scholarly journals Ezrin Phosphorylation at T567 Modulates Cell Migration, Mechanical Properties, and Cytoskeletal Organization

2020 ◽  
Vol 21 (2) ◽  
pp. 435 ◽  
Author(s):  
Xiaoli Zhang ◽  
Luis R. Flores ◽  
Michael C. Keeling ◽  
Kristina Sliogeryte ◽  
Núria Gavara
Keyword(s):  
Mechanical Properties ◽  
Plasma Membrane ◽  
Cell Migration ◽  
Intracellular Localization ◽  
Dominant Negative ◽  
Cell Stiffness ◽  
Cell Cortex ◽  
Image Quantification ◽  
Cytoskeleton Organization ◽  
Nuclear Changes

Ezrin, a member of the ERM (ezrin/radixin/moesin) family of proteins, serves as a crosslinker between the plasma membrane and the actin cytoskeleton. By doing so, it provides structural links to strengthen the connection between the cell cortex and the plasma membrane, acting also as a signal transducer in multiple pathways during migration, proliferation, and endocytosis. In this study, we investigated the role of ezrin phosphorylation and its intracellular localization on cell motility, cytoskeleton organization, and cell stiffness, using fluorescence live-cell imaging, image quantification, and atomic force microscopy (AFM). Our results show that cells expressing constitutively active ezrin T567D (phosphomimetic) migrate faster and in a more directional manner, especially when ezrin accumulates at the cell rear. Similarly, image quantification results reveal that transfection with ezrin T567D alters the cell’s gross morphology and decreases cortical stiffness. In contrast, constitutively inactive ezrin T567A accumulates around the nucleus, and although it does not impair cell migration, it leads to a significant buildup of actin fibers, a decrease in nuclear volume, and an increase in cytoskeletal stiffness. Finally, cell transfection with the dominant negative ezrin FERM domain induces significant morphological and nuclear changes and affects actin, microtubules, and the intermediate filament vimentin, resulting in cytoskeletal fibers that are longer, thicker, and more aligned. Collectively, our results suggest that ezrin’s phosphorylation state and its intracellular localization plays a pivotal role in cell migration, modulating also biophysical properties, such as membrane–cortex linkage, cytoskeletal and nuclear organization, and the mechanical properties of cells.

scholarly journals Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds

2021 ◽  
Vol 22 (19) ◽  
pp. 10515
Author(s):  
Enikő Pergel ◽  
Irén Veres ◽  
Gergely Imre Csigi ◽  
Gábor Czirják
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Protein ◽  
Intracellular Localization ◽  
Growth Factor Receptor ◽  
Direct Interaction ◽  
Dominant Negative ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Increased Risk ◽  
Inhibitor Compounds

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson’s disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.

scholarly journals The TRQQKRP motif located near the C-terminus of Rac2 is essential for Rac2 biologic functions and intracellular localization

Blood ◽  
2002 ◽  
Vol 100 (5) ◽  
pp. 1679-1688 ◽  
Author(s):  
Wen Tao ◽  
Marie-Dominique Filippi ◽  
Jeffrey R. Bailey ◽  
Simon J. Atkinson ◽  
Bret Connors ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Intracellular Localization ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Dominant Negative ◽  
Membrane Ruffling ◽  
Cytoskeleton Organization ◽  
C Terminus ◽  
Actin Cytoskeleton Organization ◽  
Mutant Phenotypes

Rac GTPases regulate a wide variety of cellular processes including actin cytoskeleton organization, gene expression, cell-cycle progression, and apoptosis. Here we report that the TRQQKRP motif of Rac2 located near the C-terminus, a region of sequence disparity among Rac proteins, is essential for complementation of Rac2 function in Rac2-deficient cells. Deletion of this sequence can also intragenically suppress the dominant-negative Rac2D57Nmutation in a variety of functional assays. In Rac2-deficient cells, expression of TRQQKRP-deleted Rac2 protein is unable to completely rescue migration and nicotinamide adenine dinucleotide phosphate oxidase deficiencies previously described in these cells. In fibroblasts, the Rac2D57N mutant phenotypes of abnormal proliferation, cell morphology, and membrane ruffling are suppressed by the TRQQKRP motif deletion. In myeloid hematopoietic cells, the deletion of the TRQQKRP motif eliminates a Rac2D57N-induced block in in vitro differentiation of neutrophils not previously described with this mutant. Mechanistically, deletion of the TRQQKRP motif results in diminished geranylgeranylation and delocalization of intracellular Rac2 protein. Taken together, these results indicate that the TRQQKRP motif in Rac2 protein is required for efficient prenylation and correct intracellular localization of Rac2 protein and is essential for Rac2 to mediate a variety of its biologic functions. These data suggest that precise localization of Rac2 protein in intracellular compartments and/or with other proteins/lipids is a prerequisite for its diverse functions.

scholarly journals Alterations in Cell Mechanics by Actin Cytoskeletal Changes Correlate with Strain-Specific Rubella Virus Phenotypes for Cell Migration and Induction of Apoptosis

Cells ◽  
2018 ◽  
Vol 7 (9) ◽  
pp. 136 ◽  
Author(s):  
Martin Kräter ◽  
Jiranuwat Sapudom ◽  
Nicole Bilz ◽  
Tilo Pompe ◽  
Jochen Guck ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Cell Migration ◽  
Cell Mechanics ◽  
Rubella Virus ◽  
Cell Movement ◽  
Cell Stiffness ◽  
Filamentous Actin ◽  
Cell Functions ◽  
Viral Spread ◽  
Induction Of Apoptosis

The cellular cytoskeleton is central for key cellular functions, and as such is a marker for diseased and infected cell states. Here we analyzed infection with rubella virus (RV) strains with respect to phenotypes in cellular mechanical properties, cell movement, and viral cytopathogenicity. Real-time deformability cytometry (RT-DC), as a high-throughput platform for the assessment of cell mechanics, revealed a correlation of an increase in cortical filamentous-actin (F-actin) with a higher cellular stiffness. The additional reduction of stress fibers noted for only some RV strains as the most severe actin rearrangement lowered cell stiffness. Furthermore, a reduced collective and single cell migration speed in a wound healing assay was detected in addition to severe changes in cell morphology. The latter was followed by activation of caspase 3/7 as a sign for induction of apoptosis. Our study emphasizes RT-DC technology as a sensitive means to characterize viral cell populations and to implicate alterations of cell mechanical properties with cell functions. These interdependent events are not only promising options to elucidate viral spread and to understand viral pathologies within the infected host. They also contribute to any diseased cell state, as exemplified by RV as a representative agent for cytoskeletal alterations involved in a cytopathological outcome.

scholarly journals LncRNA LINC00472 regulates cell stiffness and inhibits the migration and invasion of lung adenocarcinoma by binding to YBX1

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Xiangying Deng ◽  
Wei Xiong ◽  
Xianjie Jiang ◽  
Shanshan Zhang ◽  
Zheng Li ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Cell Migration ◽  
Lung Adenocarcinoma ◽  
Epithelial Mesenchymal Transition ◽  
Cell Stiffness ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Cell Migration And Invasion ◽  
Adenocarcinoma Cells ◽  
Patient Prognosis

Abstract There is increasing evidence that long non-coding RNAs (lncRNAs) play important roles in human tumorigenesis. By using publicly available expression profiling data from lung adenocarcinoma and integrating bioinformatics analysis, we screened a lncRNA, LINC00472. LINC00472 expression in lung adenocarcinoma tissues was significantly lower and tightly associated with patient prognosis and TNM clinical stages in lung adenocarcinoma. LINC00472 also inhibited lung adenocarcinoma cell migration and invasion and increased cell stiffness and adhesion. RNA pull down and RIP assays identified that LINC00472 interacted with the transcription factor Y-box binding protein 1 (YBX1), which partially reversed the inhibition of cell migration and invasion and increased LINC00472-induced cell stiffness and adhesion. LINC00472 also regulated the density and integrity of F-actin in A549 and PC-9 cells possibly via YBX1. LINC00472 inhibited the cell epithelial-mesenchymal transition (EMT) processes via the modulation of YBX1. These results indicated that LINC00472 inhibited the cell EMT process by binding to YBX1, and affected the mechanical properties of the cell, ultimately inhibited its ability to invade and metastasize. Collectively, the present study provides the first evidence that LINC00472 changes the mechanical properties and inhibits the invasion and metastasis of lung adenocarcinoma cells.

Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1

1998 ◽  
Vol 111 (16) ◽  
pp. 2433-2443 ◽  
Author(s):  
S.A. Weed ◽  
Y. Du ◽  
J.T. Parsons
Keyword(s):  
Actin Polymerization ◽  
Intracellular Localization ◽  
Adhesion Formation ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Actin Binding ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Membrane Ruffling

Small GTPases of the Rho family regulate signaling pathways that control actin cytoskeletal structures. In Swiss 3T3 cells, RhoA activation leads to stress fiber and focal adhesion formation, Rac1 to lamellipoda and membrane ruffles, and Cdc42 to microspikes and filopodia. Several downstream molecules mediating these effects have been recently identified. In this report we provide evidence that the intracellular localization of the actin binding protein cortactin, a Src kinase substrate, is regulated by the activation of Rac1. Cortactin redistributes from the cytoplasm into membrane ruffles as a result of growth factor-induced Rac1 activation, and this translocation is blocked by expression of dominant negative Rac1N17. Expression of constitutively active Rac1L61 evoked the translocation of cortactin from cytoplasmic pools into peripheral membrane ruffles. Expression of mutant forms of the serine/threonine kinase PAK1, a downstream effector of Rac1 and Cdc42 recently demonstrated to trigger cortical actin polymerization and membrane ruffling, also led to the translocation of cortactin to the cell cortex, although this was effectively blocked by coexpression of Rac1N17. Collectively these data provide evidence for cortactin as a putative target of Rac1-induced signal transduction events involved in membrane ruffling and lamellipodia formation.

Osmotic stress-induced remodeling of the cortical cytoskeleton

2002 ◽  
Vol 283 (3) ◽  
pp. C850-C865 ◽  
Author(s):  
Caterina Di Ciano ◽  
Zilin Nie ◽  
Katalin Szászi ◽  
Alison Lewis ◽  
Takehito Uruno ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
De Novo ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Signaling Mechanism ◽  
Actin Binding Domain ◽  
Cytoskeleton Remodeling

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

scholarly journals Rudhira/BCAS3 couples microtubules and intermediate filaments to promote cell migration for angiogenic remodeling

2019 ◽  
Vol 30 (12) ◽  
pp. 1437-1450 ◽  
Author(s):  
Divyesh Joshi ◽  
Maneesha S. Inamdar
Keyword(s):  
Cell Migration ◽  
Intermediate Filaments ◽  
Cross Talk ◽  
Control Cell ◽  
The Novel ◽  
Mouse Development ◽  
Cytoskeleton Organization ◽  
Sprouting Angiogenesis ◽  
Promote Cell Migration ◽  
Necessary And Sufficient

Blood vessel formation requires endothelial cell (EC) migration that depends on dynamic remodeling of the cytoskeleton. Rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) is a cytoskeletal protein essential for EC migration and sprouting angiogenesis during mouse development and is implicated in metastatic disease. Here, we report that Rudhira mediates cytoskeleton organization and dynamics during EC migration. Rudhira binds to both microtubules (MTs) and vimentin intermediate filaments (IFs) and stabilizes MTs. Rudhira depletion impairs cytoskeletal cross-talk, MT stability, and hence focal adhesion disassembly. The BCAS3 domain of Rudhira is necessary and sufficient for MT-IF cross-linking and cell migration. Pharmacologically restoring MT stability rescues gross cytoskeleton organization and angiogenic sprouting in Rudhira-depleted cells. Our study identifies the novel and essential role of Rudhira in cytoskeletal cross-talk and assigns function to the conserved BCAS3 domain. Targeting Rudhira could allow tissue-restricted cytoskeleton modulation to control cell migration and angiogenesis in development and disease.

scholarly journals RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding

2015 ◽  
Vol 309 (9) ◽  
pp. G759-G767 ◽  
Author(s):  
Hee Kyoung Chung ◽  
Navneeta Rathor ◽  
Shelley R. Wang ◽  
Jian-Ying Wang ◽  
Jaladanki N. Rao
Keyword(s):  
Cell Migration ◽  
Transient Receptor Potential ◽  
Ectopic Expression ◽  
Receptor Potential ◽  
Dominant Negative ◽  
Intestinal Epithelial ◽  
Epithelial Restitution ◽  
Epithelial Cell Migration ◽  
Transient Receptor ◽  
Wounded Area

Early mucosal restitution occurs as a consequence of epithelial cell migration to resealing of superficial wounds after injury. Our previous studies show that canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca2+ channel (SOC) in intestinal epithelial cells (IECs) and plays an important role in early epithelial restitution by increasing Ca2+ influx. Here we further reported that RhoA, a small GTP-binding protein, interacts with and regulates TRPC1, thus enhancing SOC-mediated Ca2+ entry (SOCE) and epithelial restitution after wounding. RhoA physically associated with TRPC1 and formed the RhoA/TRPC1 complexes, and this interaction increased in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1). Inactivation of RhoA by treating IEC-TRPC1 cells with exoenzyme C3 transferase (C3) or ectopic expression of dominant negative RhoA (DNMRhoA) reduced RhoA/TRPC1 complexes and inhibited Ca2+ influx after store depletion, which was paralleled by an inhibition of cell migration over the wounded area. In contrast, ectopic expression of wild-type (WT)-RhoA increased the levels of RhoA/TRPC1 complexes, induced Ca2+ influx through activation of SOCE, and promoted cell migration after wounding. TRPC1 silencing by transfecting stable WT RhoA-transfected cells with siRNA targeting TRPC1 (siTRPC1) reduced SOCE and repressed epithelial restitution. Moreover, ectopic overexpression of WT-RhoA in polyamine-deficient cells rescued the inhibition of Ca2+ influx and cell migration induced by polyamine depletion. These findings indicate that RhoA interacts with and activates TRPC1 and thus stimulates rapid epithelial restitution after injury by inducing Ca2+ signaling.

scholarly journals Pan1p, End3p, and Sla1p, Three Yeast Proteins Required for Normal Cortical Actin Cytoskeleton Organization, Associate with Each Other and Play Essential Roles in Cell Wall Morphogenesis

2000 ◽  
Vol 20 (1) ◽  
pp. 12-25 ◽  
Author(s):  
Hsin-Yao Tang ◽  
Jing Xu ◽  
Mingjie Cai
Keyword(s):  
Cell Wall ◽  
Actin Cytoskeleton ◽  
Normal Cell ◽  
Cell Cortex ◽  
Repeat Region ◽  
Cortical Actin ◽  
Cytoskeleton Organization ◽  
Eh Domain ◽  
Actin Cytoskeleton Organization ◽  
Cell Wall Morphogenesis

ABSTRACT The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Δ, andend3Δ mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast.

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