scholarly journals Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

2022 ◽  
Vol 12 ◽  
Author(s):  
Frédéric Larbret ◽  
Pierric Biber ◽  
Nicholas Dubois ◽  
Stoyan Ivanov ◽  
Laurence Lafanechere ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Leukemic Cell ◽  
Underlying Mechanism ◽  
Actin Dynamics ◽  
Deubiquitinating Enzymes ◽  
Actin Reorganization ◽  
Actin Regulatory Proteins

Actin networks are dynamically regulated through constant depolymerization and polymerization cycles. Although the fundamental mechanisms that govern these processes have been identified, the nature and role of post-translational modifications (PTMs) of actin and actin regulatory proteins are not completely understood. Here, we employed Actin CytoFRET, a method that we developed for real time detection of fluorescence resonance energy transfer (FRET) signals generated by actin dynamics, to screen a small library of PTM-interfering compounds on a biosensor leukemic T cell line. This strategy led to the identification of small molecule inhibitors of deubiquitinating enzymes (DUBs) as potent inducers of actin polymerization and blockers of chemotactic cell migration. The examination of the underlying mechanism further revealed that the actin depolymerizing protein cofilin represents a major effector of DUB inhibitor (DUBi)-induced actin reorganization. We found that DUB blockade results in the accumulation of polyubiquitinated proteins and ROS production, associated with cofilin oxidation and dephosphorylation on serine 3, which provokes uncontrolled actin polymerization impairing cell migration. Together, our study highlights DUBs as novel regulators of actin dynamics through ROS-dependent cofilin modulation, and shows that DUBi represent attractive novel tools to impede leukemic cell migration.

Actin reorganization and proplatelet formation in murine megakaryocytes: the role of protein kinase Cα

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 154-161 ◽  
Author(s):  
Ponlapat Rojnuckarin ◽  
Kenneth Kaushansky
Keyword(s):  
Protein Kinase ◽  
Actin Polymerization ◽  
Map Kinases ◽  
Molecular Mechanisms ◽  
Marrow Cell ◽  
Actin Dynamics ◽  
Actin Reorganization ◽  
Murine Bone Marrow ◽  
Proplatelet Formation

Abstract With the recent cloning and characterization of thrombopoietin, appreciation of the molecular events surrounding megakaryocyte (MK) development is growing. However, the final stages of platelet formation are less well understood. Platelet production occurs after the formation of MK proplatelet processes. In a study to explore the molecular mechanisms underlying this process, mature MKs isolated from suspension murine bone marrow cell cultures were induced to form proplatelets by exposure to plasma, and the role of various cell-signaling pathways was assessed. The results showed that (1) bis-indolylmaleimide I, which blocks protein kinase C (PKC) activation; (2) down-modulation of conventional or novel classes of PKC by phorbol myristate acetate; and (3) ribozymes specific for PKCα each inhibited proplatelet formation. Inhibition of several MAP kinases, PI3 kinase, or protein kinase A failed to affect MK proplatelet formation. To gain further insights into the function of PKCα in proplatelet formation, its subcellular localization was investigated. In cultures containing active proplatelet formation, cytoplasmic polymerized actin was highly aggregated, its subcellular distribution was reorganized, and PKCα colocalized with the cellular actin aggregates. A number of MK manipulations, including blockade of integrin signaling with a disintegrin or inhibition of actin polymerization with cytochalasin D, interrupted actin reorganization, PKC relocalization, and proplatelet formation. These findings suggest an important role for PKCα in proplatelet development and suggest that it acts by altering actin dynamics in proplatelet-forming MKs. Identification of the upstream and downstream pathways involved in proplatelet formation should provide greater insights into thrombopoiesis, potentially allowing pharmacologic manipulation of the process.

scholarly journals Mechanisms for actin reorganization in chemotactic factor-activated polymorphonuclear leukocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2750-2757
Author(s):  
RG Watts ◽  
TH Howard
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Actin Polymerization ◽  
Insoluble Fraction ◽  
Chemotactic Factor ◽  
Actin Dynamics ◽  
Cross Linking ◽  
Actin Reorganization ◽  
Cytoskeletal Structure ◽  
Cross Links ◽  
Human Pmns

Cytoskeletal structure in polymorphonuclear leukocytes (PMNs) is thought to reflect a simple equilibrium between two actin pools (globular [G]- and filamentous [F] actin). Recent description of two distinct F-actin pools in PMNs (Triton-insoluble [stable] and Triton- soluble [labile] F-actin pools) (Watts and Howard, Cell Motil Cytoskeleton, 21:25, 1992) suggest a tripartite equilibrium between these F-actin pools and G-actin and multiple possible mechanisms for polymerization. To study the contribution of each actin pool to actin dynamics in PMNs, changes in actin content of the Triton-soluble and - insoluble F-actin pools and G-actin in chemotactic factor (CTF)- activated PMNs were measured by NBDphallacidin binding and by gel scans of Triton-lysed PMNs. From 0 to 30 seconds after CTF activation, PMNs rapidly increase total (Triton-soluble + Triton-insoluble) F-actin content (maximum = 1.7- +/- 0.10-fold basal at 30 seconds). Concurrent measures of the actin content of individual actin pools (Triton-soluble and -insoluble F-actin and G-actin) show that at all times (0 to 30 seconds) only the Triton-insoluble F-actin pool grows (maximum = 2.81- +/- 0.73-fold basal at 30 seconds), whereas both the Triton-soluble and G-actin pools simultaneously decrease (50% decrease at 30 seconds). Concurrent growth of one F-actin pool (Triton-insoluble) and loss of another F-actin pool (Triton-soluble) emphasize the functional uniqueness of the F-actin pools and can occur only if the Triton- soluble F-actin anneals or cross-links filament-to-filament with the Triton-insoluble fraction or if the Triton-insoluble F-actin pool first depolymerizes to monomer, which is then added to the Triton-insoluble pool. Because from 0 to 30 seconds after FMLP activation G-actin never increases, but, like the Triton-soluble F-actin progressively decreases, the results suggest that F-actin growth results from simultaneous new filament growth by monomer addition to the Triton- insoluble F-actin and cytoskeletal remodelling by Triton-soluble F- actin annealing or cross-linking to Triton-insoluble F-actin. These findings offer important new insights into the mechanism(s) of actin polymerization in CTF-activated human PMNs.

scholarly journals Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement

2021 ◽  
Vol 22 (21) ◽  
pp. 11727
Author(s):  
Maria J. Sarmento ◽  
Luís Borges-Araújo ◽  
Sandra N. Pinto ◽  
Nuno Bernardes ◽  
Joana C. Ricardo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Functions ◽  
Cytoskeleton Organization

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

scholarly journals Podoplanin Associates with CD44 to Promote Directional Cell Migration

2010 ◽  
Vol 21 (24) ◽  
pp. 4387-4399 ◽  
Author(s):  
Ester Martín-Villar ◽  
Beatriz Fernández-Muñoz ◽  
Maddy Parsons ◽  
Maria M. Yurrita ◽  
Diego Megías ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Mesenchymal Transition ◽  
Resonance Energy Transfer ◽  
Mouse Skin ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging ◽  
Mesenchymal Transition ◽  
Tumor Cell Migration ◽  
Podoplanin Expression

Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalize at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin–CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.

scholarly journals Effects of LPLI on microglial phagocytosis in LPS-induced neuroinflammation model

2014 ◽  
Vol 07 (03) ◽  
pp. 1350049 ◽  
Author(s):  
Sheng Song ◽  
Wei Chen ◽  
Feifan Zhou
Keyword(s):  
Neurodegenerative Diseases ◽  
Microglial Activation ◽  
Actin Polymerization ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dominant Negative ◽  
Microglial Phagocytosis ◽  
Dominant Negative Form ◽  
Phagocytic Response ◽  
Negative Form

Microglial activation plays an important role in neurodegenerative diseases. Once activated, they have macrophage-like capabilities, which can be beneficial by phagocytosis and harmful by secretion of neurotoxins. However, the resident microglia always fail to trigger an effective phagocytic response to clear dead cells or Aβ deposits during the progression of neurodegeneration. Therefore, the regulation of microglial phagocytosis is considered a useful strategy in searching for neuroprotective treatments. In this study, our results showed that low-power laser irradiation (LPLI) (20 J/cm2) could enhance microglial phagocytic function in LPS-activated microglia. We found that LPLI-mediated microglial phagocytosis is a Rac-1-dependent actin-based process, that a constitutively activated form of Rac1 (Rac1Q61L) induced a higher level of actin polymerization than cells transfected with wild-type Rac1, whereas a dominant negative form of Rac1 (Rac1T17N) markedly suppressed actin polymerization. In addition, the involvement of Rac1 activation after LPLI treatment was also observed by using a Raichu fluorescence resonance energy transfer (FRET)-based biosensor. We also found that PI3K/Akt pathway was required in the LPLI-induced Rac1 activation. Our research may provide a feasible therapeutic approach to control the progression of neurodegenerative diseases.

scholarly journals Abi2-Deficient Mice Exhibit Defective Cell Migration, Aberrant Dendritic Spine Morphogenesis, and Deficits in Learning and Memory

2004 ◽  
Vol 24 (24) ◽  
pp. 10905-10922 ◽  
Author(s):  
Matthew Grove ◽  
Galina Demyanenko ◽  
Asier Echarri ◽  
Patricia A. Zipfel ◽  
Marisol E. Quiroz ◽  
...  
Keyword(s):  
Cell Migration ◽  
Dendritic Spine ◽  
Actin Polymerization ◽  
Adherens Junctions ◽  
Actin Dynamics ◽  
Long Term Memory ◽  
Cell Morphogenesis ◽  
And Migration

ABSTRACT The Abl-interactor (Abi) family of adaptor proteins has been linked to signaling pathways involving the Abl tyrosine kinases and the Rac GTPase. Abi proteins localize to sites of actin polymerization in protrusive membrane structures and regulate actin dynamics in vitro. Here we demonstrate that Abi2 modulates cell morphogenesis and migration in vivo. Homozygous deletion of murine abi2 produced abnormal phenotypes in the eye and brain, the tissues with the highest Abi2 expression. In the absence of Abi2, secondary lens fiber orientation and migration were defective in the eye, without detectable defects in proliferation, differentiation, or apoptosis. These phenotypes were consistent with the localization of Abi2 at adherens junctions in the developing lens and at nascent epithelial cell adherens junctions in vitro. Downregulation of Abi expression by RNA interference impaired adherens junction formation and correlated with downregulation of the Wave actin-nucleation promoting factor. Loss of Abi2 also resulted in cell migration defects in the neocortex and hippocampus, abnormal dendritic spine morphology and density, and severe deficits in short- and long-term memory. These findings support a role for Abi2 in the regulation of cytoskeletal dynamics at adherens junctions and dendritic spines, which is critical for intercellular connectivity, cell morphogenesis, and cognitive functions.

scholarly journals Altered Actin Dynamics in Cell Migration of GNE Mutant Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Shamulailatpam Shreedarshanee Devi ◽  
Rashmi Yadav ◽  
Ranjana Arya
Keyword(s):  
Signal Transduction ◽  
Cell Migration ◽  
Sialic Acid ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Cytoskeletal Proteins ◽  
Confocal Imaging ◽  
Actin Dynamics ◽  
Focal Adhesion Complex ◽  
Adhesion Complex

Cell migration is an essential cellular process that requires coordination of cytoskeletal dynamics, reorganization, and signal transduction. The actin cytoskeleton is central in maintaining the cellular structure as well as regulating the mechanisms of cell motility. Glycosylation, particularly sialylation of cell surface proteins like integrins, regulates signal transduction from the extracellular matrix to the cytoskeletal network. The activation of integrin by extracellular cues leads to recruitment of different focal adhesion complex proteins (Src, FAK, paxillin, etc.) and activates the signal including Rho GTPases for the regulation of actin assembly and disassembly. During cell migration, the assembly and disassembly of actin filament provides the essential force for the cell to move. Abnormal sialylation can lead to actin signaling dysfunction leading to aberrant cell migration, one of the main characteristics of cancer and myopathies. In the present study, we have reported altered F-actin to G-actin ratios in GNE mutated cells. These cells exhibit pathologically relevant mutations of GNE (UDP N-acetylneuraminic 2-epimerase/N-acetylmannosamine kinase), a key sialic acid biosynthetic enzyme. It was found that GNE neither affects the actin polymerization nor binds directly to actin. However, mutation in GNE resulted in increased binding of α-actinin to actin filaments. Further, through confocal imaging, GNE was found to be localized in focal adhesion complex along with paxillin. We further elucidated that mutation in GNE resulted in upregulation of RhoA protein and Cofilin activity is downregulated, which could be rescued with Rhosin and chlorogenic acid, respectively. Lastly, mutant in GNE reduced cell migration as implicated from wound healing assay. Our study indicates that molecules altering Cofilin function could significantly revert the cell migration defect due to GNE mutation in sialic acid-deficient cells. We propose cytoskeletal proteins to be alternate drug targets for disorders associated with GNE such as GNE myopathy.

scholarly journals Actin remodeling by Nck regulates endothelial lumen formation

2015 ◽  
Vol 26 (17) ◽  
pp. 3047-3060 ◽  
Author(s):  
Sankar P. Chaki ◽  
Rola Barhoumi ◽  
Gonzalo M. Rivera
Keyword(s):  
Antiangiogenic Therapy ◽  
Three Dimensional ◽  
Resonance Energy Transfer ◽  
Clinical Importance ◽  
Resonance Energy ◽  
Intercellular Junctions ◽  
Time Lapse ◽  
Actin Dynamics ◽  
Actin Remodeling ◽  
Lumen Formation

Multiple angiogenic cues modulate phosphotyrosine signaling to promote vasculogenesis and angiogenesis. Despite its functional and clinical importance, how vascular cells integrate phosphotyrosine-dependent signaling to elicit cytoskeletal changes required for endothelial morphogenesis remains poorly understood. The family of Nck adaptors couples phosphotyrosine signals with actin dynamics and therefore is well positioned to orchestrate cellular processes required in vascular formation and remodeling. Culture of endothelial cells in three-dimensional collagen matrices in the presence of VEGF stimulation was combined with molecular genetics, optical imaging, and biochemistry to show that Nck-dependent actin remodeling promotes endothelial cell elongation and proper organization of VE-cadherin intercellular junctions. Major morphogenetic defects caused by abrogation of Nck signaling included loss of endothelial apical-basal polarity and impaired lumenization. Time-lapse imaging using a Förster resonance energy transfer biosensor, immunostaining with phospho-specific antibodies, and GST pull-down assays showed that Nck determines spatiotemporal patterns of Cdc42/aPKC activation during endothelial morphogenesis. Our results demonstrate that Nck acts as an important hub integrating angiogenic cues with cytoskeletal changes that enable endothelial apical-basal polarization and lumen formation. These findings point to Nck as an emergent target for effective antiangiogenic therapy.

Obligatory role for phospholipase C-γ1 in villin-induced epithelial cell migration

2007 ◽  
Vol 292 (5) ◽  
pp. C1775-C1786 ◽  
Author(s):  
Yaohong Wang ◽  
Alok Tomar ◽  
Sudeep P. George ◽  
Seema Khurana
Keyword(s):  
Cell Migration ◽  
Epithelial Cell ◽  
Phospholipase C ◽  
Resonance Energy Transfer ◽  
Signal Transduction Pathway ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Actin Binding ◽  
Renal Epithelial Cell ◽  
Epithelial Cell Migration

While there is circumstantial evidence to suggest a requirement for phospholipase C-γ1 (PLC-γ1) in actin reorganization and cell migration, few studies have examined the direct mechanisms that link regulators of the actin cytoskeleton with this crucial signaling molecule. This study was aimed to examine the role that villin, an epithelial cell-specific actin-binding protein, and its ligand PLC-γ1 play in migration in intestinal and renal epithelial cell lines that endogenously or ectopically express human villin. Basal as well as epidermal growth factor (EGF)-stimulated cell migration was accompanied by tyrosine phosphorylation of villin and its association with PLC-γ1. Inhibition of villin phosphorylation prevented villin-PLC-γ1 complex formation as well as villin-induced cell migration. The absolute requirement for PLC-γ1 in villin-induced cell migration was demonstrated by measuring cell motility in PLC-γ1−/− cells and by downregulation of endogenous PLC-γ1. EGF-stimulated direct interaction of villin with the Src homology domain 2 domain of PLC-γ1 at the plasma membrane was demonstrated in living cells by using fluorescence resonance energy transfer. These results demonstrate that villin provides an important link between the activation of phosphoinositide signal transduction pathway and epithelial cell migration.

Actin reorganization contributes to loss of cell adhesion in pemphigus vulgaris

2010 ◽  
Vol 299 (3) ◽  
pp. C606-C613 ◽  
Author(s):  
Martin Gliem ◽  
Wolfgang-Moritz Heupel ◽  
Volker Spindler ◽  
Gregory S. Harms ◽  
Jens Waschke
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Pemphigus Vulgaris ◽  
Actin Dynamics ◽  
Protective Effects ◽  
Actin Reorganization ◽  
Cell Dissociation ◽  
Keratinocyte Cell

In the human autoimmune blistering skin disease pemphigus vulgaris autoantibodies (PV-IgG), which are mainly directed against keratinocyte cell adhesion molecules desmoglein (Dsg) 3 and Dsg1, cause keratinocyte cell dissociation (acantholysis). Recent studies reported that loss of keratinocyte cell adhesion was accompanied by profound alterations of the actin cytoskeleton. Nevertheless, the relevance of actin reorganization in this process is unclear at present. In this study, we provide evidence for an important role of actin reorganization in pemphigus pathogenesis. In parallel to loss of cell adhesion and fragmentation of Dsg3 staining along cell borders, PV-IgG treatment resulted in striking changes in actin cytoskeleton organization. Moreover, in experiments using fluorescence recovery after photobleaching (FRAP), PV-IgG were detected to interfere with actin dynamics. Therefore, we investigated whether pharmacological manipulation of actin polymerization modulates pathogenic effects of PV-IgG. Pharmacological stabilization of actin filaments via jasplakinolide significantly blocked cell dissociation and Dsg3 fragmentation, whereas cytochalasin D-induced actin depolymerization strongly enhanced pathogenic effects of PV-IgG. To substantiate these findings, we studied whether the protective effects of Rho GTPases, which are potent regulators of the actin cytoskeleton and were shown to be involved in pemphigus pathogenesis, were dependent on modulation of actin dynamics. Cytotoxic necrotizing factor-1 (CNF-1)-mediated activation of Rho-GTPases enhanced the cortical junction-associated actin belt and blunted PV-IgG-induced cell dissociation. However, when actin polymerization was blocked under these conditions via addition of latrunculin B, the protective effects of CNF-1 were abrogated. Taken together, these experiments indicate that reorganization of cortical actin filaments is a critical step in PV-IgG-induced keratinocyte dissociation.

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