Formin-dependent TGF-β signaling for epithelial to mesenchymal transition.

The role of distinct actin filament architectures in epithelial plasticity remains incompletely understood. We therefore determined roles for formins and the Arp2/3 complex, which are actin nucleators generating unbranched and branched actin filaments, respectively, in the process of epithelial to mesenchymal transition (EMT). In clonal lung, mammary, and renal epithelial cells, the formin activity inhibitor SMIFH2 but not the Arp2/3 complex activity inhibitor CK666 blocked EMT induced by TGF-β. SMIFH2 prevented the proximal signal of increased Smad2 phosphorylation and hence also blocked downstream EMT markers, including actin filament remodeling, decreased expression of the adherens junction protein E-cadherin, and increased expression of the matrix protein fibronectin and the transcription factor Snail. The short hairpin RNA silencing of formins DIAPH1 and DIAPH3 but not other formins phenocopied SMIFH2 effects and inhibited Smad2 phosphorylation and changes in Snail and cadherin expression. Formin activity was not necessary for the cell surface expression or dimerization of TGF-β receptors, or for nuclear translocation of TAZ, a transcription cofactor in Hippo signaling also regulated by TGF-β. Our findings reveal a previously unrecognized role for formin-dependent actin architectures in proximal TGF-β signaling that is necessary for Smad2 phosphorylation but not for cross-talk to TAZ

Tractions and stress fibers control cell shape and rearrangements in collective cell migration

Key to collective cell migration is the ability of cells to rearrange their position with respect to their neighbors. Recent theory and experiments demonstrated that cellular rearrangements are facilitated by cell shape, with cells having more elongated shapes and greater perimeters more easily sliding past their neighbors within the cell layer. Though it is thought that cell perimeter is controlled primarily by cortical tension and adhesion at each cell's periphery, experimental testing of this hypothesis has produced conflicting results. Here we studied collective cell migration in an epithelial monolayer by measuring forces, cell perimeters, and motion, and found all three to decrease with either increased cell density or inhibition of cell contraction. In contrast to previous understanding, the data suggest that cell shape and rearrangements are controlled not by cortical tension or adhesion at the cell periphery but rather by the stress fibers that produce tractions at the cell-substrate interface. This finding is confirmed by an experiment showing that increasing tractions reverses the effect of density on cell shape and rearrangements. Our study therefore reduces the focus on the cell periphery by establishing cell-substrate traction as a major physical factor controlling cell shape and motion in collective cell migration.Comment: 39 pages, 6 figure

The Role of Diaphanous in Ring Canal Development in Drosophila melanogaster

Infertility is a widespread condition that does not always have a known cause, and for which we often do not have a cure. One potential cause of infertility is defects in gametogenesis, or the formation of sperm and egg. During gametogenesis in most organisms, the developing sperm and egg are connected to each other or to supporting cells through intercellular bridges, allowing transfer of materials between cells. Defects in these connections can lead to infertility. The developing fruit fly egg is an excellent model system to study intercellular bridges, or ring canals. Rich in f-actin and actinbinding proteins, ring canals expand ~20 fold during oogenesis, and this expansion is accompanied by a 134-fold increase in the amount of actin in the structure. Ring canal expansion depends on the Arp2/3 complex; mutations in Arp2/3 complex members lead to decreased expansion and ring canal collapse. Interestingly, the Arp2/3 mutant phenotype has been reported to affect later stages of oogenesis (beginning at stage 5). This suggests that other actin nucleators could be involved in promoting ring canal growth prior to this point. I have characterized a role for the formin-family actin nucleator, Diaphanous (Dia), during oogenesis. Depletion of Dia leads to defects in normal ring canal structure and expansion, which are distinct from those observed following depletion of the Arp2/3 complex members. Future work will determine the mechanisms that promote the localization and activation of Arp2/3 and Diaphanous in the context of ring canal formation and expansion

Stress relaxation in epithelial monolayers is controlled by the actomyosin cortex

Epithelial monolayers are one-cell thick tissue sheets that separate internal and external environments. As part of their function, they have to withstand extrinsic mechanical stresses applied at high strain rates. However, little is known about how monolayers respond to mechanical deformations. Here, by subjecting suspended epithelial monolayers to stretch, we find that they dissipate stresses on a minute time-scale in a process that involves an increase in monolayer length, pointing to active remodelling of cell architecture during relaxation. Strikingly, monolayers consisting of tens of thousands of cells relax stress with similar dynamics to single rounded cells and both respond similarly to perturbations of actomyosin. By contrast, cell-cell junctional complexes and intermediate filaments do not relax tissue stress, but form stable connections between cells, allowing monolayers to behave rheologically as single cells. Taken together our data show that actomyosin dynamics governs the rheological properties of epithelial monolayers, dissipating applied stresses, and enabling changes in monolayer length.Peer ReviewedPostprint (published version

Arp2/3 complex inhibition radically alters lamellipodial actin architecture, suspended cell shape, and the cell spreading process

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 26 (2015): 887-900, doi:10.1091/mbc.E14-07-1244.Recent studies have investigated the dendritic actin cytoskeleton of the cell edge's lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network.This research was supported by National Science Foundation STEP grant 0856704 to Dickinson College, student/faculty summer research grants from the Dickinson College Research and Development Committee, Laura and Arthur Colwin Summer Research Fellowships from the Marine Biological Laboratory to J.H.H. and C.B.S., National Institutes of Health Grant EB002583 to R.O., and National Science Foundation collaborative research grants to J.H.H. (MCB-1412688) and C.B.S. (MCB-1412734)

Myosin-independent stiffness sensing by fibroblasts is regulated by the viscoelasticity of flowing actin

The stiffness of the extracellular matrix induces differential tension within integrin-based adhesions, triggering differential mechanoresponses. However, it has been unclear if the stiffness-dependent differential tension is induced solely by myosin activity. Here, we report that in the absence of myosin contractility, 3T3 fibroblasts still transmit stiffness-dependent differential levels of traction. This myosin-independent differential traction is regulated by polymerizing actin assisted by actin nucleators Arp2/3 and formin where formin has a stronger contribution than Arp2/3 to both traction and actin flow. Intriguingly, despite only slight changes in F-actin flow speed observed in cells with the combined inhibition of Arp2/3 and myosin compared to cells with sole myosin inhibition, they show a 4-times reduction in traction than cells with myosin-only inhibition. Our analyses indicate that traditional models based on rigid F-actin are inadequate for capturing such dramatic force reduction with similar actin flow. Instead, incorporating the F-actin network’s viscoelastic properties is crucial. Our new model including the F-actin viscoelasticity reveals that Arp2/3 and formin enhance stiffness sensitivity by mechanically reinforcing the F-actin network, thereby facilitating more effective transmission of flow-induced forces. This model is validated by cell stiffness measurement with atomic force microscopy and experimental observation of model-predicted stiffness-dependent actin flow fluctuation

Formin is associated with left-right asymmetry in the pond snail and the frog

While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signalling of Nodal, downstream of symmetry-breaking, may be an ancestral feature of the Bilateria. Here we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry-breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or over-expression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models, we discovered asymmetric gene expression in 2 and 4 cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro, together these results are consistent with the view that animals with diverse bodyplans may derive their asymmetries from the same intracellular chiral elements

Axon initial segment cytoskeleton, composition and plasticity regulation by formins

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-11-2019Esta tesis tiene embargado el acceso al texto completo hasta el 27-05-2021The axon initial segment (AIS) is a specialized compartment crucial for generating action potentials and maintaining neuronal polarity. An integrated structure composed of a high concentration of voltage gated ion channels, a specific cytoskeleton architecture, as well as, scaffold proteins like ankyrinG and βIV-spectrin, contributes to these functions. Recent studies have revealed that AIS is dynamically regulated in molecular composition, length and location in response to neuronal activity alterations both in physiological and pathological conditions. Some mechanisms acting on AIS plasticity have been uncovered lately, which include calcium dependent calpain or calcineurin modulation, as well as, modifications of cytoskeleton proteins. However, it is not clear how AIS is regulated in its structure and functions, and which proteins may regulate AIS cytoskeleton. In this study, using pharmacological methods we found that formins modulate AIS maintenance in vitro and in vivo. Formins are a group of proteins involved in cytoskeleton regulation. Currently, 15 formin members are identified in mammals. In neurons, different formins show differential distribution and regulation mechanisms. Only a few studies have revealed that formin members play different roles in neurons, such as axonal and dendritic development, spine morphogenesis and synapse modulation. Regarding brain diseases, some mutations in formin proteins were found in patients suffering mental retardation, Alzheimer’s disease, seizures and schizophrenia, suggesting that formins may play some important roles on neuronal activity. Our study demonstrates that formins inhibition modifies AIS cytoskeleton properties and their associated structural proteins and function. Interestingly, we found that mDia1 is at least one major formin family member that functions both in the AIS assembly and maintenance. mDia1 inhibition or suppression affects AIS length and structural and functional AIS proteins. Consistently, after blocking mDia1 activity, the action potential is affected, as well as, axonal polarity maintenance. These effects are due to the deficiency of AIS structural stability and alterations in axonal protein traffic, which can be compensated through expression or modulation of microtubules and microfilaments regulators. Taken together, our results indicate a new AIS regulator involved in AIS formation, maintenance and plasticity, which provides new insights on AIS regulation mechanisms. Further experiments will be necessary to completely understand mDia1 regulation at the AIS, and its implications in brain activity related disease

Integration of linear and dendritic actin nucleation in Nck-induced actin comets

The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails-dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, form-in-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens.Fil: Surtayeva, Sofya. University of Connecticut School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Velle, Katrina B.. University of Connecticut; Estados UnidosFil: Campellone, Kenneth G.. University of Connecticut; Estados UnidosFil: Talman, Arthur. Yale School of Medicine; Estados UnidosFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Agaisse, Hervé. Yale School of Medicine; Estados UnidosFil: Wu, Yi I.. University of Connecticut School of Medicine; Estados UnidosFil: Loew, Leslie M.. University of Connecticut School of Medicine; Estados UnidosFil: Mayer, Bruce J.. University of Connecticut School of Medicine; Estados Unido