity and plasticity of epithelial cells.  This review also describes how the true diversity of actin bundling proteins must be considered to predict the far-reaching significance and versatile functions of filopodia in epithelial cells.