Selective control of membrane ruffling and actin plaque assembly by the Rho GTPases Rac1 and CDC42 in FcepsilonRI-activated rat basophilic leukemia (RBL-2H3) cells

1997 ◽  
Vol 110 (18) ◽  
pp. 2215-2225 ◽  
Author(s):  
J.C. Guillemot ◽  
P. Montcourrier ◽  
E. Vivier ◽  
J. Davoust ◽  
P. Chavrier
Keyword(s):  
Mast Cells ◽  
Rho Gtpases ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Membrane Ruffling ◽  
Cytoskeletal Responses ◽  
High Affinity Ige Receptor ◽  
Mutant Forms ◽  
Basophilic Leukemia

Engagement of the high affinity IgE receptor (FcepsilonRI) in mast cells elicits a series of intracellular signalling events including cytoskeletal reorganization and granule exocytosis. To analyze the coupling of receptor activation to specific cytoskeletal responses, we expressed dominant negative mutant forms of the Rho GTPases CDC42 and Rac1 in rat RBL-2H3 tumor mast cells. We show here that dominant inhibition of CDC42 function decreases cell adhesion, interferes with Fc(epsilon)RI-induced actin plaque assembly and reduced the recruitment of vinculin at the cell-substratum interface, while the inhibitory Rac1 mutant abolishes Fc(epsilon)RI-mediated membrane ruffling. The expression of trans-dominant inhibitory forms of either CDC42 or Rac1 significantly inhibited antigen-induced degranulation. Altogether, our results demonstrate that CDC42 and Rac1 control distinct pathways downstream of FcepsilonRI engagement leading either to the induction of actin plaques, or to the production of membrane ruffles. These two pathways are critically involved during the degranulation response induced by Fc(epsilon)RI aggregation.

scholarly journals Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid.

1997 ◽  
Vol 17 (3) ◽  
pp. 1201-1211 ◽  
Author(s):  
R Kozma ◽  
S Sarner ◽  
S Ahmed ◽  
L Lim
Keyword(s):  
Lysophosphatidic Acid ◽  
Growth Cones ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Neurite Retraction ◽  
Rho Family Gtpases ◽  
C3 Exoenzyme ◽  
Rho Family ◽  
Neurite Development

Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells. Here we show that microinjection of Cdc42Hs and Rac1 promoted formation of filopodia and lamellipodia in N1E-115 neuroblastoma growth cones and along neurites. These actin-containing structures were also induced by injection of Clostridium botulinum C3 exoenzyme, which abolishes RhoA-mediated functions such as neurite retraction. The C3 response was inhibited by coinjection with the dominant negative mutant Cdc42Hs(T17N), while the Cdc42Hs response could be competed by coinjection with RhoA. We also demonstrate that the neurotransmitter acetylcholine (ACh) can induce filopodia and lamellipodia on neuroblastoma growth cones via muscarinic ACh receptor activation, but only when applied in a concentration gradient. ACh-induced formation of filopodia and lamellipodia was inhibited by preinjection with the dominant negative mutants Cdc42Hs(T17N) and Rac1(T17N), respectively. Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, was inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth was inhibited by injection with Cdc42Hs(T17N) or Rac1(T17N). Together these results suggest that there is competition between the ACh- and LPA-induced morphological pathways mediated by Cdc42Hs and/or Rac1 and by RhoA, leading to either neurite development or collapse.

scholarly journals Mutant Rab7 Causes the Accumulation of Cathepsin D and Cation-independent Mannose 6–Phosphate Receptor in an Early Endocytic Compartment

1998 ◽  
Vol 140 (5) ◽  
pp. 1075-1089 ◽  
Author(s):  
Barry Press ◽  
Yan Feng ◽  
Bernard Hoflack ◽  
Angela Wandinger-Ness
Keyword(s):  
Cathepsin D ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Lysosomal Hydrolases ◽  
Endocytic Compartment ◽  
Lysosome Biogenesis ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Lysosomal Membrane Glycoprotein ◽  
Mutant Forms

Stable BHK cell lines inducibly expressing wild-type or dominant negative mutant forms of the rab7 GTPase were isolated and used to analyze the role of a rab7-regulated pathway in lysosome biogenesis. Expression of mutant rab7N125I protein induced a dramatic redistribution of cation-independent mannose 6–phosphate receptor (CI-MPR) from its normal perinuclear localization to large peripheral endosomes. Under these circumstances ∼50% of the total receptor and several lysosomal hydrolases cofractionated with light membranes containing early endosome and Golgi markers. Late endosomes and lysosomes were contained exclusively in well-separated, denser gradient fractions. Newly synthesized CI-MPR and cathepsin D were shown to traverse through an early endocytic compartment, and functional rab7 was crucial for delivery to later compartments. This observation was evidenced by the fact that 2 h after synthesis, both markers were more prevalent in fractions containing light membranes. In addition, both were sensitive to HRP-DAB– mediated cross-linking of early endosomal proteins, and the late endosomal processing of cathepsin D was impaired. Using similar criteria, the lysosomal membrane glycoprotein 120 was not found accumulated in an early endocytic compartment. The data are indicative of a post-Golgi divergence in the routes followed by different lysosome-directed molecules.

scholarly journals Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the Trans-Golgi Network

2000 ◽  
Vol 151 (6) ◽  
pp. 1207-1220 ◽  
Author(s):  
Mona Wilcke ◽  
Ludger Johannes ◽  
Thierry Galli ◽  
Véronique Mayau ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Intracellular Compartments ◽  
Golgi Network ◽  
Mutant Forms ◽  
In Cells

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20°C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

scholarly journals The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

1998 ◽  
Vol 143 (4) ◽  
pp. 1087-1099 ◽  
Author(s):  
Angeliki Malliri ◽  
Marc Symons ◽  
Robert F. Hennigan ◽  
Adam F.L. Hurlstone ◽  
Richard F. Lamb ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cortical Actin ◽  
A431 Cells ◽  
Membrane Ruffling ◽  
In Vitro Invasion ◽  
Cytoskeletal Rearrangements ◽  
Lamellipodia Formation

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

scholarly journals The HTR1 Gene Is a Dominant Negative Mutant Allele of MTH1 and Blocks Snf3- and Rgt2-Dependent Glucose Signaling in Yeast

2000 ◽  
Vol 182 (2) ◽  
pp. 540-542 ◽  
Author(s):  
Frank Schulte ◽  
Roman Wieczorke ◽  
Cornelis P. Hollenberg ◽  
Eckhard Boles
Keyword(s):  
Gene Expression ◽  
Mutant Allele ◽  
Transcriptional Regulator ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Hexose Transporter ◽  
Transporter Gene ◽  
Glucose Signaling ◽  
Mutant Forms ◽  
Negative Mutant

ABSTRACT Saccharomyces cerevisiae HTR1 mutants are severely impaired in the uptake of glucose. We have cloned dominantHTR1 mutant alleles and show that they encode mutant forms of the Mth1 protein. Mth1 is shown to be involved in carbon source-dependent regulation of its own, invertase and hexose transporter gene expression. The mutant forms block the transduction of the Snf3- and Rgt2-mediated glucose signals upstream of the Rgt1 transcriptional regulator.

Regulation of FcεRI-mediated degranulation by an adaptor protein 3BP2 in rat basophilic leukemia RBL-2H3 cells

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2138-2144 ◽  
Author(s):  
Kiyonao Sada ◽  
S. M. Shahjahan Miah ◽  
Koichiro Maeno ◽  
Shinkou Kyo ◽  
Xiujuan Qu ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Calcium Influx ◽  
Adaptor Protein ◽  
Cellular Protein ◽  
Sh2 Domain ◽  
External Sources ◽  
High Affinity Ige Receptor ◽  
Basophilic Leukemia

Abstract Aggregation of high-affinity IgE receptor FcεRI induces sequential activation of nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, leading to degranulation in mast cells. A hematopoietic cell–specific adaptor protein, 3BP2, that was originally identified as an Abl SH3-binding protein was rapidly tyrosine phosphorylated by the aggregation of FcεRI on rat basophilic leukemia RBL-2H3 cells. Tyrosine phosphorylation of 3BP2 did not depend on calcium influx from external sources. To examine the role of 3BP2 in mast cells, we overexpressed the SH2 domain of 3BP2 in the RBL-2H3 cells. Overexpression of 3BP2-SH2 domain resulted in a suppression of antigen-induced degranulation as assessed by β-hexosaminidase release. Even though overall tyrosine phosphorylation of cellular protein was not altered, antigen-mediated tyrosine phosphorylation of phospholipase C-γ (PLC-γ) and calcium mobilization were significantly suppressed in the cells overexpressing the 3BP2-SH2 domain. Furthermore, antigen stimulation induced the association of 3BP2-SH2 domain with LAT and other signaling molecule complexes in the RBL-2H3 cells. FcεRI-mediated phosphorylation of JNK and ERK was not affected by the overexpression of 3BP2-SH2 domain. These data indicate that 3BP2 functions to positively regulate the FcεRI-mediated tyrosine phosphorylation of PLC-γ and thereby the signals leading to degranulation.

scholarly journals The Membrane Dynamics of Pexophagy Are Influenced by Sar1p in Pichia pastoris

2008 ◽  
Vol 19 (11) ◽  
pp. 4888-4899 ◽  
Author(s):  
Laura A. Schroder ◽  
Michael V. Ortiz ◽  
William A. Dunn
Keyword(s):  
Pichia Pastoris ◽  
Membrane Dynamics ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Guanosine Triphosphate ◽  
Guanosine Diphosphate ◽  
Mutant Forms ◽  
Negative Mutant

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy.

scholarly journals A Role for Centrin 3 in Centrosome Reproduction

2000 ◽  
Vol 148 (3) ◽  
pp. 405-416 ◽  
Author(s):  
Sandrine Middendorp ◽  
Thomas Küntziger ◽  
Yann Abraham ◽  
Simon Holmes ◽  
Nicole Bordes ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Protein S ◽  
Spindle Pole ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Cell Stage ◽  
Ef Hand ◽  
Mutant Forms ◽  
Xenopus Laevis Embryos

Centrosome reproduction by duplication is essential for the bipolarity of cell division, but the molecular basis of this process is still unknown. Mutations in Saccharomyces cerevisiae CDC31 gene prevent the duplication of the spindle pole body (SPB). The product of this gene belongs to the calmodulin super-family and is concentrated at the half bridge of the SPB. We present a functional analysis of HsCEN3, a human centrin gene closely related to the CDC31 gene. Tran- sient overexpression of wild-type or mutant forms of HsCen3p in human cells demonstrates that centriole localization depends on a functional fourth EF-hand, but does not produce mitotic phenotype. However, injection of recombinant HsCen3p or of RNA encoding HsCen3p in one blastomere of two-cell stage Xenopus laevis embryos resulted in undercleavage and inhibition of centrosome duplication. Furthermore, HsCEN3 does not complement mutations or deletion of CDC31 in S. cerevisiae, but specifically blocks SPB duplication, indicating that the human protein acts as a dominant negative mutant of CDC31. Several lines of evidence indicate that HsCen3p acts by titrating Cdc31p-binding protein(s). Our results demonstrate that, in spite of the large differences in centrosome structure among widely divergent species, the centrosome pathway of reproduction is conserved.

scholarly journals High resolution mapping of mast cell membranes reveals primary and secondary domains of FcϵRI and LAT

2001 ◽  
Vol 154 (3) ◽  
pp. 645-658 ◽  
Author(s):  
Bridget S. Wilson ◽  
Janet R. Pfeiffer ◽  
Zurab Surviladze ◽  
Elizabeth A. Gaudet ◽  
Janet M. Oliver
Keyword(s):  
Mast Cells ◽  
Receptor Activation ◽  
Pi3 Kinase ◽  
Cross Linking ◽  
Phosphatidylinositol 3 Kinase ◽  
Coated Pits ◽  
High Resolution Mapping ◽  
Inositol Phospholipids ◽  
Resolution Mapping ◽  
High Affinity Ige Receptor

In mast cells, cross-linking the high-affinity IgE receptor (FcϵRI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting FcϵRI colocalize loosely with Lyn, whereas cross-linked FcϵRI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of FcϵRI β is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCγ isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCγ1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCγ2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCγ2, Gab2, and a portion of p85 colocalize with FcϵRI β in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCγ1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, FcϵRI cross-linking increases PI3-kinase activity in anti-LAT, anti-FcεRIβ, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around FcεRIβ and from secondary domains, including one organized around LAT.

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