scholarly journals Single-component optogenetic tools for inducible RhoA GTPase signaling

2021 ◽  
Author(s):  
Erin E. Berlew ◽  
Ivan A. Kuznetsov ◽  
Keisuke Yamada ◽  
Lukasz J. Bugaj ◽  
Joel D. Boerckel ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Cell Polarization ◽  
Single Component ◽  
Membrane Recruitment ◽  
Actin Organization ◽  
Precise Control ◽  
Binding Partners ◽  
Actomyosin Contractility ◽  
Rhoa Gtpase ◽  
Rhoa Signaling

ABSTRACTWe created optogenetic tools to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activating GEF effectors, were fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these effectors induced potent contractile signaling sufficient to separate adherens junctions in response to as little as one pulse of blue light. Cytoskeletal morphology changes were dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation induced YAP nuclear localization within minutes and subsequent mechanotransduction, verified by YAP-TEAD transcriptional activity. These single-component tools, which do not require protein binding partners, offer spatiotemporally precise control over RhoA signaling that will advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

scholarly journals RhoA-GTPase Modulates Neurite Outgrowth by Regulating the Expression of Spastin and p60-Katanin

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 230
Author(s):  
Dandan Tan ◽  
Haowen Zhang ◽  
Junyao Deng ◽  
Jingmin Liu ◽  
Jinkun Wen ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Signaling Pathway ◽  
Lentiviral Vector ◽  
Coiled Coil ◽  
Current Data ◽  
Microtubule Dynamics ◽  
Microtubule Severing ◽  
Downstream Effector ◽  
Rhoa Gtpase ◽  
Rhoa Signaling

RhoA-GTPase (RhoA) is widely regarded as a key molecular switch to inhibit neurite outgrowth by rigidifying the actin cytoskeleton. However, during neurite outgrowth, whether and how microtubule dynamics are regulated by RhoA remains to be elucidated. Herein, CT04 and Y27632 were used to inactivate RhoA and its downstream effector Rho-associated coiled coil-forming kinase (ROCK), while the RhoAQ63L lentiviral vector was utilized to overexpress the constitutively activated RhoA in dorsal root ganglion (DRG) neurons or neuronal differentiated PC12 cells. The current data illustrate that the RhoA signaling pathway negatively modulates neurite outgrowth and elevates the expression of Glu-tubulin (a marker for a stabilized microtubule). Meanwhile, the microtubule-severing proteins spastin and p60-katanin were downregulated by the RhoA signaling pathway. When spastin and p60-katanin were knocked down, the effects of RhoA inhibition on neurite outgrowth were significantly reversed. Taken together, this study demonstrates that the RhoA pathway-mediated inhibition of neurite outgrowth is not only related to the modulation of microfilament dynamics but is also attributable to the regulation of the expression of spastin and p60-katanin and thus influences microtubule dynamics.

Post-Translational Modification and Differential Protein Binding Partners of CCAAT Enhancer Binding Protein Alpha (C/EBPα) p42 Versus p30 May Contribute to Aberrant C/EBPα Activity in Acute Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3577-3577
Author(s):  
Matthew Silver ◽  
Nirmalee Abayasekara ◽  
Dylan Perry ◽  
Hong Sun ◽  
Nancy Berliner ◽  
...  
Keyword(s):  
Protein Binding ◽  
Transcriptional Activity ◽  
Binding Protein ◽  
Myeloid Cells ◽  
Leukemic Cells ◽  
Myeloid Differentiation ◽  
Differential Protein ◽  
Binding Partners ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is the founding member of a family of basic region/leucine zipper (bzip) transcription factors and has been shown to be a master regulator of granulopoiesis It is expressed at high levels throughout myeloid differentiation and has been shown to bind to the promoters of multiple myeloid- specific gene promoters at different stages of myeloid maturation. Profound hematopoietic abnormalities have been reported for mice nullizygous for including a selective early block in the differentiation of C/EBPα, granulocytes. Mutations in C/EBPα have been demonstrated in a subset of patients with AML presenting with a normal karyotype. These mutations can result in the expression of a 30kD dominant negative C/EBPα isoform which contributes to loss of C/EBPα function. We have sought to understand the molecular basis for this observation. We and others have demonstrated that C/EBPα is post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) that lies within a region of the C/EBPα protein that can negatively affect transcriptional activity. We have demonstrated that the levels of sumoylated p42C/EBPα decrease upon normal neutrophil maturation and that transactivation of the myeloid-specific lactoferrin (LF) promoter reporter is significantly enhanced by a p42 sumoylation mutant of C/EBPα (K159A). Additionally, in oligonucleotide pull down assays, we show that sumoylated p42C/EBPα binds to the C/EBP site in the LF promoter in immature myeloid cells (which do not express LF) while loss binding and LF of sumoylation correlates with loss of p42C/EBPα expression in more mature cells. Based on these observations we is associated with the negative conclude that sumoylated p42C/EBPα regulation of LF in early myeloid cells. We further demonstrate that sumoylated p42C/EBPα remains bound to the LF promoter following ATRA induction of the leukemic NB4 cells, which do not express LF despite induction of morphologic maturation. Based on these observations we conclude that during normal myeloid differentiation, sumoylated p42C/EBPα is associated with the negative regulation of LF in early myeloid cells, and that LF expression upon maturation is associated with loss of binding of sumoylated p42 C/EBPα In leukemic cells induced toward mature neutrophils, sumoylated p42C/EBPα remains bound to the LF promoter, contributing to the lack of expression of LF in these cells. We show in addition, that p30 C/EBPα can also be sumoylated. In transactivation assays, however, sumoylated p42C/EBPα suppresses LF promoter activity more efficiently than p30C/EBPα in 293 cells. In order to identify differential protein binding partners of p30 and p42C/EBPα that could account for the differential transcriptional activity of the two isoforms, we have used a one step purification method that allows isolation of biotinylated C/EBPα p30 and p42- containing complexes using magnetic-streptavidin beads. The K562 myelomonocytic cell line stably expressing a biotin ligase (BirA) plasmid was transfected with p30C/EBPα or p42C/EBPα each containing a 23 amino acid tag at the N-terminus that allows for in vivo biotinylation. Proteins complexed with the two C/EBP isofoms have been isolated and are currently being identified by LC- MS MS analysis. Their differential association with the two isofoms of C/EBPα will be confimed by coimmunoprecipitation assays in normal myeloid and in leukemic cells. The identification of differentially bound proteins to p30 and p42 C/EBPα may identify molecular targets for future drug development.

scholarly journals A Novel and Conserved Protein-Protein Interaction Domain of Mammalian Lin-2/CASK Binds and Recruits SAP97 to the Lateral Surface of Epithelia

2002 ◽  
Vol 22 (6) ◽  
pp. 1778-1791 ◽  
Author(s):  
Seonok Lee ◽  
Shuling Fan ◽  
Olya Makarova ◽  
Samuel Straight ◽  
Ben Margolis
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Mdck Cells ◽  
Cell Polarization ◽  
Dominant Negative ◽  
Mammalian Development ◽  
Interaction Domain ◽  
Membrane Recruitment ◽  
Protein Protein Interaction ◽  
N Terminus

ABSTRACT Mammalian Lin-2 (mLin-2)/CASK is a membrane-associated guanylate kinase (MAGUK) and contains multidomain modules that mediate protein-protein interactions important for the establishment and maintenance of neuronal and epithelial cell polarization. The importance of mLin-2/CASK in mammalian development is demonstrated by the fact that mutations in mLin-2/CASK or SAP97, another MAGUK protein, lead to cleft palate in mice. We recently identified a new protein-protein interaction domain, called the L27 domain, which is present twice in mLin-2/CASK. In this report, we further define the binding of the L27C domain of mLin-2/CASK to the L27 domain of mLin-7 and identify the binding partner for L27N of mLin-2/CASK. Biochemical analysis reveals that this L27N domain binds to the N terminus of SAP97, a region that was previously reported to be essential for the lateral membrane recruitment of SAP97 in epithelia. Our colocalization studies, using dominant-negative mLin-2/CASK, show that the association with mLin-2/CASK is crucial for lateral localization of SAP97 in MDCK cells. We also report the identification of a novel isoform of Discs Large, a Drosophila melanogaster orthologue of SAP97, which contains a region highly related to the SAP97 N terminus and which binds Camguk, a Drosophila orthologue of mLin-2/CASK. Our data identify evolutionarily conserved protein-protein interaction domains that link mLin-2/CASK to SAP97 and account for their common phenotype when mutated in mice.

scholarly journals STAT3 noncell-autonomously controls planar cell polarity during zebrafish convergence and extension

2004 ◽  
Vol 166 (7) ◽  
pp. 975-981 ◽  
Author(s):  
Chiemi Miyagi ◽  
Susumu Yamashita ◽  
Yusuke Ohba ◽  
Hisayoshi Yoshizaki ◽  
Michiyuki Matsuda ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Planar Cell Polarity ◽  
Cell Polarization ◽  
Rhoa Activity ◽  
Stat3 Signaling ◽  
Convergence And Extension ◽  
Pcp Signaling ◽  
Autonomous Activity ◽  
Rhoa Signaling ◽  
Transcription 3

Zebrafish signal transducer and activator of transcription 3 (STAT3) controls the cell movements during gastrulation. Here, we show that noncell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cell polarity (Wnt-PCP) pathway. In STAT3-depleted embryos, although all the known molecules in the Wnt-PCP pathway were expressed normally, the RhoA activity in lateral mesendodermal cells was down-regulated, resulting in severe cell polarization defects in convergence and extension movements identical to Strabismus-depleted embryos. Cell-autonomous activation of Wnt-PCP signaling by ΔN-dishevelled rescued the defect in cell elongation, but not the orientation of lateral mesendodermal cells in STAT3-depleted embryos. The defect in the orientation could be rescued by transplantation of shield cells having noncell-autonomous activity of STAT3 signaling. These results suggest that the cells undergoing convergence and extension movement may sense the gradient of signaling molecules, which are expressed in gastrula organizer by STAT3 and noncell-autonomously activate PCP signaling in neighboring cells during zebrafish gastrulation.

scholarly journals Id1 Overexpression Induces Tetraploidization and Multiple Abnormal Mitotic Phenotypes by Modulating Aurora A

2008 ◽  
Vol 19 (6) ◽  
pp. 2389-2401 ◽  
Author(s):  
Cornelia Man ◽  
Jack Rosa ◽  
Y. L. Yip ◽  
Annie Lai-Man Cheung ◽  
Y. L. Kwong ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Epithelial Cells ◽  
Transcriptional Activity ◽  
Dna Amplification ◽  
Centrosome Amplification ◽  
Aurora A ◽  
Cell Models ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Binding Partners

The basic helix-loop-helix transcription factor, Id1, was shown to induce tetraploidy in telomerase-immortalized nasopharyngeal epithelial cells in this study. Using both transient and stable Id1-expressing cell models, multiple mitotic aberrations were detected, including centrosome amplification, binucleation, spindle defects, and microtubule perturbation. Many of these abnormal phenotypes have previously been reported in cells overexpressing Aurora A. Further experiments showed that Id1 could stabilize Aurora A, whereas knocking down Aurora A expression in Id1-expressing cells could rescue some of the mitotic defects. The mechanisms by which Aurora A could be modulated by Id1 were explored. DNA amplification of the Aurora A locus was not involved. Id1 could only weakly activate the transcriptional activity of the Aurora A promoter. We found that Id1 overexpression could affect Aurora A degradation, leading to its stabilization. Aurora A is normally degraded from mitosis exit by the APC/CCdh1-mediated proteasomal proteolysis pathway. Our results revealed that Id1 and Cdh1 are binding partners. The association of Id1 and Cdh1 was found to be dependent on the canonical destruction box motif of Id1, the increased binding of which may compete with the interaction between Cdh1 and Aurora A, leading to stabilization of Aurora A in Id1-overexpressing cells.

scholarly journals Infectious Bursal Disease Virus Activates c-Src To Promote α4β1 Integrin-Dependent Viral Entry by Modulating the Downstream Akt-RhoA GTPase-Actin Rearrangement Cascade

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Chengjin Ye ◽  
Xinpeng Han ◽  
Zhaoli Yu ◽  
Enli Zhang ◽  
Lijuan Wang ◽  
...  
Keyword(s):  
Disease Virus ◽  
Infectious Bursal Disease Virus ◽  
Dominant Negative ◽  
Bursa Of Fabricius ◽  
Infectious Bursal Disease ◽  
Dominant Negative Mutant ◽  
Rhoa Gtpase ◽  
Bursal Disease ◽  
Rhoa Signaling ◽  
Actin Rearrangement

ABSTRACT While the entry of infectious bursal disease virus (IBDV) is initiated by the binding of the virus to the two major receptors integrin and HSP90, the signaling events after receptor binding and how they contribute to virus entry remain elusive. We show here that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src both in DF-1 chicken fibroblasts and in vivo in the bursa of Fabricius from specific-pathogen-free (SPF) chickens. Importantly, inactivated IBDV fails to stimulate c-Src Y416 phosphorylation, and a very virulent IBDV strain induces a much higher level of c-Src Y416 phosphorylation than does an attenuated strain. Inhibition of c-Src activation by an Src kinase inhibitor or expression of a c-Src dominant negative mutant results in a significant decrease in the internalization of IBDV but has little effect on virus adhesion. Furthermore, short hairpin RNA (shRNA) downregulation of integrin, either the α4 or β1 subunit, but not HSP90 remarkably attenuates IBDV-induced c-Src Y416 phosphorylation, resulting in a decrease in IBDV internalization but not virus adhesion. Moreover, interestingly, inhibition of either c-Src downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt-RhoA signaling cascade or actin rearrangement leads to a significant decrease in IBDV internalization irrespective of the IBDV-induced high levels of c-Src phosphorylation. Cumulatively, our results suggest a novel feed-forward model whereby IBDV activates c-Src for benefiting its cell entry via an integrin-mediated pathway by the activation of downstream PI3K/Akt-RhoA signaling and cytoskeleton actin rearrangement. IMPORTANCE While IBDV-caused immunosuppression is highly related to viral invasion, the molecular basis of the cellular entry of IBDV remains elusive. In this study, we demonstrate that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src to promote virus internalization but not virus adhesion. The ability to induce the level of c-Src Y416 phosphorylation correlates with the pathogenicity of an IBDV strain. IBDV-induced c-Src Y416 activation is α4β1 integrin but not HSP90 dependent and involves the activation of the downstream PI3K/Akt-RhoA GTPase-actin rearrangement cascade. Thus, our findings provide new insights into the IBDV infection process and the potential for c-Src as a candidate target for the development of IBDV therapeutic drugs.

scholarly journals GPCR-mediated Activation of PLCγ2 Initiates Dysregulated Recruitment of Neutrophils in Cold-induced Urticaria in PLAID Patients

2020 ◽  
Author(s):  
Xuehua Xu ◽  
Xi Wen ◽  
Smit Bhimani ◽  
Amer Moosa ◽  
Dustin Parsons ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Tyrosine Kinases ◽  
Actin Polymerization ◽  
Cell Polarization ◽  
G Protein Coupled Receptors ◽  
Gain Of Function ◽  
Membrane Recruitment ◽  
Akt Activation ◽  
And Migration ◽  
G Protein Coupled

AbstractThe current dogma is that chemoattractants G protein coupled receptors (GPCRs) activate β phospholipase C (PLCβ) while receptor tyrosine kinases (RTKs) activate γ phospholipase C (PLCγ). Here, we show that chemoattractant/GPCR-mediated membrane recruitment of PLCγ2 constitutes GPCR-mediated phospholipase C (PLC) signaling and is essential for neutrophil polarization and migration during GPCR-mediated chemotaxis. In response to a chemoattractant stimulation, cells lacking PLCγ2 (plcg2kd) displayed altered dynamics of diacylglycerol (DAG) production and calcium response; increased Ras/PI3K/Akt activation; elevated GSK3 phosphorylation and cofilin activation; impaired dynamics of actin polymerization; and consequently defects in cell polarization and migration during chemotaxis. At low temperature, neutrophils expressing the gain-of-function mutant of PLCγ2 (Δ686) displayed better chemotaxis than the cells expressing wild-type PLCγ2. The study provides a molecular mechanism for the dysregulated recruitment and activation of neutrophils in cold-induced urticaria in PLCγ2-associated antibody deficiency and immune dysregulation (PLAID) patients bearing gain-of-function mutations of PLCγ2.

Designing Single-Component Optogenetic Membrane Recruitment Systems: The Rho-Family GTPase Signaling Toolbox

2022 ◽  
Author(s):  
Erin E. Berlew ◽  
Keisuke Yamada ◽  
Ivan A. Kuznetsov ◽  
Eleanor A. Rand ◽  
Chandler C. Ochs ◽  
...  
Keyword(s):  
Single Component ◽  
Membrane Recruitment ◽  
Rho Family

scholarly journals Cdc42 regulates junctional actin but not cell polarization in the Caenorhabditis elegans epidermis

2017 ◽  
Vol 216 (11) ◽  
pp. 3729-3744 ◽  
Author(s):  
Yuliya Zilberman ◽  
Joshua Abrams ◽  
Dorian C. Anderson ◽  
Jeremy Nance
Keyword(s):  
Caenorhabditis Elegans ◽  
Epidermal Cell ◽  
Cell Shape ◽  
Time Lapse ◽  
Cell Polarization ◽  
Epithelial Morphogenesis ◽  
Actin Organization ◽  
Protein Levels ◽  
Guanosine Triphosphatase ◽  
Shape Changes

During morphogenesis, adherens junctions (AJs) remodel to allow changes in cell shape and position while preserving adhesion. Here, we examine the function of Rho guanosine triphosphatase CDC-42 in AJ formation and regulation during Caenorhabditis elegans embryo elongation, a process driven by asymmetric epidermal cell shape changes. cdc-42 mutant embryos arrest during elongation with epidermal ruptures. Unexpectedly, we find using time-lapse fluorescence imaging that cdc-42 is not required for epidermal cell polarization or junction assembly, but rather is needed for proper junctional actin regulation during elongation. We show that the RhoGAP PAC-1/ARHGAP21 inhibits CDC-42 activity at AJs, and loss of PAC-1 or the interacting linker protein PICC-1/CCDC85A-C blocks elongation in embryos with compromised AJ function. pac-1 embryos exhibit dynamic accumulations of junctional F-actin and an increase in AJ protein levels. Our findings identify a previously unrecognized molecular mechanism for inhibiting junctional CDC-42 to control actin organization and AJ protein levels during epithelial morphogenesis.

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