scholarly journals A module for Rac temporal signal integration revealed with optogenetics

2017 ◽  
Vol 216 (8) ◽  
pp. 2515-2531 ◽  
Author(s):  
Brian R. Graziano ◽  
Delquin Gong ◽  
Karen E. Anderson ◽  
Anne Pipathsouk ◽  
Anna R. Goldberg ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Dynamic Range ◽  
Signaling Cascades ◽  
Signaling Networks ◽  
Nucleotide Exchange ◽  
Adaptive Circuits ◽  
Guanine Nucleotide Exchange ◽  
Transient Activation ◽  
Nucleotide Exchange Factor ◽  
Complex Signaling

Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation. We find that persistent, optogenetically driven phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production results in only transient activation of Rac, a hallmark feature of adaptive circuits. We further identify the guanine nucleotide exchange factor P-Rex1 as the primary PIP3-stimulated Rac activator, whereas actin polymerization and the GTPase-activating protein ArhGAP15 are essential for proper Rac turnoff. This circuit is masked by feedback and redundancy when chemoattractant is used as the input, highlighting the value of probing signaling networks at intermediate nodes to deconvolve complex signaling cascades.

EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity

2011 ◽  
Vol 439 (3) ◽  
pp. 433-445 ◽  
Author(s):  
Sigi Benjamin ◽  
Hilla Weidberg ◽  
Debora Rapaport ◽  
Olga Pekar ◽  
Marina Nudelman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Inhibitory Effect ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
System A ◽  
Nucleotide Exchange Factor ◽  
Rac1 Activity ◽  
The Impact

EHDs [EH (Eps15 homology)-domain-containing proteins] participate in different stages of endocytosis. EHD2 is a plasma-membrane-associated EHD which regulates trafficking from the plasma membrane and recycling. EHD2 has a role in nucleotide-dependent membrane remodelling and its ATP-binding domain is involved in dimerization, which creates a membrane-binding region. Nucleotide binding is important for association of EHD2 with the plasma membrane, since a nucleotide-free mutant (EHD2 T72A) failed to associate. To elucidate the possible function of EHD2 during endocytic trafficking, we attempted to unravel proteins that interact with EHD2, using the yeast two-hybrid system. A novel interaction was found between EHD2 and Nek3 [NIMA (never in mitosis in Aspergillus nidulans)-related kinase 3], a serine/threonine kinase. EHD2 was also found in association with Vav1, a Nek3-regulated GEF (guanine-nucleotide-exchange factor) for Rho GTPases. Since Vav1 regulates Rac1 activity and promotes actin polymerization, the impact of overexpression of EHD2 on Rac1 activity was tested. The results indicated that wt (wild-type) EHD2, but not its P-loop mutants, reduced Rac1 activity. The inhibitory effect of EHD2 overexpression was partially rescued by co-expression of Rac1 as measured using a cholera toxin trafficking assay. The results of the present study strongly indicate that EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity.

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 Myosin II–interacting guanine nucleotide exchange factor promotes bleb retraction via stimulating cortex reassembly at the bleb membrane

2018 ◽  
Vol 29 (5) ◽  
pp. 643-656 ◽  
Author(s):  
Meng Jiao ◽  
Di Wu ◽  
Qize Wei
Keyword(s):  
Actin Polymerization ◽  
Myosin Ii ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Nonmuscle Myosin Ii

Blebs are involved in various biological processes such as cell migration, cytokinesis, and apoptosis. While the expansion of blebs is largely an intracellular pressure-driven process, the retraction of blebs is believed to be driven by RhoA activation that leads to the reassembly of the actomyosin cortex at the bleb membrane. However, it is still poorly understood how RhoA is activated at the bleb membrane. Here, we provide evidence demonstrating that myosin II–interacting guanine nucleotide exchange factor (MYOGEF) is implicated in bleb retraction via stimulating RhoA activation and the reassembly of an actomyosin network at the bleb membrane during bleb retraction. Interaction of MYOGEF with ezrin, a well-known regulator of bleb retraction, is required for MYOGEF localization to retracting blebs. Notably, knockout of MYOGEF or ezrin not only disrupts RhoA activation at the bleb membrane, but also interferes with nonmuscle myosin II localization and activation, as well as actin polymerization in retracting blebs. Importantly, MYOGEF knockout slows down bleb retraction. We propose that ezrin interacts with MYOGEF and recruits it to retracting blebs, where MYOGEF activates RhoA and promotes the reassembly of the cortical actomyosin network at the bleb membrane, thus contributing to the regulation of bleb retraction.

scholarly journals PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front

2016 ◽  
Vol 113 (36) ◽  
pp. 10091-10096 ◽  
Author(s):  
Trang Thi Thu Nguyen ◽  
Wei Sun Park ◽  
Byung Ouk Park ◽  
Cha Yeon Kim ◽  
Yohan Oh ◽  
...  
Keyword(s):  
Positive Feedback ◽  
Actin Polymerization ◽  
Leading Edge ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cell Front ◽  
Control Protein

Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.

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