Publications by authors named "Perihan Nalbant"

23 Publications

  • Page 1 of 1

Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis.

Cell Rep 2021 Nov;37(8):110056

Department of Medical Oncology, West German Cancer Center, University Hospital Essen at the University Duisburg-Essen, Duisburg, Germany; German Cancer Consortium (DKTK) partner site Essen, Essen, Germany. Electronic address:

Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.
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http://dx.doi.org/10.1016/j.celrep.2021.110056DOI Listing
November 2021

Optogenetic Tuning Reveals Rho Amplification-Dependent Dynamics of a Cell Contraction Signal Network.

Cell Rep 2020 12;33(9):108467

Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany; Faculty of Chemistry and Chemical Biology, TU Dortmund University, 44227 Dortmund, Germany. Electronic address:

Local cell contraction pulses play important roles in tissue and cell morphogenesis. Here, we improve a chemo-optogenetic approach and apply it to investigate the signal network that generates these pulses. We use these measurements to derive and parameterize a system of ordinary differential equations describing temporal signal network dynamics. Bifurcation analysis and numerical simulations predict a strong dependence of oscillatory system dynamics on the concentration of GEF-H1, an Lbc-type RhoGEF, which mediates the positive feedback amplification of Rho activity. This prediction is confirmed experimentally via optogenetic tuning of the effective GEF-H1 concentration in individual living cells. Numerical simulations show that pulse amplitude is most sensitive to external inputs into the myosin component at low GEF-H1 concentrations and that the spatial pulse width is dependent on GEF-H1 diffusion. Our study offers a theoretical framework to explain the emergence of local cell contraction pulses and their modulation by biochemical and mechanical signals.
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http://dx.doi.org/10.1016/j.celrep.2020.108467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710677PMC
December 2020

Exploratory cell dynamics: a sense of touch for cells?

Biol Chem 2018 07;399(8):809-819

Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, and Dortmund University of Technology, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Str. 4a, D-44227 Dortmund, Germany.

Cells need to process multifaceted external cues to steer their dynamic behavior. To efficiently perform this task, cells implement several exploratory mechanisms to actively sample their environment. In particular, cells can use exploratory actin-based cell protrusions and contractions to engage and squeeze the environment and to actively probe its chemical and mechanical properties. Multiple excitable signal networks were identified that can generate local activity pulses to control these exploratory processes. Such excitable signal networks offer particularly efficient mechanisms to process chemical or mechanical signals to steer dynamic cell behavior, such as directional migration, tissue morphogenesis and cell fate decisions.
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http://dx.doi.org/10.1515/hsz-2017-0341DOI Listing
July 2018

An excitable Rho GTPase signaling network generates dynamic subcellular contraction patterns.

J Cell Biol 2017 12 20;216(12):4271-4285. Epub 2017 Oct 20.

Department of Molecular Cell Biology, Center for Medical Biotechnology, University of Duisburg-Essen, Essen, Germany

Rho GTPase-based signaling networks control cellular dynamics by coordinating protrusions and retractions in space and time. Here, we reveal a signaling network that generates pulses and propagating waves of cell contractions. These dynamic patterns emerge via self-organization from an activator-inhibitor network, in which the small GTPase Rho amplifies its activity by recruiting its activator, the guanine nucleotide exchange factor GEF-H1. Rho also inhibits itself by local recruitment of actomyosin and the associated RhoGAP Myo9b. This network structure enables spontaneous, self-limiting patterns of subcellular contractility that can explore mechanical cues in the extracellular environment. Indeed, actomyosin pulse frequency in cells is altered by matrix elasticity, showing that coupling of contractility pulses to environmental deformations modulates network dynamics. Thus, our study reveals a mechanism that integrates intracellular biochemical and extracellular mechanical signals into subcellular activity patterns to control cellular contractility dynamics.
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http://dx.doi.org/10.1083/jcb.201706052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716289PMC
December 2017

Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression.

PLoS One 2015 2;10(9):e0137043. Epub 2015 Sep 2.

Pediatric Nephrology, Pediatrics II, University Hospital Essen, Essen, Germany.

Background: Glomerular podocytes are highly differentiated cells that are key components of the kidney filtration units. The podocyte cytoskeleton builds the basis for the dynamic podocyte cytoarchitecture and plays a central role for proper podocyte function. Recent studies implicate that immunosuppressive agents including the mTOR-inhibitor everolimus have a protective role directly on the stability of the podocyte actin cytoskeleton. In contrast, a potential stabilization of microtubules by everolimus has not been studied so far.

Methods: To elucidate mechanisms underlying mTOR-inhibitor mediated cytoskeletal rearrangements, we carried out microarray gene expression studies to identify target genes and corresponding pathways in response to everolimus. We analyzed the effect of everolimus in a puromycin aminonucleoside experimental in vitro model of podocyte injury.

Results: Upon treatment with puromycin aminonucleoside, microarray analysis revealed gene clusters involved in cytoskeletal reorganization, cell adhesion, migration and extracellular matrix composition to be affected. Everolimus was capable of protecting podocytes from injury, both on transcriptional and protein level. Rescued genes included tubulin beta 2B class IIb (TUBB2B) and doublecortin domain containing 2 (DCDC2), both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Validating gene expression data, Western-blot analysis in cultured podocytes demonstrated an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Interestingly, Tubb2bbrdp/brdp mice revealed a delay in glomerular podocyte development as showed by podocyte-specific markers Wilm's tumour 1, Podocin, Nephrin and Synaptopodin.

Conclusions: Taken together, our study suggests that off-target, non-immune mediated effects of the mTOR-inhibitor everolimus on the podocyte cytoskeleton might involve regulation of microtubules, revealing a potential novel role of TUBB2B and DCDC2 in glomerular podocyte development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137043PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4557973PMC
May 2016

FHOD1 regulates stress fiber organization by controlling the dynamics of transverse arcs and dorsal fibers.

J Cell Sci 2014 Apr 30;127(Pt 7):1379-93. Epub 2014 Jan 30.

Department of Molecular Cell Biology, Center for Medical Biotechnology, University of Duisburg-Essen, 45141 Essen, Germany.

The formin FHOD1 (formin homology 2 domain containing protein 1) can act as a capping and bundling protein in vitro. In cells, active FHOD1 stimulates the formation of ventral stress fibers. However, the cellular mechanisms by which this phenotype is produced and the physiological relevance of FHOD1 function are not currently understood. Here, we first show that FHOD1 controls the formation of two distinct stress fiber precursors differentially. On the one hand, it inhibits dorsal fiber growth, which requires the polymerization of parallel bundles of long actin filaments. On the other hand, it stimulates transverse arcs that are formed by the fusion of short antiparallel actin filaments. This combined action is crucial for the maturation of stress fibers and their spatio-temporal organization, and a lack of FHOD1 function perturbs dynamic cell behavior during cell migration. Furthermore, we show that the GTPase-binding and formin homology 3 domains (GBD and FH3) are responsible for stress fiber association and colocalization with myosin. Surprisingly, a version of FHOD1 that lacks these domains nevertheless retains its full capacity to stimulate arc and ventral stress fiber formation. Based on our findings, we propose a mechanism in which FHOD1 promotes the formation of short actin filaments and transiently associates with transverse arcs, thus providing tight temporal and spatial control of the formation and turnover of transverse arcs into mature ventral stress fibers during dynamic cell behavior.
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http://dx.doi.org/10.1242/jcs.134627DOI Listing
April 2014

Protective effects of the mTOR inhibitor everolimus on cytoskeletal injury in human podocytes are mediated by RhoA signaling.

PLoS One 2013 13;8(2):e55980. Epub 2013 Feb 13.

Pediatric Nephrology, Pediatrics II, University of Duisburg-Essen, Essen, Germany.

Podocytes are highly differentiated kidney cells playing an important role in maintaining the glomerular filtration barrier. Particularly, the integrity of the actin cytoskeleton is crucial as cytoskeletal damage associated with foot process effacement and loss of slit diaphragms constitutes a major aspect of proteinuria. Previously, the mammalian target of rapamycin (mTOR) was linked to actin regulation and aberrant activity of the kinase was associated with renal disease. In this study, actin-related effects of mTOR inhibition by the immunosuppressant everolimus (EV) were investigated in human podocytes using an in vitro model of puromycin aminonucleoside (PAN) induced proteinuria. EV substantially recovered aberrant podocyte behavior by re-establishing a stationary phenotype with decreased migration efficiency, enhanced cell adhesion and recovery of actin stress fibers. Biochemical studies revealed substantial increase in the activity of RhoA and the effector pathway Rho-associated protein kinase (ROCK) and myosin light chain (MLC) by EV, all known regulators of stress fiber generation. Taken together, we show for the first time cytoskeleton stabilizing effects of the mTOR inhibitor EV and establish RhoA signaling as a key mediator in this process.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055980PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572151PMC
August 2013

Coordination of Rho GTPase activities during cell protrusion.

Nature 2009 Sep 19;461(7260):99-103. Epub 2009 Aug 19.

Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.

The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8), and Rac1 and RhoA are mutually inhibitory. However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. Here we examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a 'computational multiplexing' approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 micro-m behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.
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http://dx.doi.org/10.1038/nature08242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2885353PMC
September 2009

Guanine nucleotide exchange factor-H1 regulates cell migration via localized activation of RhoA at the leading edge.

Mol Biol Cell 2009 Sep 22;20(18):4070-82. Epub 2009 Jul 22.

Departments of Immunology and Microbial Science, and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Cell migration involves the cooperative reorganization of the actin and microtubule cytoskeletons, as well as the turnover of cell-substrate adhesions, under the control of Rho family GTPases. RhoA is activated at the leading edge of motile cells by unknown mechanisms to control actin stress fiber assembly, contractility, and focal adhesion dynamics. The microtubule-associated guanine nucleotide exchange factor (GEF)-H1 activates RhoA when released from microtubules to initiate a RhoA/Rho kinase/myosin light chain signaling pathway that regulates cellular contractility. However, the contributions of activated GEF-H1 to coordination of cytoskeletal dynamics during cell migration are unknown. We show that small interfering RNA-induced GEF-H1 depletion leads to decreased HeLa cell directional migration due to the loss of the Rho exchange activity of GEF-H1. Analysis of RhoA activity by using a live cell biosensor revealed that GEF-H1 controls localized activation of RhoA at the leading edge. The loss of GEF-H1 is associated with altered leading edge actin dynamics, as well as increased focal adhesion lifetimes. Tyrosine phosphorylation of focal adhesion kinase and paxillin at residues critical for the regulation of focal adhesion dynamics was diminished in the absence of GEF-H1/RhoA signaling. This study establishes GEF-H1 as a critical organizer of key structural and signaling components of cell migration through the localized regulation of RhoA activity at the cell leading edge.
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http://dx.doi.org/10.1091/mbc.e09-01-0041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743625PMC
September 2009

RhoA GTPase activation by TLR2 and TLR3 ligands: connecting via Src to NF-kappa B.

J Immunol 2009 Mar;182(6):3522-9

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA.

Rho GTPases are essential regulators of signaling networks emanating from many receptors involved in innate or adaptive immunity. The Rho family member RhoA controls cytoskeletal processes as well as the activity of transcription factors such as NF-kappaB, C/EBP, and serum response factor. The multifaceted host cell activation triggered by TLRs in response to soluble and particulate microbial structures includes rapid stimulation of RhoA activity. RhoA acts downstream of TLR2 in HEK-TLR2 and monocytic THP-1 cells, but the signaling pathway connecting TLR2 and RhoA is still unknown. It is also not clear if RhoA activation is dependent on a certain TLR adapter. Using lung epithelial cells, we demonstrate TLR2- and TLR3-triggered recruitment and activation of RhoA at receptor-proximal cellular compartments. RhoA activity was dependent on TLR-mediated stimulation of Src family kinases. Both Src family kinases and RhoA were required for NF-kappaB activation, whereas RhoA was dispensable for type I IFN generation. These results suggest that RhoA plays a role downstream of MyD88-dependent and -independent TLR signaling and acts as a molecular switch downstream of TLR-Src-initiated pathways.
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http://dx.doi.org/10.4049/jimmunol.0802280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2684960PMC
March 2009

Processing of naturally occurring sense/antisense transcripts of the vertebrate Slc34a gene into short RNAs.

Physiol Genomics 2008 Jun 15;34(1):95-100. Epub 2008 Apr 15.

RNA Biology Group, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK.

Overlapping sense/antisense RNAs transcribed in opposite directions from the same genomic locus are common in vertebrates. The impact of antisense transcription on gene regulation and cell biology is largely unknown. We show that sense/antisense RNAs of an evolutionarily conserved phosphate transporter gene (Slc34a2a) are coexpressed in a short time window during embryonic development of zebrafish at 48 hours postfertilization (hpf). To address the mechanism by which coexpressed sense/antisense transcripts are processed, we injected sense/antisense RNAs in various combinations into Xenopus oocytes. In the cytoplasm RNAs were stable in whatever combination expressed. In the nucleus coinjected sense/antisense transcripts were degraded into short RNAs of approximately 23 bases within 4 h. A homologous transcript from toad or another isoform (Slc34a2b) from zebrafish failed to trigger processing. In oocytes that were primed with nuclear sense/antisense RNA coinjections, a reporter RNA was rapidly degraded. We produced evidence that the observed processing of complementary transcripts was not restricted to Xenopus oocytes, because Slc34a-related short RNAs were detected in zebrafish embryos by Northern blotting. Signals were observed at stages that showed coexpression of sense/antisense transcripts. Remarkably, strand-specific probes revealed that the orientation of short RNAs was developmentally regulated. In addition, RNA from zebrafish embryos 48 hpf was able to induce degradation of reporter constructs in Xenopus oocytes. Our findings may give important clues to understanding the physiological role of the widespread antisense transcription.
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http://dx.doi.org/10.1152/physiolgenomics.00004.2008DOI Listing
June 2008

Cellular functions of GEF-H1, a microtubule-regulated Rho-GEF: is altered GEF-H1 activity a crucial determinant of disease pathogenesis?

Trends Cell Biol 2008 May 3;18(5):210-9. Epub 2008 Apr 3.

Direvo Biotech AG, Nattermannallee 1, D-50829 Cologne, Germany.

The Rho guanine nucleotide exchange factor GEF-H1 is uniquely regulated by microtubule binding and is crucial in coupling microtubule dynamics to Rho-GTPase activation in a variety of normal biological situations. Here, we review the roles of GEF-H1 in epithelial barrier permeability, cell motility and polarization, dendritic spine morphology, antigen presentation, leukemic cell differentiation, cell cycle regulation, and cancer. GEF-H1 might also contribute to pathophysiological signaling involved in leukemias, and in cancers associated with mutated p53 tumor suppressor gene, epithelial and endothelial cell dysfunction, infectious disease, and cardiac hypertrophy. We suggest that GEF-H1 could be a novel therapeutic target in multiple human diseases.
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http://dx.doi.org/10.1016/j.tcb.2008.02.006DOI Listing
May 2008

GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA.

Mol Biol Cell 2008 May 20;19(5):2147-53. Epub 2008 Feb 20.

Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

The RhoA GTPase plays a vital role in assembly of contractile actin-myosin filaments (stress fibers) and of associated focal adhesion complexes of adherent monolayer cells in culture. GEF-H1 is a microtubule-associated guanine nucleotide exchange factor that activates RhoA upon release from microtubules. The overexpression of GEF-H1 deficient in microtubule binding or treatment of HeLa cells with nocodazole to induce microtubule depolymerization results in Rho-dependent actin stress fiber formation and contractile cell morphology. However, whether GEF-H1 is required and sufficient to mediate nocodazole-induced contractility remains unclear. We establish here that siRNA-mediated depletion of GEF-H1 in HeLa cells prevents nocodazole-induced cell contraction. Furthermore, the nocodazole-induced activation of RhoA and Rho-associated kinase (ROCK) that mediates phosphorylation of myosin regulatory light chain (MLC) is impaired in GEF-H1-depleted cells. Conversely, RhoA activation and contractility are rescued by reintroduction of siRNA-resistant GEF-H1. Our studies reveal a critical role for a GEF-H1/RhoA/ROCK/MLC signaling pathway in mediating nocodazole-induced cell contractility.
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http://dx.doi.org/10.1091/mbc.e07-12-1269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2366883PMC
May 2008

PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility.

J Cell Biol 2008 Jan;180(1):187-203

Department of Reproductive Medicine, Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK(-/-) or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK-p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.
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http://dx.doi.org/10.1083/jcb.200708194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2213606PMC
January 2008

Positive feedback between Cdc42 activity and H+ efflux by the Na-H exchanger NHE1 for polarity of migrating cells.

J Cell Biol 2007 Nov;179(3):403-10

Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA.

A fundamental feature of cell polarity in response to spatial cues is asymmetric amplification of molecules generated by positive feedback signaling. We report a positive feedback loop between the guanosine triphosphatase Cdc42, a central determinant in eukaryotic cell polarity, and H(+) efflux by Na-H(+) exchanger 1 (NHE1), which is necessary at the front of migrating cells for polarity and directional motility. In response to migratory cues, Cdc42 is not activated in fibroblasts expressing a mutant NHE1 that lacks H(+) efflux, and wild-type NHE1 is not activated in fibroblasts expressing mutationally inactive Cdc42-N17. H(+) efflux by NHE1 is not necessary for release of Cdc42-guanosine diphosphate (GDP) from Rho GDP dissociation inhibitor or for the membrane recruitment of Cdc42 but is required for GTP binding by Cdc42 catalyzed by a guanine nucleotide exchange factor (GEF). Data indicate that GEF binding to phosphotidylinositol 4,5-bisphosphate is pH dependent, suggesting a mechanism for how H(+) efflux by NHE1 promotes Cdc42 activity to generate a positive feedback signal necessary for polarity in migrating cells.
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http://dx.doi.org/10.1083/jcb.200704169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064788PMC
November 2007

GEF-H1 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases.

Dev Cell 2007 May;12(5):699-712

Departments of Immunology and Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP loading on RhoA. Using a live cell biosensor, we demonstrate distinct roles for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. Our results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases.
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http://dx.doi.org/10.1016/j.devcel.2007.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1965589PMC
May 2007

Signaling requirements for translocation of P-Rex1, a key Rac2 exchange factor involved in chemoattractant-stimulated human neutrophil function.

J Leukoc Biol 2007 Apr 16;81(4):1127-36. Epub 2007 Jan 16.

Department of Immunology, IMM14, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

PI 3,4,5-trisphosphate [PI(3,4,5)P3; PIP3]-dependent Rac exchanger 1 (P-Rex1) is a Rac-specific guanine nucleotide exchange factor abundant in neutrophils and myeloid cells. As a selective catalyst for Rac2 activation, P-Rex1 serves as an important regulator of human neutrophil NADPH oxidase activity and chemotaxis in response to a variety of extracellular stimuli. The exchange activity of P-Rex1 is synergistically activated by the binding of PIP3 and betagamma subunits of heterotrimeric G proteins in vitro, suggesting that the association of P-Rex1 with membranes is a prerequisite for cellular activation. However, the spatial regulation of endogenous P-Rex1 has not been well defined, particularly in human neutrophils activated through G protein-coupled receptors. Upon stimulation of neutrophil chemoattractant receptors, we observed that P-Rex1 translocated from cytoplasm to the leading edge of polarized cells in a G protein betagamma subunit- and PIP3-dependent manner, where it colocalized with F-actin and its substrate, Rac2. Redistribution of P-Rex1 to the leading edge was also dependent on tyrosine kinase activity and was modulated by cell adhesion. Furthermore, we observed that activation of cAMP-dependent protein kinase A (PKA), which phosphorylates and inactivates P-Rex1, inhibited its translocation. Our data indicate that endogenous P-Rex1 translocates to areas of Rac2 and cytoskeletal activation at the leading edge in response to chemoattractant stimuli in human neutrophils and that this translocation can be negatively modulated by activation of PKA and by cell adhesion.
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http://dx.doi.org/10.1189/jlb.0406251DOI Listing
April 2007

Integrin-linked kinase regulates Bergmann glial differentiation during cerebellar development.

Mol Cell Neurosci 2006 Oct 17;33(2):109-25. Epub 2006 Aug 17.

The Scripps Research Institute, Department of Cell Biology, Institute for Childhood and Neglected Disease, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

We demonstrate here that integrin-linked kinase (ILK), a serine/threonine kinase that binds to the beta1 integrin cytoplasmic domain, regulates cerebellar development. Mice with a CNS-restricted knock-out of the Ilk gene show perturbations in the laminar structure of the cerebellar cortex that are associated with defects in Bergmann glial fibers and the formation of meningeal basement membranes. Similar defects have been observed in mice lacking beta1 integrins in the CNS. ILK and beta1 integrins are coexpressed in Bergmann glial cells, and studies with primary cells in culture demonstrate that ILK and CDC42 are required for beta1-integrin-dependent glial process outgrowth. Consistent with these findings, the amount of GTP-bound CDC42 is impaired in the cerebellum of Ilk-deficient mice. We conclude that beta1 integrin, ILK and CDC42 are components of the signaling machinery that regulates glial process outgrowth in the cerebellum. We also show that granule cell precursor proliferation is affected in ILK-deficient mice, but our findings provide strong evidence that proliferative defects are a secondary consequence of ILK function in glia.
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http://dx.doi.org/10.1016/j.mcn.2006.06.013DOI Listing
October 2006

Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation.

Methods Enzymol 2006 ;406:140-56

Department of Pharmacology, University of North Carolina, Chapel Hill, USA.

This chapter details quantitative imaging of the Mero-CBD biosensor, which reports activation of endogenous Cdc42 in living cells. The procedures described are appropriate for imaging any biosensor that uses two different fluorophores on a single molecule, including FRET biosensors. Of particular interest is an algorithm to correct for fluorophore photobleaching, useful when quantitating activity changes over time. Specific topics include procedures and caveats in production of the Mero-CBD sensor, image acquisition, motion artifacts, shading correction, background subtraction, registration, and ratio imaging.
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http://dx.doi.org/10.1016/S0076-6879(06)06012-5DOI Listing
March 2006

Vinculin controls PTEN protein level by maintaining the interaction of the adherens junction protein beta-catenin with the scaffolding protein MAGI-2.

J Biol Chem 2005 Feb 5;280(7):5676-81. Epub 2004 Dec 5.

Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.

PTEN is a frequently mutated tumor suppressor in malignancies. Interestingly, some malignancies exhibit undetectable PTEN protein without mutations or loss of PTEN mRNA. The cause(s) for this reduction in PTEN is unknown. Cancer cells frequently exhibit loss of cadherin, beta-catenin, alpha-catenin and/or vinculin, key elements of adherens junctions. Here we show that F9 vinculin-null (vin(-/-)) cells lack PTEN protein despite normal PTEN mRNA levels. Their PTEN protein expression was restored by transfection with vinculin or by inhibition of PTEN degradation. F9 vin(-/-) cells express PTEN protein upon transfection with a vinculin fragment (amino acids 243-1066) that is capable of interacting with alpha-catenin but unable to target into focal adhesions. On the other hand, disruption of adherens junctions with an E-cadherin blocking antibody reduced PTEN protein to undetectable levels in wild-type F9 cells. PTEN protein levels were restored in F9 vin(-/-) cells upon transfection with an E-cadherin-alpha-catenin fusion protein, which targets into adherens junctions and interacts with beta-catenin in F9 vin(-/-) cells. beta-Catenin is known to interact with MAGI-2. MAGI-2 interaction with PTEN in the cell membrane is known to prevent PTEN protein degradation. Thus, MAGI-2 overexpression in F9 vin(-/-) cells restored PTEN protein levels. Moreover, expression of vinculin mutants that reinstated the disrupted interactions of beta-catenin with MAGI-2 in F9 vin(-/-) cells also restored PTEN protein levels. These studies indicate that PTEN protein levels are dependent on the maintenance of beta-catenin-MAGI-2 interaction, in which vinculin plays a critical role.
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http://dx.doi.org/10.1074/jbc.M405561200DOI Listing
February 2005

Activation of endogenous Cdc42 visualized in living cells.

Science 2004 Sep;305(5690):1615-9

Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7365, USA.

Signaling proteins are tightly regulated spatially and temporally to perform multiple functions. For Cdc42 and other guanosine triphosphatases, the subcellular location of activation is a critical determinant of cell behavior. However, current approaches are limited in their ability to examine the dynamics of Cdc42 activity in living cells. We report the development of a biosensor capable of visualizing the changing activation of endogenous, unlabeled Cdc42 in living cells. With the use of a dye that reports protein interactions, the biosensor revealed localized activation in the trans-Golgi apparatus, microtubule-dependent Cdc42 activation at the cell periphery, and activation kinetics precisely coordinated with cell extension and retraction.
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http://dx.doi.org/10.1126/science.1100367DOI Listing
September 2004

Regulation of the NPT gene by a naturally occurring antisense transcript.

Cell Biochem Biophys 2002 ;36(2-3):241-52

Department of Physiological Sciences, University of Newcastle upon Tyne, UK.

The epithelial Na/Pi cotransporter (NaPi-II) is instrumental in maintaining phosphate (Pi) homeostasis in vertebrates. Hormones and metabolic factors (PTH, Pi availability) that acutely influence renal Pi excretion have been demonstrated to target NaPi-II expression. Upon stimulation, newly synthesized transporter molecules become integrated into the brush-border membrane to increase the Vmax of Pi uptake; reduction of Pi reabsorption is achieved by endocytosis of NaPi-II followed by lysosomal degradation of the protein. The long-term regulation of the protein is less well studied. Only recently, regulatory elements for vitamin D3 and Pi have been identified in the promoter region of the npt gene. However, signaling pathways leading to the activation of these regulatory sequences need to be established. Other reports suggested messenger RNA stability to play a role in the medium range regulation of NaPi-II expression. Recent findings in our laboratory added to the complex picture of npt gene regulation. We have identified npt-related endogenous antisense transcripts from mouse, zebrafish, and winter flounder. The two fish transcripts have been cloned and characterized; the mouse homolog has only very recently been detected. The transcripts are devoid of an open reading frame and appear in different splice forms. The evolutionary conservation of bidirectional transcription of the npt gene implies a regulatory function for the antisense transcript. In order to test the functional consequences of bidirectional transcription, we coexpressed sense and the antisense transcripts from zebrafish in Xenopus oocytes. Pi transport activity was reduced as a result of the presence of antisense RNA. Re-extraction of the RNA from injected oocytes followed by Northern blot revealed that the coexpression had no significant effect on the stability of either transcript. We concluded that the antisense mRNA interfered with the translation of the transporter if coexpressed in the Xenopus system. However, the regulatory mechanism(s) involving the npt-related antisense transcript is expected to be much more complicated in vivo, (i.e., requiring supplementary factors like double-stranded RNA recognizing proteins or specific RNases). It is planned to test this hypothesis by a transgenic zebrafish approach and/or knockout mice.
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http://dx.doi.org/10.1385/CBB:36:2-3:241DOI Listing
January 2003

Facile synthesis of thiol-reactive Cy3 and Cy5 derivatives with enhanced water solubility.

Bioconjug Chem 2002 May-Jun;13(3):387-91

Department of Cell Biology, Scripps Research Institute, 10550, N. Torrey Pines Road, BCC162, La Jolla, California 92037, USA.

The cyanine dyes Cy3 and Cy5 have proven valuable in numerous applications involving conjugation with proteins. Practical syntheses of lysine-selective, succinimidyl ester derivatives of these dyes have been published, and succinimidyl esters are commercially available. However, the published syntheses of cysteine-selective derivatives produce relatively low yields from expensive starting materials, or produce molecules with marginal water solubility for protein labeling. We report here facile syntheses (four steps, >50% overall yield) of iodoacetamide, sulfhydryl-reactive derivatives of the Cy3 and Cy5 fluorophores. These novel derivatives have good water solubility (>2.5 mM) and bear only one reactive side chain, reducing possible protein cross-linking encountered with previous derivatives.
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http://dx.doi.org/10.1021/bc015558qDOI Listing
December 2002
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