scholarly journals Thymosin β4 is essential for adherens junction stability and epidermal planar cell polarity

Development ◽  
2020 ◽  
Vol 147 (23) ◽  
pp. dev193425
Author(s):  
Krishnanand Padmanabhan ◽  
Hanna Grobe ◽  
Jonathan Cohen ◽  
Arad Soffer ◽  
Adnan Mahly ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Polarity ◽  
Actin Polymerization ◽  
Planar Cell Polarity ◽  
Adherens Junction ◽  
Actin Binding ◽  
Thymosin Β4 ◽  
Eyelid Closure

ABSTRACTPlanar cell polarity (PCP) is essential for tissue morphogenesis and homeostasis; however, the mechanisms that orchestrate the cell shape and packing dynamics required to establish PCP are poorly understood. Here, we identified a major role for the globular (G)-actin-binding protein thymosin-β4 (TMSB4X) in PCP establishment and cell adhesion in the developing epidermis. Depletion of Tmsb4x in mouse embryos hindered eyelid closure and hair-follicle angling owing to PCP defects. Tmsb4x depletion did not preclude epidermal cell adhesion in vivo or in vitro; however, it resulted in abnormal structural organization and stability of adherens junction (AJ) due to defects in filamentous (F)-actin and G-actin distribution. In cultured keratinocytes, TMSB4X depletion increased the perijunctional G/F-actin ratio and decreased G-actin incorporation into junctional actin networks, but it did not change the overall actin expression level or cellular F-actin content. A pharmacological treatment that increased the G/F-actin ratio and decreased actin polymerization mimicked the effects of Tmsb4x depletion on both AJs and PCP. Our results provide insights into the regulation of the actin pool and its involvement in AJ function and PCP establishment.

scholarly journals Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

Endocrinology ◽  
2014 ◽  
Vol 155 (10) ◽  
pp. 3981-3995 ◽  
Author(s):  
N. Ece Gungor-Ordueri ◽  
Elizabeth I. Tang ◽  
Ciler Celik-Ozenci ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Adherens Junction ◽  
Actin Binding ◽  
Permeability Barrier ◽  
Tight Junction Permeability ◽  
Residual Bodies ◽  
Cell Interface

Abstract During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.

scholarly journals Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function, the Actin Cytoskeleton, and Cell Adhesion

2013 ◽  
Vol 288 (29) ◽  
pp. 20966-20977 ◽  
Author(s):  
Haitao Zhang ◽  
Pooja Ghai ◽  
Huhehasi Wu ◽  
Changhui Wang ◽  
Jeffrey Field ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Hela Cells ◽  
Actin Polymerization ◽  
Actin Dynamics ◽  
Ras Signaling ◽  
Filamentous Actin ◽  
Camp Pathway

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

scholarly journals EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

2007 ◽  
Vol 179 (6) ◽  
pp. 1247-1259 ◽  
Author(s):  
Jacco van Rheenen ◽  
Xiaoyan Song ◽  
Wies van Roosmalen ◽  
Michael Cammer ◽  
Xiaoming Chen ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Carcinoma Cells ◽  
Actin Binding ◽  
Temporal Regulation ◽  
Vitro Binding ◽  
Epidermal Growth ◽  
Membrane Bound ◽  
Regulation Of Actin Cytoskeleton

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP2; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP2 in living cells. We show that EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

scholarly journals Tissue fluidity mediated by adherens junction dynamics promotes planar cell polarity-driven ommatidial rotation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nabila Founounou ◽  
Reza Farhadifar ◽  
Giovanna M. Collu ◽  
Ursula Weber ◽  
Michael J. Shelley ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Cell Polarity ◽  
Planar Cell Polarity ◽  
Adherens Junction ◽  
Live Imaging ◽  
Cellular Migration ◽  
Imaging Analysis ◽  
Ommatidial Rotation ◽  
Eye Morphogenesis

AbstractThe phenomenon of tissue fluidity—cells’ ability to rearrange relative to each other in confluent tissues—has been linked to several morphogenetic processes and diseases, yet few molecular regulators of tissue fluidity are known. Ommatidial rotation (OR), directed by planar cell polarity signaling, occurs during Drosophila eye morphogenesis and shares many features with polarized cellular migration in vertebrates. We utilize in vivo live imaging analysis tools to quantify dynamic cellular morphologies during OR, revealing that OR is driven autonomously by ommatidial cell clusters rotating in successive pulses within a permissive substrate. Through analysis of a rotation-specific nemo mutant, we demonstrate that precise regulation of junctional E-cadherin levels is critical for modulating the mechanical properties of the tissue to allow rotation to progress. Our study defines Nemo as a molecular tool to induce a transition from solid-like tissues to more viscoelastic tissues broadening our molecular understanding of tissue fluidity.

scholarly journals Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

2008 ◽  
Vol 295 (5) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Anne E. Kruchten ◽  
Eugene W. Krueger ◽  
Yu Wang ◽  
Mark A. McNiven
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Control Cell ◽  
Focal Adhesions ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Actin Assembly

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

scholarly journals The actin-binding protein EPS8 binds VE-cadherin and modulates YAP localization and signaling

2015 ◽  
Vol 211 (6) ◽  
pp. 1177-1192 ◽  
Author(s):  
Costanza Giampietro ◽  
Andrea Disanza ◽  
Luca Bravi ◽  
Miriam Barrios-Rodiles ◽  
Monica Corada ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Adherens Junction ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Intracellular Signals ◽  
Transcriptional Cofactors

Vascular endothelial (VE)–cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14–3-3–YAP associates with the VE–cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity.

scholarly journals Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides.

1996 ◽  
Vol 135 (1) ◽  
pp. 169-179 ◽  
Author(s):  
D A Schafer ◽  
P B Jennings ◽  
J A Cooper
Keyword(s):  
Protein Binding ◽  
Rate Constant ◽  
Half Life ◽  
Actin Polymerization ◽  
Beta Subunit ◽  
Actin Binding ◽  
Capping Protein ◽  
Subunit Isoforms

Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on-rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro.

scholarly journals The LRRK2-related Roco kinase Roco2 is regulated by Rab1A and controls the actin cytoskeleton

2011 ◽  
Vol 22 (13) ◽  
pp. 2198-2211 ◽  
Author(s):  
Sebastian Kicka ◽  
Zhouxin Shen ◽  
Sarah J. Annesley ◽  
Paul R. Fisher ◽  
Susan Lee ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Polarity ◽  
Actin Polymerization ◽  
Kinase Activity ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Actin Binding ◽  
Leucine Rich Repeat ◽  
Mediate Cell

We identify a new pathway that is required for proper pseudopod formation. We show that Roco2, a leucine-rich repeat kinase 2 (LRRK2)-related Roco kinase, is activated in response to chemoattractant stimulation and helps mediate cell polarization and chemotaxis by regulating cortical F-actin polymerization and pseudopod extension in a pathway that requires Rab1A. We found that Roco2 binds the small GTPase Rab1A as well as the F-actin cross-linking protein filamin (actin-binding protein 120, abp120) in vivo. We show that active Rab1A (Rab1A-GTP) is required for and regulates Roco2 kinase activity in vivo and that filamin lies downstream from Roco2 and controls pseudopod extension during chemotaxis and random cell motility. Therefore our study uncovered a new signaling pathway that involves Rab1A and controls the actin cytoskeleton and pseudopod extension, and thereby, cell polarity and motility. These findings also may have implications in the regulation of other Roco kinases, including possibly LRRK2, in metazoans.

scholarly journals Shear Stress Regulates Forward and Reverse Planar Cell Polarity of Vascular Endothelium In Vivo and In Vitro

2006 ◽  
Vol 98 (7) ◽  
pp. 939-946 ◽  
Author(s):  
Shannon McCue ◽  
Dorota Dajnowiec ◽  
Feng Xu ◽  
Ming Zhang ◽  
Moira R. Jackson ◽  
...  
Keyword(s):  
Shear Stress ◽  
Cell Polarity ◽  
Vascular Endothelium ◽  
Planar Cell Polarity

Metalloprotease-disintegrin ADAM 12 interacts with α-actinin-1

2001 ◽  
Vol 357 (2) ◽  
pp. 353-361 ◽  
Author(s):  
Yi CAO ◽  
Qing KANG ◽  
Anna ZOLKIEWSKA
Keyword(s):  
Cell Adhesion ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Cytoplasmic Domain ◽  
C2c12 Cells ◽  
Actin Binding ◽  
Myoblast Differentiation ◽  
Adam 12

ADAM 12, a member of the ADAM family of proteins (containing ADisintegrin And Metalloprotease domain), has been implicated in differentiation and fusion of myoblasts. While the extracellular domain of ADAM 12 contains an active metalloprotease and a region involved in cell adhesion, the function of the cytoplasmic tail of ADAM 12 has been less clear. Here we show that the cytoplasmic domain of ADAM 12 interacts in vitro and in vivo with α-actinin-1, an actin-binding and cross-linking protein. Green fluorescent protein fused to ADAM 12 cytoplasmic domain co-localizes with α-actinin-1-containing actin stress fibres in C2C12 cells. The interaction between ADAM 12 and α-actinin-1 is direct and involves the 58-amino acid C-terminal fragment of ADAM 12 and the 27kDa N-terminal domain of α-actinin-1. Consistently, expression of the 27kDa fragment of α-actinin-1 in C2C12 cells using a mitochondrial targeting system results in recruitment of the co-expressed ADAM 12 cytoplasmic domain to the mitochondrial surface. Moreover, α-actinin-1 co-purifies with a transmembrane, His6-tagged form of ADAM 12 expressed in C2C12 myoblasts, indicating that the transmembrane ADAM 12 forms a complex with α-actinin-1 in vivo. These results indicate that the actin cytoskeleton may play a critical role in ADAM 12-mediated cell–cell adhesion or cell signalling during myoblast differentiation and fusion.

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