San Martín
4.2 C ONFLICTOS SOCIALES DESARROLLADOS EN MÁS DE UN DEPARTAMENTO
317
Open Microfluidics for Passive Chemoattractant Gradient Generation.
G. Wright1, D. Jowhar1, A. Terekhov2, L. Costa2, W. Hofmeister2, C. J. Janetopoulos1;
1Biological Sciences, Vanderbilt University, Nashville, TN, 2Center for Laser Applications,
University of Tennessee Space Institute
The ability to elicit chemoattractant gradients in 3 dimensional channels has provided investigators with a whole new set of experimental platforms for cell migration studies. However, a major drawback with these devices is that it is difficult to load cells. In addition, chemoattractant gradients are generally provided by laminar flow and expose the cells to shear
forces that influences cell migration. We have developed several platforms in both polydimethylsiloxane (PDMS) and glass to overcome both of these problems. First, we have combined PDMS technology with a micropipette system and can provide a passive gradient to cells traversing 3D microfluidic channels. Using this system allows us to perform highly quantitative experiments on a cell’s response to chemical gradients with varying slopes and mean concentrations. We have also developed a device that allows us to break down and re- establish the polarity of cells while migrating through a channel. Lastly, we have developed devices that are made entirely of etched glass. These glass devices are durable, optically superior, easy to unclog and fill, and are reusable. We have developed several devices in both bulk glass and in cover slips that have incorporated 3D microfluidic channels and have used them to image migrating Dictyostelium discoideum cells. Channels were fabricated using a laser ablation system which consists of a diode-pumped frequency doubled Neodymium-doped Yttrium Orthovanadate (Nd:YVO4) laser pump (Verdi), a Ti:Sapphire laser oscillator (Tsunami) and a regenerative amplifier (RegA9000). These on-chip devices produce passive gradients for migration assays and are useful for confocal and epifluorescent microscopes that are outfitted with environmental chambers or bulky equipment that do not permit the use of a micromanipulator and micropipette system as is typically used to create a passive gradient. We have etched 3D channels in a variety of configurations that range in diameter from several hundred nanometers to tens of microns and have elicited passive gradients of cAMP from single and multiple channels that lead to the successful recruitment of D. discoideum cells. Like the PDMS devices, these platforms are created so that the system is “open”. An open system provides easy cell loading and allows the careful control of passive chemoattractant gradients for many hours and potentially days.
318
Unraveling G-protein signaling mechanisms during chemotaxis. G. A. Wright1, K. Qu1, C. Janetopoulos1,2;
1Biological Sciences, Vanderbilt University, Nashville, TN, 2Cell and Developmental Biology,
Vanderbilt University
Many of the signaling pathways controlling chemotaxis are identical in mammalian cells and in the social amoeba, Dictyostelium discoideum. D. discoideum exhibits chemotaxis toward folic acid during vegetative growth and to cAMP during aggregation and multicellular development. Since the majority of signaling components are conserved between folic acid and cAMP chemotaxis, mutants defective for folic acid chemotaxis should also exhibit a defect cAMP chemotaxis. While some mutants can have severe effects on gene regulation during cAMP development we find that we can typically rescue these developmental defects by co-developing mutants with wild-type cells. In this study, polarity and adhesion mutants (rckA and rap1) were assayed for their ability to chemotax under a variety of conditions. RckA is a Regulator of G- protein Signaling (RGS) domain-containing kinase implicated in managing cell polarity. Rap1 is a Ras-like small G protein that is involved in adhesion, chemotaxis, and polarity. The Rap1-CA and Rap1-DN mutations varied in their effects on cAMP chemotaxis depending on whether the mutants were co-developed with wild-type cells or were chemotaxing to folic acid during vegetative growth. RckA null mutants displayed a dramatic gain-of-function phenotype in their ability to chemotax to cAMP when assayed under these conditions. However, rckA mutants did not show a chemotaxis defect towards the chemoattractant folic acid. When the Rap1-CA or Rap1-DN mutations were expressed in a rckA null strain, cAMP chemotaxis was normal when these cells were co-developed with wild-type cells. However, these same cells displayed dramatic defects when tested for their ability to chemotax toward folic acid. These results suggest that RckA is functioning upstream of Rap1 during cAMP chemotaxis. However, during folic acid chemotaxis RckA and Rap1 seem to be regulated in different pathways and only effect
folic acid chemotaxis in the rckA null:Rap1 double mutants. The results also demonstrate that folic acid chemotaxis is not regulated similar to cAMP chemotaxis upstream of the small G- proteins. To our knowledge this is the first evidence of a heterotrimeric G-protein associated protein regulating downstream small-G proteins in D. discoideum.
319
Inhibition of Glutaminyl Cyclase Attenuates Cell Migration Modulated by Monocyte Chemoattractant Proteins.
Y-M. Lee1, Y-L. Chen1, W-L. Hwu2, K-F. Huang1, A. H. Wang1; 1Academia Sinica, Nankang, Taiwan, 2Pediatrics, National Taiwan University Hospital, Taiwan
Glutaminyl cyclase (QC) catalyzes the formation of N-terminal pyroglutamate (pGlu) in peptides and proteins. pGlu formation in chemoattractants may participate in the regulation of macrophage activation and migration. However, a clear molecular mechanism for the regulation is lacking. In this study, we explored the role of QC-mediated pGlu formation on monocyte chemoattractant proteins (MCPs) in inflammation. We first demonstrated in vitro the pGlu formation on MCPs by QC using mass spectrometry. With purified N-terminal uncyclized MCPs precursor (preMCPs) and pyroglutamate-containing MCPs (pMCPs), we showed that MCPs- stimulated macrophage migration is dependent on the pGlu formation. A potent QC inhibitor, PBD150, significantly decreased the effect of preMCPs on macrophage migration. QC siRNA revealed a similar inhibitory effect. Lastly, we tested whether inhibiting QC can modulate LPS- stimulated macrophage activation. We demonstrated that, in both U937 cells and human peripheral blood-derived monocytes, QC activity is increased by LPS stimulation. Furthermore, PBD150 significantly decreases the effect of LPS. These results strongly suggest that QC- catalyzed N-terminal pGlu formation of MCPs is required for macrophage migration and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a target for anti-inflammatory drug.
320
Polar localization of chemotactic proteins in Vibrio parahaemolyticus.
M. Zepeda Rivera1, S. Ringgaard2,3, M. K. Waldor2,3; 1University of Washington, Seattle, WA,
2Channing Laboratory, Brigham and Women’s Hospital, 3Microbiology and Molecular Genetics,
Harvard Medical School
Chemotaxis is the process by which bacteria bias their flagella-assisted swimming in response to their environment. Recent work in the rod-shaped bacterium Vibrio cholerae, revealed that ParC plays a key role in promoting the polar localization and segregation of chemotaxis proteins. The purpose of our work is to study ParC and chemotaxis protein localization in Vibrio parahaemolyticus, another enteric pathogen, which unlike V. cholerae, has lateral as well as polar flagellae. To detect the subcellular localization of ParC and chemotaxis proteins within V. parahaemolyticus, we fused genes encoding fluorescent proteins to the genes encoding ParC and to genes encoding other chemotaxis proteins, including VP2226 and CheW. In wild-type V. parahaemolyticus time-lapse fluorescence microscopy revealed that ParC and VP2226 localize to the old flagellated pole in newborn cells forming a unipolar focus. Later in the cell cycle, each is recruited to the new pole resulting in bipolar foci. Consequently, after cell division, each daughter cell inherits a ParC/VP2226 focus at the old pole. However a new CheW focus does not form at the pre-divisional proximal pole until post-cell division. This shows a hierarchy in the protein recruitment to the new pole suggesting that ParC, as in V. cholerae, is important for recruitment of chemotaxis proteins to the developing pole, but that unlike V. cholerae, not all proteins are recruited in the same time frame. We have constructed a V. parahaemolyticus parC deletion mutant and analyses of this strain will reveal if ParC is required for proper localization
and segregation of chemotaxis proteins. Our parC deletion mutant exhibited a swimming defect, suggesting that ParC is essential for optimal chemotaxis. Preliminary swarmer assays using our parC deletion mutant suggest that ParC deficiency does not impair the lateral flagellar system. Finally, a bacteria-two-hybrid assay, revealed that there are multiple inter and intra protein- protein interactions among V. parahaemolyticus chemotaxis proteins. Future work includes analyses of requirement for ParC in both flagellar systems, understanding the nature of chemotaxis protein-protein interactions, and utilizing an infant rabbit model to test the effect of ParC deficiency on the intestinal colonization ability of V. parahaemolyticus.
321
Nociceptin is a Chemorepellent in Tetrahymena thermophila.
N. Braun1, T. Lampert2, B. Gibson1, C. Nugent1, H. G. Kuruvilla1; 1Science and Mathematics, Cedarville University, Cedarville, OH, 2Biological Sciences, SUNY at Buffalo, Buffalo, NY
Tetrahymena thermophila are free-living, ciliated, eukaryotic organisms that respond to stimuli by moving toward chemoattractants and avoiding chemorepellents. Chemoattractant responses involve faster ciliary beating, which propels the organisms forward more rapidly. Chemorepellent signaling involves ciliary reversal, which disrupts forward swimming, and causes the organism to jerk back and forth, swim in small circles, or spin in an attempt to get away from the repellent. Many food sources, such as proteins, are chemoattractants for Tetrahymena, while a variety of compounds are repellents. Repellents in nature are thought to come from the secretions of predators, or from ruptured organisms, which may serve as “danger” signals. Several hormones involved in human pain signaling have been shown to be chemorepellents in Tetrahymena, including substance P, ACTH, PACAP, VIP, and nociceptin.
We have been studying the response of Tetrahymena to nociceptin, using pharmacological inhibitors in order to elucidate components of the nociceptin signaling pathway. We have found that G-protein inhibitors and a number of mammalian tyrosine kinase inhibitors have no effect on nociceptin avoidance. However, the tyrosine kinase inhibitor, genistein, inhibits avoidance to nociceptin, likely by an unrelated mechanism. Nociceptin avoidance is also inhibited by the calcium chelator, EGTA, and partially inhibited by the ER calcium ATPase inhibitor, thapsigargin. Whole cell electrophysiology experiments in a calcium-containing buffer show that addition of 50 μM nociceptin to the buffer causes a sustained depolarization of approximately 30 mV. This depolarization is nearly eliminated in the presence of EGTA, further supporting the hypothesis that calcium is involved in nociceptin signaling.
J-113397, an inhibitor of the human nociceptin receptor, also inhibits nociceptin avoidance in Tetrahymena, though other nociceptin antagonists we tested had no effect on avoidance. Further experimentation on this organism will give a more complete picture of the signaling pathway, as well as allowing greater comparison between nociceptin avoidance in Tetrahymena and nociceptin signaling in vertebrates.
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NANIVID: A new technology to analyze shallow gradient chemotaxis in vitro and in vivo. J. K. Williams1, M. Padgen1, Y. Wang2, F. Gertler3, J. Condeelis2, J. Castracane4; 1College of Nanoscale Science and Engineering, University at Albany, 2Albert Einstein College of Medicine, Yeshiva University, 3Massachusetts Institute of Technology, 4College of Nanoscale Science and Engineering, University at Albany, Albany, NY
Cancer cells create a unique microenvironment in vivo that enables tumor cell migration and dissemination to distant organs. To better understand the role of the tumor microenvironment in dissemination, special tools and devices are required to monitor the interactions between different cell types and the role of chemotaxis in migration and dissemination in vivo. Here we
describe the design and optimization of a new, versatile chemotaxis device called the NANIVID (NANo IntraVItal Device). The device is fabricated using BioMEMS techniques and consists of etched and bonded glass substrates, a soluble factor reservoir, fluorescent tracking beads and a microelectrode array for cell quantification. The reservoir contains a customized hydrogel blend that is loaded with epidermal growth factor (EGF). Upon hydration, EGF will diffuse out of the hydrogel to create a well-defined chemotactic gradient that can be sustained for many hours in order to attract tumor cells to the device. Here we show that mammary tumor cells in vitro and in vivo are chemotactic to the NANIVID. A new insight derived from this study is that tumor cells are capable of following much shallower gradients than previously thought possible. In particular, tumor cells can chemotax up a gradient that is as shallow as 0.7% (Fit R^2 =0.55), indicating that migratory tumor cells found in shallow gradients around co-migratory stromal cells are sufficient for initiating chemotaxis and migration in tumors.
323
The Role of CXCR4 and CXCR7-Mediated Chemokine Signaling in Zebrafish Keratocyte Motility.
A. D. Burke1, A. G. Van1, E. E. Hull1, K. J. Leyva2; 1Biomedical Sciences, Midwestern University, Glendale, AZ, 2Microbiology & Immunology, AZCOM, Midwestern University, Glendale, AZ The role of CXCR4 in regulating immunity, cell growth, cancer, angiongenesis, and development has been examined in many systems. Although it is known that the cognate, and only known, ligand for CXCR4 is CXCL12 (SDF-1) and that this cytokine is a known promoter of cellular migration, the role of this receptor in regulating non-immune cell migration is a recent development and has not been previously examined in fish epidermal keratocytes. Our data show that both CXCR4 and CXCL12a are differentially expressed during primary keratocyte explant culture. In our cell sheet migration assays, addition of CXCL12a to explant culture media increases keratocyte motility while addition of AMD3100 (a CXCR4b antagonist) appears to inhibit cell migration. The recently discovered chemokine receptor, CXCR7, binds CXCL11 as well as CXCL12a, although no reports to date have linked an effect of CXCR7 to cell motility. Both CXCR7 and CXCL11 appear to be differentially expressed during explant culture and in our migration assays, the addition of CXCL11 increases keratocyte motility. In order to determine the relative contribution of these two receptors on keratocyte motility, we performed the same migration assays on zebrafish homozygous for nonsense mutations in either CXCR4b or CXCR7b. Our data show that CXC receptors may have a more prominent role in regulating non-immune cell motility than previously thought, providing a first look at how cytokine signaling and the acute inflammatory response affect keratocyte cell migration.
324
N-3-oxo-dodecanoyl-L-homoserine lactone inhibits epithelial cells migration via Rac1/Cdc42, IQGAP1 and actin remodeling.
L. Yakymenko1, M. Turkina1, T. Karlsson1, K-E. Magnusson1, E. Vikstrom1; 1Department of Clinical and Experimental Medicine, Linkoping University, Sweden
In gram-negative bacteria, cell-to-cell communication based on N-acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human immune cell behavior, including cell motility. The aim of this study was to investigate the effect of 3-oxo-C12-HSL from Pseudomonas aeruginosa on the migration of human intestinal epithelial Caco-2 cells. Using wound-healing and migration assays we found that 3-oxo-C12-HSL inhibits Caco-2 cells migration in a dose- and time-dependent manner. The changes in cell migration were paralleled by F-actin cytoskeleton reorganization as evidenced by phalloidin staining and confocal
microscopy. Moreover, for the first time we demonstrated by proteome analysis, Western blot, immunostaining and confocal imaging that the 3-oxo-C12-HSL down-regulates expression levels of the Rho GTPases (Rac1 and Cdc42) as well as their effector, IQGAP1, which binds and stabilizes the active forms of Rho GTPases. Taken together, our findings provide a novel insight on modulation mechanisms between bacterial quorum sensing molecule 3-oxo-C12-HSL and epithelial cells at the sites of bacterial infection.
325
Ca++ chemotaxis in Dictyostelium discoideum.
D. J. Wessels1, A. Scherer2, D. Lusche2, S. Kuhl2, K. Wood2, K. Daniels2, P. Steimle3, T. Egelhoff4, D. R. Soll2; 1Univ Iowa, Iowa City, IA, 2Biology, University of Iowa, Iowa City, IA,
3Biology, University of North Carolina, Greensboro, Greensboro, NC, 4Cell Biology, Cleveland
Clinic Foundation, Cleveland, OH
Using a newly developed microfluidic chamber, we have demonstrated in vitro that Ca++ also functions as a chemoattractant of aggregation-competent D. discoideum amoebae. Effective Ca++ gradients are extremely steep compared to effective cAMP gradients. Given that Ca++ chemotaxis is co-acquired with cAMP chemotaxis during development of this organism, we speculated on the role Ca++ chemotaxis might play, notably the possibility that steep, transient Ca++ gradients may be generated during natural aggregation in the interstitial region between neighboring cells. In searching for a potential Ca++ chemotaxis receptor, we discovered that deletion of IplA (inositol triphosphate receptor-like protein), a putative Ca++ channel, resulted in loss of Ca++, but not loss of cAMP chemotaxis. We also found that iplA- cells could not accurately orient towards the aggregation center at the onset of each normal cAMP wave generated and relayed by a majority population of wild-type cells. In support of the independence of two chemotactic systems, we found that of the four myosin II heavy chain kinases, deletion of either myosin heavy chain kinase A (MHCKA) or myosin heavy chain kinase C (MHCKC), blocked Ca++, but not cAMP, chemotaxis. These mutant phenotypes were similar to that of the iplA- mutant. Furthermore, we show that neither Ca++ nor Ca++/calmodulin directly regulates these kinases intracellularly, a further indication that Ca++ chemotaxis is mediated through a surface receptor, potentially IplA. These discoveries increase the complexity of the contextual framework, a paradigm in place for forty years, for interpreting how chemotaxis functions in the natural aggregation of the model organism D. discoideum. Finally, we are investigating the role Ca++ chemotaxis may play in mammalian cell migration, especially during cancer cell metastasis.
326
HS1-dependent Rac activation is necessary for neutrophil chemotaxis.
P. Cavnar1, K. Mogen1, A. Huttenlocher1; 1Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI
HS1 is a 75-kDa protein that is primarily expressed in hematopoietic cells and is heavily phosphorylated in response to B- and T-cell receptor signaling. HS1 is important in antigen- induced proliferation of lymphocytes and like its homologue cortactin, is thought to stabilize F- actin branching through an N-terminal domain that binds Arp2/3 and tandem repeat domains that bind F-actin. HS1 contains a C-terminal proline-rich region and an SH3 domain that are important for a variety of protein-protein interactions including Src family kinases and Vav1. Despite progress in understanding HS1 function in lymphocytes, the function of HS1 during neutrophil chemotaxis remains unknown. Neutrophil chemotaxis is dependent on dynamic actin turnover at the leading edge. Here we investigate the function of HS1 during neutrophil chemotaxis using the neutrophil-like cell line, PLB-985 cells. We show that HS1 co-localizes
with F-actin at the leading edge of PLB-985 cells and primary neutrophils during chemotaxis. HS1 is tyrosine phosphorylated in response to fMLP stimulation and this is dependent on both adhesion and the activation of Src family kinases. Moreover, HS1-deficient cells show impaired activation of Rac GTPases in response to fMLP, suggesting that HS1-mediated Rac activity is necessary for neutrophil chemotaxis. Finally, we also show that phosphorylation of the RhoGEF Vav1 is impaired in HS1-deficient cells providing a link between HS1 and Rac activation. Taken together, our findings suggest that HS1 is necessary for Rac activation through the modulation of Vav1 GEF activity to allow for efficient neutrophil chemotaxis to fMLP.
327
Actin Crosslinking Proteins, Cortexillin I and II, are Required for cAMP-signaling During Dictyostelium Chemotaxis.
S. Shu1, X. Liu1, P. Kriebel2, M. Daniels3, E. Korn4; 1Laboratory of Cell Biology, NHLBI, NIH, Bethesda, MD, 2NCI, NIH, 3NHLBI, NIH, Bethesda, MD, 4NIH/NHLBI, Bethesda, MD
Dictyostelium has long been an excellent model for understanding the molecular basis of chemotaxis and intracellular and extracellular cAMP signaling. Binding of cAMP to G-protein- coupled cAMP receptors (cAR1) on the cell surface of Dictyostelium amoebae initiates a series of molecular responses. Expression of cAR1 and adenylyl cyclase (ACA) is markedly increased. Gß? released from the heterotrimeric G-protein coupled to cAR1 activates myosin II (via