LIMA PROVINCIAS

J. R. Beach1, G. Hussey2, T. E. Miller3, A. Chaudhury4, P. Patel5, J. Monslow3, Q. Zheng3, R. Keri6, O. Reizes3, A. Bresnick7, P. Howe8, T. T. Egelhoff3; 1Cell Biology, NC10, Cleveland Clinic, Cleveland, OH, 2Cancer Biology, Hollins Cancer Center, Charleston, SC, 3Cell Biology,

Cleveland Clinic, Cleveland, OH, 4Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, 5Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, 6Pharmacology, Case Western Reserve, Cleveland, OH, 7Department of Biochemistry, Albert Einstein School of Medicine, Bronx, NY, 8Hollins Cancer Center, Medical University of South Carolina, Charleston, SC

An epithelial-mesenchymal transition (EMT) occurs when epithelial cells acquire the morphological and phenotypic properties of a mesenchymal cell, including increased migratory and invasive characteristics. TGFβ is a known inducer of EMT in many settings. EMT and EMT- like events are suggested to precede cancer cell metastasis. Despite functional significance of non-muscle myosin II in cell migration and invasion, its role in EMT or TGFβ signaling is unknown. The goal of these studies was to determine the roles and regulation of nonmuscle myosin II during TGFβ-induced EMT in mammary epithelial cells. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells while myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGFβ induction of EMT in non-transformed murine mammary gland (NMuMG) epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (Ser1916, Ser1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGFβ signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state, and concomitantly upregulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that

transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.

334

Galectin 3 modulates the mast cells migration.

V. D. Toso1, M. de Cássia Campos1, D. A. de Souza Junior1, M. Dias Bariffi2, M. Roque Antunes Barreira1, C. Oliver1, M. Jamur1; 1Departamento de biologia celular e molecular e biagentes patogenicos, Faculdade de Medicina de Ribeirão Preto - USP, Ribeirão preto, Brazil,

2_{Departamento de Biociências Aplicadas à Farmácia, Faculdade de Ciências Farmaucêuticas}

de Ribeirão Preto - USP

Mast cells are imunorregulatory cells that participate in the defense of the organism and are known for exerting a fundamental role in asthma, allergy and inflammatory reactions, as well as expulsion of parasites. Galectins have a modulatory role in various cell types by regulating growth, adhesion, migration and cell proliferation. The aim of this study was to investigate the role of galectins 1 and 3 in mast cell function, using knockout mice for these galectins. The distribution of mast cells in the spleen, lungs and skin was characterized. The role of galectins in cell adhesion and migration was studied in vitro with bone marrow derived mast cells. The results showed that there were no differences in the number of mast cells in the spleen, skin and lungs between or between the knockout mice and the wild-type mice. Using Transwell assays, bone marrow derived mast cells from galectin-3 knockout mice migrate less in comparison with mast cells from galectin-1 knockout mice or wild-type mice, even in presence of the chemotactic factors IL-3 and SCF. On the other hand, no differences were observed in the adhesion of bone marrow derived mast cells from galectin-1 and 3 knockout mice as well as wild-type mice to components of the extracellular matrix such as fibronectin, laminin, collagen type I and IV. These results indicate that galectin-3 is important for the migration of mast cells, but the absence of galectin-3 does not alter the adhesion of these cells to extracellular matrix components.

335

EB1-dependent microtubule +TIP complexes coordinate protrusion dynamics during 3D epithelial remodeling.

S. Gierke1, T. Wittmann1; 1Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA

Epithelial remodeling, in which apical-basal polarized cells switch to a migratory phenotype, plays essential roles in development and cancer progression. How microtubules are controlled or contribute to epithelial remodeling in a physiological three-dimensional (3D) environment is not understood. We analyzed microtubule function and dynamics using an epithelial cyst cell culture system in which polarized MDCK epithelial cells undergo a partial epithelial-to- mesenchymal transition (EMT) in response to hepatocyte growth factor (HGF), and cells extend, and migrate into the surrounding extracellular matrix (ECM). Using high-resolution confocal microscopy, we found that extensions at the basal surface of HGF-treated cysts were filled with dense microtubule bundles. Computational tracking of EB1-EGFP showed large numbers of microtubules growing persistently from the apical domain into these extensions, often deforming the extension tip, and an increase in microtubule growth rate in response to HGF before morphological changes were evident. Next, we tested the role of microtubule plus-end tracking protein (+TIP) complexes in HGF-induced migration in 3D by depleting cells of EB1, the central adaptor that mediates association of other +TIP proteins with growing microtubule plus-ends. In EB1-depleted cells, microtubules displayed rapid lateral and retrograde movements demonstrating that EB1 is required to anchor and stabilize microtubules in HGF-induced

extensions. EB1-depleted cysts formed shorter, more branched extensions further suggesting that EB1 is required for productive HGF-induced extension outgrowth. Analysis of cell-matrix interactions and F-actin dynamics revealed that control extensions progressively pulled on and deformed the ECM typically with one F-actin-rich protrusion near the tip. However, EB1- depleted cells produced multiple highly dynamic F-actin-rich protrusions that did not productively engage the matrix. This resulted in extensions that rapidly protruded, retracted, and changed direction. The inability to engage the matrix and stabilize a dominant protrusion was also associated with defects in cell-matrix adhesions. Although EB1-depleted extensions formed nascent adhesions, they never matured, were mislocalized, and were uncoordinated, in contrast to the highly coordinated adhesions of control cells. Together our findings indicate that EB1- dependent microtubule +TIP complexes are required to coordinate protrusion dynamics in migrating cells during 3D epithelial remodeling.

336

Phosphoinositide Signaling Regulates the Exocyst Complex and Polarized Integrin Trafficking in Directionally Migrating Cells.

N. Thapa1, Y. Sun1, S. Choi1, R. A. Anderson1; 1University of Wisconsin-Madison, Madison, WI Cell migration is essential for many biological processes. Cell migration hinges on the ability of cells to traffic signaling molecules and proteins toward the direction of migration, a process that requires the tight regulation of cytoskeletal and vesicle trafficking machineries. The trafficking of both newly synthesized and recycled integrin molecules to and from the leading edge plasma membrane is critical for directional cell migration. Here, we describe a novel role for the phosphatidylinositol-4,5-biphosphate (PIP2) synthesizing enzyme, PIPKI(i2, in modulating polarized integrin trafficking during cell migration. PIPKI(i2 knockdown impaired directional migration in multiple assays. Loss of PIPKI(i2 impaired Golgi and microtubule orientation towards the direction of cell migration and trafficking of (1 integrin to the leading edge.

PIP2 signaling specificity is defined by the interaction of PIP kinases with PIP2 effectors or compartments containing PIP2 effectors. We show that PIPKI(i2 interacts with the exocyst an evolutionarily conserved vesicle-trafficking protein complex required for polarized trafficking. In vitro binding study showed Sec6 and Exo70 as PIPKI(i2-interacting subunits of the exocyst complex. Upon initiation of directional migration, PIPKI(i2 associated with both the exocyst complex and ?1 integrin and this was dependent both on kinase activity of PIPKI(i2 and its ability to associate with talin.

The integrity of exocyst complex was pivotal for PIPKI(i2-regulated cell migration as knockdown of any exocyst complex components impaired cell migration including polarized trafficking of ?1 integrin to leading edge. Furthermore, the expression of Exo70 mutant defective in PIP2-binding abrogated the PIPKI(i2-regulated cell migration emphasizing the role of PIP2. PIPKI(i2 was specific isoforms to promote integrin-dependent cell migration as its expression specifically promoted the integration of exocyst complex into ?1 integrin-containing vesicle like intracellular compartment. We have discovered a fundamental role for PIP2 in polarized integrin trafficking required for directionally migrating cells.

337

Myosin-II-mediated contractility coordinates cell movements during collective migration through cell-cell mechanotransduction.

M. Ng1, A. Besser1, G. Danuser1, J. Brugge1; 1Cell Biology, Harvard Medical School, Boston, MA

Collective migration of epithelial sheets is an important process in morphogenesis, tissue repair and tumor invasion. To achieve collective migration, individual cells must communicate with one

another and coordinate their migratory processes. The mechanisms underlying such coordination are not well understood. Using an automated, high-throughput time-lapse microscopy and cell tracking platform, we monitored the migration trajectories of ~0.5 million MCF10A cells undergoing collective migration in wound-healing assays. We used several parameters to assess coordination in cell movements, and found that migration persistence, as well as the correlation in migration velocities and polarization directions between individual cells, is dependent on myosin-II-mediated cell contractility. Decreasing cell contractility by decreasing substrate stiffness or by RNAi-mediated knockdown of myosin-IIA and myosin-IIB reduces coordination in cell motion during collective migration. Importantly, we discovered that contractility can affect coordinated motion by modulating mechanical forces communicated between cells through cadherin-mediated cell-cell adhesions. Using a novel traction force microscopy-based method to measure tension exerted on adherens junctions, we found that the forces transmitted across individual cell-cell junctions positively correlate with cellular myosin-II activity. Interestingly, a high level of actomyosin activity can also disrupt mechanical communication across multiple adjacent cells. These results reveal a complex relationship between actomyosin contractility, cell-cell mechanotransduction and coordination in cell movements during collective migration. Further characterization of this relationship will be an important step towards understanding collective migration, and may shed light on pathological developments such as cancer invasion, which is often associated with altered microenvironmental stiffness, cellular contractility and cell-cell adhesions.

338

F-actin dynamics and actomyosin contraction during neutrophil motility in live zebrafish. P-Y. Lam1, R. S. Fischer2, C. M. Waterman2, A. Huttenlocher1; 1Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI, 2Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health

Neutrophils are the most abundant white blood cell and are the first cells to respond to sites of tissue injury. The in vivo migration of neutrophils is highly dynamic and involves the generation of multiple leading edge pseudopodia. For directed motility, neutrophils maintain a dominant pseudopodium in the direction of movement while retracting other pseudopodia. Although uropod retraction is known to require myosin II contractility, the cytoskeletal mechanisms that govern pseudopodia selection at the front of the cell are not well understood. To investigate pseudopodia selection in vivo, we used time-lapse spinning-disk confocal microscopy of neutrophils with dual channel imaging of bioprobes specific for all (Lifeact-Ruby) and more stable (GFP-UtrCH) populations of F-actin. We show that dynamic F-actin is localized in the dominant pseudopodium while the more stable F-actin accumulates in the retracting pseudopodia and uropod during neutrophil migration. To determine the role of myosin II in pseudopodia selection, we performed ratiometric imaging of myosin regulatory light chain (MRLC) to a volume marker to allow visualization specifically of cytoskeleton-associated myosin II. This revealed localization of MRCL with the cytoskeleton in the uropod, as expected. However, MRLC also accumulated and localized to the leading edge of pseudopodia precisely at the time of retraction, and this accumulation increased as the retraction progressed. Taken together, we demonstrated that in an in vivo context, F-actin stabilization and myosin II accumulation occur in retracting pseudopodia, suggesting that actin stabilization and myosin II activity are regionally regulated during pseudopodia selection to guide directed neutrophil motility. Further studies will be needed to test if actomyosin contraction is required for biased pseudopodia retraction in directed motility during the immune response in vivo.

339

Dynamic Cytoskeleton Organization Couple Cell Shape Variations with Migration Phenotypes in HL-60 Cells.

T. Tsai1, M. Davidson2, J. Ferrell1, J. Theriot3; 1Chemical and Systems Biology, Stanford, Stanford, CA, 2Florida State University, 3Biochemistry, Stanford University

The neutrophil-like HL60 cell is a common model system for the study of human neutrophil migration. A population of HL60 cells under constant conditions exhibits highly heterogeneous migration phenotypes, as characterized by their speed, shape, and frequency of polarity switching. These natural variations allow us to investigate the mechanism underlying shape determination and its correlation with migration phenotypes.

Well-differentiated HL-60 cells, stably expressing fluorescent protein-tagged actin or myosin, are migrating within uniform field of chemoattractant, and imaged with high spatiotemporal resolution. To mimic the neutrophil’s native confined environment, the cells are plated in between the fibronectin-coated coverslip and an agarose pad. To identify important shape parameters, we apply principal component analysis on hundreds of cell contours. Interestingly, the first few principal modes are biologically meaningful. Variations in cell length, leading edge width, and left-right asymmetry can explain close to 70% of total shape variation. These three shape modes are also preserved in migrating human primary neutrophils. Cells with relatively wider leading edge migrate faster and switch polarity less frequently. Left-right asymmetry of cell shape correlates well with asymmetric distribution of myosin, as well as turns in cell trajectory, although interestingly asymmetric myosin accumulation lags behind cell turning. Inhibition of actin polymerization by latrunculin reduces cell length and leading edge width, while inhibition of myosin by blebbistatin increases cell length and reduces leading edge width. This observation is consistent with our model using membrane tension as a limiting factor for extension of leading edge width and cell length.

Finally, we use the movement and position of cell nucleus relative to the leading edge and cell rear to probe for the cell’s internal mechanical properties. Nocodazole treatment increases persistence of protrusion, and reduces the distance between leading edge and the nucleus, while taxol treatment decreases the persistence of protrusion. Variations in microtubule dynamics might affect the cell-nuclear coupling and contribute to variations in migration persistence.

340

An in vivo tracking system to investigate the migration of bone marrow stem cells in an osteochondral defect model.

G-I. Im1, C-N. Im1, J-M. Lee1; 1Orthopaedics, Dongguk University Ilsan Hospital, Goyang, Korea An in vivo system was developed to monitor the migration of human bone marrow-derived MSCs (BMSCs) transplanted within the marrow cavity of athymic nude rats. The in vitro studies confirmed that PDGF (platelet-derived growth factor)-AA had the most potent chemotactic effect of the factors tested, and possessed the greatest number of receptors in BMSCs. In the in vivo study, the BMSCs were labeled with fluorescent nanoparticles and injected into the marrow cavity through an osteochondral defect created in the distal femur of rats. The defect was sealed with HCF (heparin-conjugated fibrin) or PDGF-AA-loaded HCF. In the HCF-only control group without PDGF-AA, the nanoparticle-labeled BMSCs were dispersed outside the marrow cavity within 3 days after the injection. In the PDGF-AA-loaded HCF group, the labeled cells moved time-dependently for 14 days toward the osteochondral defect. The in vivo tracking

patterns differed depending on the PDGF-AA concentration 21 days after injection: HCF-PDGF in low dose (8.5 ng/µl) was more effective than HCF-PDGF in high dose (17 ng/µl) in recruiting the BMSCs to the osteochondral defect. When the osteochondral defect was evaluated macroscopically and histologically, the defects treated with PDGF and TGF-β1-loaded HCF showed excellent cartilage repair compared with other groups. Further in vivo studies confirmed that this in vivo osteochondral BMSCs tracking system (IOBTS) worked for other chemo- attractants (CCL-2 and PDGF-BB). In conclusion, IOBTS can be a very useful tool for examining the effect of growth factors or chemokines on BMSC migration and cartilage repair.

341

Manganese Superoxide Dismutase Inhibited Gastric Cancer Cellular Invasion.

H. Matsui1, T. Tomita2, M. Tamura1, Y. Nagano1, T. Kaneko1, I. Hyodo1; 1Graduate school of

comprehensive human sciences, Univ Tsukuba, Tsukuba, Japan, 2Graduate School of Pure and

Applied Sciences, Univ Tsukuba, Tsukuba, Japan

Reactive oxygen species (ROS) have been reported to play an important role for cancer cellular invasion. Recently, we have established a new cancerous cell-line RGK-1 from a rat gastric normal epithelial cell-line RGM-1. We also established a stable clone RGK-MnSOD, which overexpressed a manganese superoxide disumutase. In this study, we elucidated a role of ROS for cancer cellular invasion. [Methods] The ROS concentration and the cell membrane lipid peroxidation were determined using fluorescence probes. The kind of ROS was clarified with EPR. The invasion abilities were determined by a matrigel assay. [Results and Discussion] The ROS from cancer cell was O2-. Cancer cellular O2- concentration was significantly higher than in

normal, and MnSOD significantly decreased the O2- concentration. The depth of cancer cellular

invasion into matrigel was significantly longer than that of normal, and MnSOD also significantly inhibited the invasion. We concluded that cancer specific ROS derived from mitochondria involved the cell invasion ability because MnSOD is a mitochondrion-particular enzyme.