Cytoskeletal Crosslinking Proteins Research Articles

Contrasting effects of filamin A and B proteins in modulating filovirus entry.

Ebola (EBOV) and Marburg viruses (MARV) cause severe hemorrhagic fever associated with high mortality rates in humans. A better understanding of filovirus-host interactions that regulate the EBOV and MARV lifecycles can provide biological and mechanistic insight critical for therapeutic development. EBOV glycoprotein (eGP) and MARV glycoprotein (mGP) mediate entry into host cells primarily by actin-dependent macropinocytosis. Here, we identified actin-binding cytoskeletal crosslinking proteins filamin A (FLNa) and B (FLNb) as important regulators of both EBOV and MARV entry. We found that entry of pseudotype psVSV-RFP-eGP, infectious recombinant rVSV-eGP-mCherry, and live authentic EBOV and MARV was inhibited in filamin A knockdown (FLNaKD) cells, but was surprisingly enhanced in filamin B knockdown (FLNbKD) cells. Mechanistically, our findings suggest that differential regulation of macropinocytosis by FLNa and FLNb likely contributes to their specific effects on EBOV and MARV entry. This study is the first to identify the filamin family of proteins as regulators of EBOV and MARV entry. These findings may provide insight into the development of new countermeasures to prevent EBOV and MARV infections.

Ezrin immunoexpression in trophoblast cells of the equine placenta

Ezrin expression in microcotyledons and trophoblastic cells plays an important role in vascularization and maternal-fetal exchange. Ezrin is a membrane cytoskeletal crosslinker protein that helps regulate several cytoskeletal-related functions, including cell adhesion (Souza.et.al. Research in Veterinary Science.2023. 154: 84-88). Here, we describe the histopathological findings and expression as detected by an ezrin-specific antibody in pregnant and non-pregnant uterine horns of normal equine placentas. Eighteen placentas from embryo recipient mares of the Mangalarga Machador breed were collected at delivery (n=18; age 4-8 years). All placentas were evaluated for macroscopic characteristics. As part of the histopathological evaluation, pregnant and non-pregnant horn samples underwent hematoxylin-eosin staining to grade and compare the arrangement and vascularity of microcotyledons and allantochorions. Silanized slides were immunostained with an ezrin-specific antibody (clone: H-276; rabbit polyclonal-IgG; Santa Cruz; dilution: 1:500μl; LSAB+ Dako, Carpenteria, California, USA). A sample of human breast ductal adenocarcinoma was used as the positive control. For the negative control, the primaryantibody was replaced with a primary antibody isotype. In the microscopic analysis, the microcotyledonary arrangement in the pregnant horn was more intense than in the non-pregnant horn, except for one case. In terms of vascularization, the horns did not differ in the number or intensity of vessels in the microcotyledons and allantochorion. Ezrin antibody expression in placental horns was more intense in the proximal region of the microcotyledons compared with the basal lamina of the allantochorion and was diffused throughout the microcotyledonary network. The pregnant horn showed greater ezrin expression in trophoblasts compared to the non-pregnant horn. The two horn types showed similar color intensity in most samples however, ezrin was expressed mildly in the pregnant horn, while the non-pregnant horn from the same placenta showed moderate to intense ezrin expression. These observations may reflect a way of modulating maternal–fetal exchange, because trophoblast cells have this function. In summary, ezrin was expressed in trophoblast cells located in the microcotyledons of equine placentas, suggesting that this protein may play a role at the maternal–fetal interface.

Visualizing Actin and Microtubule Coupling Dynamics <em>In Vitro</em> by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Actin Crosslinking Family Protein 7 Deficiency Does Not Impair Hearing in Young Mice.

To enable hearing, the sensory hair cell contains specialized subcellular structures at its apical region, including the actin-rich cuticular plate and circumferential band. ACF7 (actin crosslinking family protein 7), encoded by the gene Macf1 (microtubule and actin crosslinking factor 1), is a large cytoskeletal crosslinking protein that interacts with microtubules and filamentous actin to shape cells. ACF7 localizes to the cuticular plate and the circumferential band in the hair cells of vertebrates. The compelling expression pattern of ACF7 in hair cells, combined with conserved roles of this protein in the cytoskeleton of various cell types in invertebrates and vertebrates, led to the hypothesis that ACF7 performs a key function in the subcellular architecture of hair cells. To test the hypothesis, we conditionally target Macf1 in the inner ears of mice. Surprisingly, our data show that in young, but mature, conditional knockout mice cochlear hair cell survival, planar cell polarity, organization of the hair cells within the organ of Corti, and capacity to hear are not significantly impacted. Overall, these results fail to support the hypothesis that ACF7 is an essential hair cell protein in young mice, and the purpose of ACF7 expression in the hair cell remains to be understood.

Modeling spatiotemporally varying protein-protein interactions in CyLaKS, the Cytoskeleton Lattice-based Kinetic Simulator.

Interaction of cytoskeletal filaments, motor proteins, and crosslinking proteins drives important cellular processes such as cell division and cell movement. Cytoskeletal networks also exhibit nonequilibrium self-assembly in reconstituted systems. An emerging problem in cytoskeletal modeling and simulation is spatiotemporal alteration of the dynamics of filaments, motors, and associated proteins. This can occur due to motor crowding, obstacles along the filament, motor interactions and direction switching, and changes, defects, or heterogeneity in the filament binding lattice. How such spatiotemporally varying cytoskeletal filaments and motor interactions affect their collective properties is not fully understood. We developed the Cytoskeleton Lattice-based Kinetic Simulator (CyLaKS) to investigate such problems. The simulation model builds on previous work by incorporating motor mechanochemistry into a simulation with many interacting motors and/or associated proteins on a discretized lattice. CyLaKS also includes detailed balance in binding kinetics, movement, and lattice heterogeneity. The simulation framework is flexible and extensible for future modeling work and is available on GitHub for others to freely use or build upon. Here we illustrate the use of CyLaKS to study long-range motor interactions, microtubule lattice heterogeneity, motion of a heterodimeric motor, and how changing crosslinker number affects filament separation.

Mutations of MACF1, Encoding Microtubule-Actin Crosslinking-Factor 1, Cause Spectraplakinopathy

As a member of spectraplakin family of cytoskeletal crosslinking proteins, microtubule-actin crosslinking factor 1 (MACF1) controls cytoskeleton network dynamics. Knockout of Macf1 in mice resulted in the developmental retardation and embryonic lethality. Spectraplakinopathy type I, a novel neuromuscular condition characterized by periodic hypotonia, lax muscles, joint contracture, and diminished motor skill, was reported to be associated with heterozygous genomic duplication involving the MACF1 loci, with incomplete penetrance and highly variable clinical presentation in a single pedigree. In this study, parental-derived compound heterozygous novel missense mutations of MACF1, c.1517C>T (p.Thr506Ile) and c.11654T>C (p.Ile3885Thr), were found to co-segregate with disease status in two affected brothers presenting with progressive spastic tetraplegia, dystonia, joint contracture, feeding difficulty and developmental delay. We speculated that MACF1 mutations cause spectraplakinopathy inherited in an autosomal recessive manner. Our clinical findings expanded the phenotype of this neuromuscular disorder and provided new insights into the function of MACF1.

Inhibition of the Spectraplakin Protein Microtubule Actin Crosslinking Factor 1 Sensitizes Glioblastomas to Radiation.

BackgroundMicrotubule actin crosslinking factor 1 (MACF1) is a spectraplakin cytoskeletal crosslinking protein whose function and role in cancer biology has lacked investigation. Recent studies have identified MACF1 as a novel target in glioblastomas expressed in tissue from tumor patient explants but not normal brain tissue and when silenced has an antitumorigenic impact on these tumors. Radiation as a single agent therapy to treat glioblastomas has been used for decades and has done little to improve survival of individuals diagnosed with this disease. However, contemporary clinical radiotherapy protocols have provided evidence that combinatorial radiotherapy approaches confer a therapeutic benefit in glioblastoma patients. In this study MACF1 was investigated as a radiosensitization target in glioblastomas.MethodsTo provide context of MACF1 in glioblastomas, The Cancer Genome Atlas expression analyses were performed in conjunction with genes associated with glioblastoma evolution, while a genetic inhibitory approach, cell migratory assays, and immunofluorescence procedures were used to evaluate responses to MACF1 suppression with radiation. Additionally, expression analyses were conducted to assess co-expression of mTOR signaling pathway regulators and MACF1 in glioblastoma patient samples.ResultsOur amalgamation approach demonstrated that negative regulation of MACF1, which was positively correlated with epidermal growth factor receptor and p70s6k expression, enhanced the sensitivity of glioblastoma cells to radiation as a consequence of reducing glioblastoma cell viability and migration. Mechanistically, the antitumorigenic effects on glioblastoma cell behaviors after radiation and impairing MACF1 function were associated with decreased expression of ribosomal protein S6, a downstream effector of p70s6k.ConclusionMACF1 represents a diagnostic marker with target specificity in glioblastomas that can enhance the efficacy of radiation while minimizing normal tissue toxicity. This approach could potentially expand combinatorial radiation strategies for glioblastoma treatments via impairment of translational regulatory processes that contribute to poor patient survival.

The cytoskeletal crosslinking protein MACF1 is dispensable for thrombus formation and hemostasis

Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.

Regulation of ezrin tension by S-nitrosylation mediates non-small cell lung cancer invasion and metastasis.

Cancer invasion and metastasis depend on accurate and rapid modulation of both chemical and mechanical activities. The S-nitrosylation (SNO) of membrane cytoskeletal cross-linker protein ezrin may regulate the malignant process in a tension-dependent manner.Methods: The level of nitrosylated ezrin in non-small cell lung cancer (NSCLC) tissues and A549 cell line were evaluated by biotin-switch assay. A few cysteine mutated plasmids of ezrin were used to identify active site for SNO. Newly designed ezrin or mutated-ezrin tension probes based on Förster resonance energy transfer (FRET) theory were applied to visually observe real-time tension changes. Cytoskeleton depolymerizing and motor molecular inhibiting experiments were performed to reveal the alternation of the mechanical property of ezrin after SNO. Transwell assays and xenograft mouse model were used to assess aggressiveness of A549 cells in different groups. Fluorescent staining was also applied to examine cellular location and structures.Results: High inducible nitric oxide synthase (iNOS) levels were observed to induce ezrin-SNO, and then promote malignant behaviors of NSCLC cells both in vitro and in vivo. Cys117 was identified as the only active site for ezrin-SNO. Meanwhile, an increased level of ezrin tension was observed after iNOS-induced SNO. Enhanced ezrin tension was positively correlated with aggressiveness of NSCLC. Moreover, Microfilament (MF) forces instead of microtubule (MT) forces played dominant roles in modulating ezrin tension, especially after ezrin nitrosylation.Conclusion: This study revealed a SNO-associated mechanism underlying the mechanical tension of ezrin. Ezrin-SNO promotes NSCLC cells invasion and metastasis through facilitating mechanical transduction from the cytoskeleton to the membrane. These studies implicate the therapeutic potential by targeting ezrin in the inhibition NSCLC invasion and metastasis.

A Missense Variant in PLEC Increases Risk of Atrial Fibrillation

BackgroundGenome-wide association studies (GWAS) have yielded variants at >30 loci that associate with atrial fibrillation (AF), including rare coding mutations in the sarcomere genes MYH6 and MYL4. ObjectivesThe aim of this study was to search for novel AF associations and in doing so gain insights into the mechanisms whereby variants affect AF risk, using electrocardiogram (ECG) measurements. MethodsThe authors performed a GWAS of 14,255 AF cases and 374,939 controls, using whole-genome sequence data from the Icelandic population, and tested novel signals in 2,002 non-Icelandic cases and 12,324 controls. They then tested the AF variants for effect on cardiac electrical function by using measurements in 289,297 ECGs from 62,974 individuals. ResultsThe authors discovered 2 novel AF variants, the intergenic variant rs72700114, between the genes LINC01142 and METTL11B (risk allele frequency = 8.1%; odds ratio [OR]: 1.26; p = 3.1 × 10−18), and the missense variant p.Gly4098Ser in PLEC (frequency = 1.2%; OR: 1.55; p = 8.0 × 10−10), encoding plectin, a cytoskeletal cross-linking protein that contributes to integrity of cardiac tissue. The authors also confirmed 29 reported variants. p.Gly4098Ser in PLEC significantly affects various ECG measurements in the absence of AF. Other AF variants have diverse effects on the conduction system, ranging from none to extensive. ConclusionsThe discovery of a missense variant in PLEC affecting AF combined with recent discoveries of variants in the sarcomere genes MYH6 and MYL4 points to an important role of myocardial structure in the pathogenesis of the disease. The diverse associations between AF variants and ECG measurements suggest fundamentally different categories of mechanisms contributing to the development of AF.

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity.

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial-mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation.

Shear Stress Stimulated MSC Activities: Direct Changes of Membrane Tension or Cytoskeletal Stress?

Fluid shear stress induced membrane transport such as Ca2+ influx and the activation of ion channels has been widely reported. The shear stress can be mediated by a direct change in bilipid membrane tension and/or by a change in cytoskeletal stress via binding proteins that link channels to actin, but how the shear stress is coupled to ion channels is unclear. Using narrow shear pulse stimuli generated by a pressure servo in a microfluidic chamber, we measured the changes of membrane tension and cytoskeletal protein stress simultaneously in astrocytes. The membrane tension was reported using molecular motor probes (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ) and cytoskeletal tension reported by genetically encoded force probe actinin-cpst-FRET. Our results show that the changes of membrane tension are highly localized and the gradient is relevant to the flow directions. A shear stress pulse (23 dyn/cm2, 400 ms duration) caused a rapid increase in membrane tension at the front edge of the cell with respect to the flow and a decrease in tension (compression) at the distal edge. The rise time was less than 30 ms, and the tension dropped to the initial state within ∼30 ms post stimulus, showing a typical elastic behavior. In contrast, the same shear pulse generated profound and long-lasting tension in cytoskeletal cross-linking proteins α-actinin at the front edge of the cell, and the tension persisted for the entire experimental duration of 60 s. In situ Ca2+ imaging showed that the initial Ca2+ influx was strongly correlated with the region having high cytoskeletal tension, but weakly linked to the bilayer tension. The results suggest the cytoskeletal tension plays primary role in shear stress activated Ca2+ influx. This work was funded by NINDS.

The CAMSAP3-ACF7 Complex Couples Noncentrosomal Microtubules with Actin Filaments to Coordinate Their Dynamics

For adaptation to complex cellular functions, dynamic cytoskeletal networks are required. There are two major components of the cytoskeleton, microtubules and actin filaments, which form an intricate network maintaining an exquisite cooperation to build the physical basis for their cellular function. However, little is known about the molecular mechanism underlying their synergism. Here, we show that in Caco2 epithelial cells, noncentrosomal microtubules crosstalk with F-actin through their minus ends and contribute to the regulation of focal adhesion size and cell migration. We demonstrate that ACF7, a member of the spectraplakin family of cytoskeletal crosslinking proteins, interacts with Nezha (also called CAMSAP3) at the minus ends of noncentrosomal microtubules and anchors them to actin filaments. Those noncentrosomal microtubules cooperate with actin filaments through retrograde flow to keep their length and orientation perpendicular to the cell edge as well as regulate focal adhesion size and cell migration.

Abstract 3269: Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level

Abstract Ezrin is a member of the ezrin-radixin-moesin (ERM) family of actin-membrane linker proteins that play key roles in regulating cell shape, movement, adhesion and signal transduction pathways. The expression of ezrin is linked to the metastatic progression in several cancers including osteosarcoma (OS). We discovered a small molecule, NSC305787, that inhibits ezrin activity and metastatic phenotype both in vitro and in vivo. We hypothesized that the anti-metastatic effects of NSC308787 could be mediated through preventing specific protein-protein interactions involving ezrin. In this study, we used affinity pull-down coupled with mass spectrometry-based proteomic approach to unravel putative ezrin interactors that are competed away by NSC305787. We identified a number of candidate ezrin binding proteins that are associated with metastatic behavior and implicated in the regulation of stress granule dynamics and protein translation initiation. We selected DDX3, a DEAD-box RNA helicase, as a candidate for further analysis. We confirmed that ezrin directly binds to DDX3. Depletion of ezrin protein expression by RNA interference in several cancer cell lines resulted in substantial reduction in DDX3 protein levels without affecting its transcription, which suggested that ezrin is required for post-transcriptional maintenance of DDX3 in the cell. Paradoxically, recombinant ezrin specifically inhibited the RNA duplex unwinding activity and stimulated the ATPase activity of DDX3. Our data suggest that ezrin regulates the translation of mRNAs with 5′ secondary structure through DDX3, at least in part, through maintaining its intracellular protein level and/or modulating its unwinding and ATPase activities. Therefore, our findings suggest that a novel function of ezrin regarding the regulation of mRNA translation exists that is independent from its classical role as a cytoskeletal cross-linker protein at the plasma membrane. Citation Format: Haydar Celik, Kamal P. Sajwan, Amrita V. Pai, Ben J. Marsh, Yasemin Saygideger Kont, Said Rahim, Jenny Han, Tsion Minas, Jeffrey A. Toretsky, Aykut Uren. Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3269. doi:10.1158/1538-7445.AM2015-3269

Abstract 54: Ezrin enhances signaling and nuclear translocation of the epidermal growth factor receptor in non-small cell lung cancer cells

Abstract The cytoskeletal cross linker protein ezrin is a member of the ezrin-radixin-moesin (ERM) family and plays important roles not only in cell motility, cell adhesion, and apoptosis, but also in various cell-signaling pathways. Ezrin interacts with EGFR in the cell membrane and involves in cell motility events, but little is known about the effects of this interaction on the EGFR signaling pathway. We investigated the role of Ezrin in EGFR signaling and nuclear trafficking in non-small cell lung cancer (NSCLC) cell lines. The ligand induced interaction between Ezrin and EGFR was evaluated by immunoprecipitation (IP) and immunofluorescence (IF) in H292 and A549 cells. Ezrin levels were reduced using siRNA in these two cell lines. Downstream signaling protein phosphorylation and nuclear localization of EGFR were detected after EGF treatment. Expressions of nuclear EGFR target genes were evaluated by qPCR. Endogenous Ezrin was found in a complex with EGFR in IP and IF. When Ezrin protein expression was inhibited, phosphorylation levels of EGFR at Y1068, Y1101 and Y845 were reduced as well as phosphorylation levels of downstream signaling pathway proteins ERK and STAT3. Cell fractionation revealed that EGFR nuclear translocation after EGF treatment significantly reduced in Ezrin-knockdown cells. Further, mRNA levels of EGFR target genes AuroraK-A, COX2, Cyclin D1 and iNOS were decreased in Ezrin-knockdown A549 cells. Small molecule ezrin inhibitors showed strong synergy with EGFR inhibitors in cytotoxicity assays. These results suggest that Ezrin has a role as an enhancer in the EGFR pathway and targeting ezrin may potentiate anti-EGFR based therapies in NSCLC. Citation Format: Yasemin Saygideger Kont, Haydar Celik, Hayriye V. Erkizan, Tsion Minas, Jenny Han, Jeffrey Toretsky, Aykut Uren. Ezrin enhances signaling and nuclear translocation of the epidermal growth factor receptor in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 54. doi:10.1158/1538-7445.AM2015-54

Knockdown of ezrin causes intrahepatic cholestasis by the dysregulation of bile fluidity in the bile duct epithelium in mice.

Cholangiopathies share common features, including bile duct proliferation, periportal fibrosis, and intrahepatic cholestasis. Damage of biliary epithelium by autoimunne disorder, virus infection, toxic compounds, and developmental abnormalities causes severe progressive hepatic disorders responsible for high mortality. However, the etiologies of these cholestatic diseases remain unclear because useful models to study the pathogenic mechanisms are not available. In the present study, we have found that ezrin knockdown (Vil2(kd/kd) ) mice develop severe intrahepatic cholestasis characterized by extensive bile duct proliferation, periductular fibrosis, and intrahepatic bile acid accumulation without developmental defects of bile duct morphology and infiltration of inflammatory cells. Ezrin is a membrane cytoskeletal cross-linker protein, which is known to interact with transporters, scaffold proteins, and actin cytoskeleton at the plasma membrane. We found that the normal apical membrane localizations of several transport proteins including cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE-2), aquaporin 1 (AQP1), and Na(+) /H(+) exchanger regulatory factor were disturbed in bile ducts of Vil2(kd/kd) mice. Stable expression of a dominant negative form of ezrin in immortalized mouse cholangiocytes also led to the reduction of the surface expression of CFTR, AE-2, and AQP1. Reduced surface expression of these transport proteins was accompanied by reduced functional expression, as evidenced by the fact these cells exhibited decreased CFTR-mediated Cl(-) efflux activity. Furthermore, bile flow and biliary HCO3 (-) concentration were also significantly reduced in Vil2(kd/kd) mice. Dysfunction of ezrin mimics important aspects of the pathological mechanisms responsible for cholangiopathies. The Vil2(kd/kd) mouse may be a useful model to exploit in the development and testing of potential therapies for cholangiopathies.

Kinetics of Mechanoresponse in the Mammalian Actin Cytoskeleton

Cells have a remarkable ability to sense and respond to their mechanical environment; this ability directs a host of cellular and global processes, such as metastasis, engulfment, blood vessel constriction, and tissue development. However, the direct mechanism by which the dynamic network of actin filaments, myosin motors, and actin crosslinking proteins assemble and interact to exert force on the membrane in response to mechanical perturbations remains unclear. Here, we define the in vivo kinetics of 20 cytoskeletal and signaling proteins in response an applied external stress and identify a number of structural elements that accumulate to regions of the cell under shear or dilational forces. Prominent among these are the three non-muscle myosin II paralogs, the actin bundling protein α-actinin 4, and the orthogonal actin crosslinking protein filamin B. All three myosin paralogs, as well as α-actinin 4, accumulate to the tip region of the aspirated cell, which experiences primarily dilational force. Filamin B, however, accumulates to the tip and the neck region, demonstrating that it responds to both sheared and dilated microenvironments. These differences can be directly related to the actin binding geometries and distinct mechanism of force response for each protein. In support of a proposed model for an increased actin affinity state under load, a disease-relevant point mutation K255E in α-actinin 4, which abolishes the structural low affinity state, greatly slows its accumulation kinetics to dilational stress. The accumulation of cytoskeletal motors and crosslinking proteins under mechanical load represents a mechanism by which cells can concentrate structural components to reduce strain or retract from a local environment of high stress. Understanding how cortical protein dynamics intrinsically give rise to the self-tuning nature of the cell will offer insight into the nature of mechanically-dependent cell processes.

Cortical and Cytoskeletal Structural Network regulates the Three-Dimensional Traction Forces Exerted by Migrating Amoeboid Cells

Cells exert traction forces, which are necessary to perform their migration. There is still a lack of information regarding the mechanical aspects of cell migration and in particular the quantification of the three-dimensional traction forces exerted during migration and invasion.We use a novel traction force cytometry method to measure the three-dimensional traction forces exerted by Dictyostelium cells chemotaxing over flat elastic substrates. Our measurements of the 3-D traction forces show that there are two independent mechanisms in their generation, one that relies on the cytoskeletal crosslinking and contractility, and another based on the cortical stiffness. In particular, we examine the roles that specific cortical and cytoskeletal crosslinking proteins and those liking the cell's cortex and the cytoskeletal F-actin play in the development of the spatial and temporal distribution of three-dimensional traction forces during migration. We investigate mutants with cortical integrity defects, such as cells lacking the protein cortexillin to study the cortical function. We use mutants with cytoskeletal crosslinking defects, such as cells lacking the proteins Myosin II and Abp120 to quantify the function of the cytoskeletal crosslinking. In addition we examine the proteins Myosin IA and B, regarding their role liking the cell's cortex and the cytoskeletal F-actin network.The effects on the magnitude and distribution of the tangential and perpendicular components of the traction forces to the substrate are shown to be different depending on the specific cytoskeletal and cortical mutations. Our 3-D traction measurements indicate that the vertical and horizontal traction forces are regulated by the cortical and cytoskeletal crosslinking. These two mechanisms are interconnected since the time evolution of the vertical and horizontal forces is well correlated.

Collective Dynamics of Active Cytoskeletal Networks

Self organization mechanisms are essential for the cytoskeleton to adapt to the requirements of living cells. They rely on the intricate interplay of cytoskeletal filaments, crosslinking proteins and molecular motors. Here we present an in vitro minimal model system consisting of actin filaments, fascin and myosin-II filaments exhibiting pulsatile collective dynamics and superdiffusive transport properties. Both phenomena rely on the complex competition of crosslinking molecules and motor filaments in the network. They are only observed if the relative strength of the binding of myosin-II filaments to the actin network allows exerting high enough forces to unbind actin/fascin crosslinks. This is shown by varying the binding strength of the acto-myosin bond and by combining the experiments with phenomenological simulations based on simple interaction rules.