scholarly journals Aldolase directly interacts with ARNO and modulates cell morphology and acidic vesicle distribution

2011 ◽  
Vol 300 (6) ◽  
pp. C1442-C1455 ◽  
Author(s):  
Maria Merkulova ◽  
Andrés Hurtado-Lorenzo ◽  
Hiroyuki Hosokawa ◽  
Zhenjie Zhuang ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Hela Cells ◽  
Morphological Changes ◽  
Direct Interaction ◽  
Pleckstrin Homology Domain ◽  
High Avidity ◽  
Cell Fractionation ◽  
Nucleotide Exchange ◽  
Early Endosomes ◽  
Acidic Vesicle

Previously, we demonstrated that the vacuolar-type H+-ATPase (V-ATPase) a2-subunit functions as an endosomal pH sensor that interacts with the ADP-ribosylation factor (Arf) guanine nucleotide exchange factor, ARNO. In the present study, we showed that ARNO directly interacts not only with the a2-subunit but with all a-isoforms (a1–a4) of the V-ATPase, indicating a widespread regulatory interaction between V-ATPase and Arf GTPases. We then extended our search for other ARNO effectors that may modulate V-ATPase-dependent vesicular trafficking events and actin cytoskeleton remodeling. Pull-down experiments using cytosol of mouse proximal tubule cells (MTCs) showed that ARNO interacts with aldolase, but not with other enzymes of the glycolytic pathway. Direct interaction of aldolase with the pleckstrin homology domain of ARNO was revealed by pull-down assays using recombinant proteins, and surface plasmon resonance revealed their high avidity interaction with a dissociation constant: KD = 2.84 × 10−10 M. MTC cell fractionation revealed that aldolase is also associated with membranes of early endosomes. Functionally, aldolase knockdown in HeLa cells produced striking morphological changes accompanied by long filamentous cell protrusions and acidic vesicle redistribution. However, the 50% knockdown we achieved did not modulate the acidification capacity of endosomal/lysosomal compartments. Finally, a combination of small interfering RNA knockdown and overexpression revealed that the expression of aldolase is inversely correlated with gelsolin levels in HeLa cells. In summary, we have shown that aldolase forms a complex with ARNO/Arf6 and the V-ATPase and that it may contribute to remodeling of the actin cytoskeleton and/or the trafficking and redistribution of V-ATPase-dependent acidic compartments via a combination of protein-protein interaction and gene expression mechanisms.

scholarly journals Interaction between Tiam1 and the Arp2/3 complex links activation of Rac to actin polymerization

2006 ◽  
Vol 397 (1) ◽  
pp. 39-45 ◽  
Author(s):  
Jean Paul ten Klooster ◽  
Eva E. Evers ◽  
Lennert Janssen ◽  
Laura M. Machesky ◽  
Frits Michiels ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Morphological Changes ◽  
Coiled Coil ◽  
Pleckstrin Homology Domain ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Coiled Coil Region ◽  
T Lymphoma ◽  
Cell Cell

The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 (T-lymphoma invasion and metastasis 1) regulates migration, cell–matrix and cell–cell adhesion by modulating the actin cytoskeleton through the GTPase, Rac1. Using yeast two-hybrid screening and biochemical assays, we found that Tiam1 interacts with the p21-Arc [Arp (actin-related protein) complex] subunit of the Arp2/3 complex. Association occurred through the N-terminal pleckstrin homology domain and the adjacent coiled-coil region of Tiam1. As a result, Tiam1 co-localizes with the Arp2/3 complex at sites of actin polymerization, such as epithelial cell–cell contacts and membrane ruffles. Deletion of the p21-Arc-binding domain in Tiam1 impairs its subcellular localization and capacity to activate Rac1, suggesting that binding to the Arp2/3 complex is important for the function of Tiam1. Indeed, blocking Arp2/3 activation with a WASP (Wiskott–Aldrich syndrome protein) inhibitor leads to subcellular relocalization of Tiam1 and decreased Rac activation. Conversely, functionally active Tiam1, but not a GEF-deficient mutant, promotes activation of the Arp2/3 complex and its association with cytoskeletal components, indicating that Tiam1 and Arp2/3 are mutually dependent for their correct localization and signalling. Our data suggests a model in which the Arp2/3 complex acts as a scaffold to localize Tiam1, and thereby Rac activity, which are both required for activation of the Arp2/3 complex and further Arp2/3 recruitment. This ‘self-amplifying’ signalling module involving Tiam1, Rac and the Arp2/3 complex could thus drive actin polymerization at specific sites in cells that are required for dynamic morphological changes.

scholarly journals Effects of innate immune receptor stimulation on extracellular α-synuclein uptake and degradation by brain resident cells

2021 ◽  
Author(s):  
Changyoun Kim ◽  
Somin Kwon ◽  
Michiyo Iba ◽  
Brian Spencer ◽  
Edward Rockenstein ◽  
...  
Keyword(s):  
Degradation Kinetics ◽  
Morphological Changes ◽  
Direct Interaction ◽  
Innate Immune ◽  
Pathological Feature ◽  
Tlr2 Expression ◽  
Immune Receptors ◽  
Age Related ◽  
Stimulation Of

AbstractSynucleinopathies are age-related neurological disorders characterized by the progressive deposition of α-synuclein (α-syn) aggregates and include Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Although cell-to-cell α-syn transmission is thought to play a key role in the spread of α-syn pathology, the detailed mechanism is still unknown. Neuroinflammation is another key pathological feature of synucleinopathies. Previous studies have identified several immune receptors that mediate neuroinflammation in synucleinopathies, such as Toll-like receptor 2 (TLR2). However, the species of α-syn aggregates varies from study to study, and how different α-syn aggregate species interact with innate immune receptors has yet to be addressed. Therefore, we investigated whether innate immune receptors can facilitate the uptake of different species of α-syn aggregates. Here, we examined whether stimulation of TLRs could modulate the cellular uptake and degradation of α-syn fibrils despite a lack of direct interaction. We observed that stimulation of TLR2 in vitro accelerated α-syn fibril uptake in neurons and glia while delaying the degradation of α-syn in neurons and astrocytes. Internalized α-syn was rapidly degraded in microglia regardless of whether TLR2 was stimulated. However, cellular α-syn uptake and degradation kinetics were not altered by TLR4 stimulation. In addition, upregulation of TLR2 expression in a synucleinopathy mouse model increased the density of Lewy-body-like inclusions and induced morphological changes in microglia. Together, these results suggest that cell type-specific modulation of TLR2 may be a multifaceted and promising therapeutic strategy for synucleinopathies; inhibition of neuronal and astroglial TLR2 decreases pathogenic α-syn transmission, but activation of microglial TLR2 enhances microglial extracellular α-syn clearance.

scholarly journals Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

1997 ◽  
Vol 8 (3) ◽  
pp. 533-545 ◽  
Author(s):  
T Harder ◽  
R Kellner ◽  
R G Parton ◽  
J Gruenberg
Keyword(s):  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Annexin Ii ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Independent Manner ◽  
Low Concentrations ◽  
Early Endosomes ◽  
Associated Proteins ◽  
Cytoskeletal Elements

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

scholarly journals Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export

2014 ◽  
Vol 206 (6) ◽  
pp. 751-762 ◽  
Author(s):  
Kota Saito ◽  
Koh Yamashiro ◽  
Noriko Shimazu ◽  
Tomoya Tanabe ◽  
Kenji Kontani ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Secretory Proteins ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Transport Vesicles ◽  
Nucleotide Exchange Factor ◽  
Receptor Component ◽  
Er Exit Sites ◽  
Guanosine Triphosphatase ◽  
Trimeric Structure

Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60–90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate–bound Sar1 generated by Sec12 localized at ER exit sites.

scholarly journals Coxiella burnetii Induces Reorganization of the Actin Cytoskeleton in Human Monocytes

1998 ◽  
Vol 66 (11) ◽  
pp. 5527-5533 ◽  
Author(s):  
Sonia Meconi ◽  
Véronique Jacomo ◽  
Patrice Boquet ◽  
Didier Raoult ◽  
Jean-Louis Mege ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Coxiella Burnetii ◽  
Actin Polymerization ◽  
Q Fever ◽  
Morphological Changes ◽  
Critical Role ◽  
Filamentous Actin ◽  
Cytoskeleton Reorganization ◽  
Membrane Protrusions ◽  
Actin Protrusions

ABSTRACT Coxiella burnetii, an obligate intracellular bacterium which survives in myeloid cells, causes Q fever in humans. We previously demonstrated that virulent C. burnetiiorganisms are poorly internalized by monocytes compared to avirulent variants. We hypothesized that a differential mobilization of the actin cytoskeleton may account for this distinct phagocytic behavior. Scanning electron microscopy demonstrated that virulent C. burnetii stimulated profound and polymorphic changes in the morphology of THP-1 monocytes, consisting of membrane protrusions and polarized projections. These changes were transient, requiring 5 min to reach their maximum extent and vanishing after 60 min of incubation. In contrast, avirulent variants of C. burnetii did not induce any significant changes in cell morphology. The distribution of filamentous actin (F-actin) was then studied with a specific probe, bodipy phallacidin. Virulent C. burnetii induced a profound and transient reorganization of F-actin, accompanied by an increase in the F-actin content of THP-1 cells. F-actin was colocalized with myosin in cell protrusions, suggesting that actin polymerization and the tension of actin-myosin filaments play a role in C. burnetii-induced morphological changes. In addition, contact between the cell and the bacterium seems to be necessary to induce cytoskeleton reorganization. Bacterial supernatants did not stimulate actin remodeling, and virulent C. burnetii organisms were found in close apposition with F-actin protrusions. The manipulation of the actin cytoskeleton by C. burnetiimay therefore play a critical role in the internalization strategy of this bacterium.

scholarly journals Selectivity of Guanine Nucleotide Exchange Factor-mediated Cdc42 activation in primary human endothelial cells

2019 ◽  
Author(s):  
Nathalie R. Reinhard ◽  
Sanne van der Niet ◽  
Anna Chertkova ◽  
Marten Postma ◽  
Peter L. Hordijk ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Actin Dynamics ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Dh Domain

AbstractThe Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signaling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the full-length GEFs. Our data reveal a specific GEF dependent activation profile, with most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and pRex1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may activate Cdc42, which will be of great value for the field of vascular biology.Abstract FigureGraphical Abstract

scholarly journals ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration

2008 ◽  
Vol 181 (2) ◽  
pp. 351-365 ◽  
Author(s):  
Junji Yamauchi ◽  
Yuki Miyamoto ◽  
Jonah R. Chan ◽  
Akito Tanoue
Keyword(s):  
Cell Migration ◽  
Schwann Cell ◽  
Rho Gtpase ◽  
Morphological Changes ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Schwann Cell Migration ◽  
Subsequent Activation

The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118–to–Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.

scholarly journals Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors.

1995 ◽  
Vol 15 (2) ◽  
pp. 943-953 ◽  
Author(s):  
R I Scheinman ◽  
A Gualberto ◽  
C M Jewell ◽  
J A Cidlowski ◽  
A S Baldwin
Keyword(s):  
Gene Expression ◽  
Hela Cells ◽  
Transcriptional Activation ◽  
Glucocorticoid Receptors ◽  
Interleukin 1 ◽  
Direct Interaction ◽  
Significant Loss ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Important Regulator

Glucocorticoids are potent immunosuppressants which work in part by inhibiting cytokine gene transcription. We show here that NF-kappa B, an important regulator of numerous cytokine genes, is functionally inhibited by the synthetic glucocorticoid dexamethasone (DEX). In transfection experiments, DEX treatment in the presence of cotransfected glucocorticoid receptor (GR) inhibits NF-kappa B p65-mediated gene expression and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF-kappa B subunits p65 and p50. In addition, we demonstrate that the ability of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX and GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-kappa B at the levels of both DNA binding and transcriptional activation. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data reveal a second mechanism by which NF-kappa B activity may be regulated by DEX. We also report that RU486 treatment of wild-type GR and DEX treatment of a transactivation mutant of GR each can significantly inhibit p65 activity. In addition, we found that the zinc finger domain of GR is necessary for the inhibition of p65. This domain is also required for GR repression of AP-1. Surprisingly, while both AP-1 and NF-kappa B can be inhibited by activated GR, synergistic NF-kappa B/AP-1 activity is largely unaffected. These data suggest that NF-kappa B, AP-1, and GR interact in a complex regulatory network to modulate gene expression and that cross-coupling of NF-kappa B and GR plays an important role in glucocorticoid-mediated repression of cytokine transcription.

Sign in / Sign up

Export Citation Format

Share Document