Matthew E. Berginski

Arp2/3 Is Critical for Lamellipodia and Response to Extracellular Matrix Cues but Is Dispensable for Chemotaxis

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.

The Vinculin C-terminal Hairpin Mediates F-actin Bundle Formation, Focal Adhesion, and Cell Mechanical Properties

Vinculin is an essential and highly conserved cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples integrins or cadherins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility, and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) contains determinants necessary for binding and bundling of actin filaments. Actin binding to Vt has been proposed to induce formation of a Vt dimer that is necessary for cross-linking actin filaments. Results from this study provide additional support for actin-induced Vt self-association. Moreover, the actin-induced Vt dimer appears distinct from the dimer formed in the absence of actin. To better characterize the role of the Vt strap and carboxyl terminus (CT) in actin binding, Vt self-association, and actin bundling, we employed smaller amino-terminal (NT) and CT deletions that do not perturb the structural integrity of Vt. Although both NT and CT deletions retain actin binding, removal of the CT hairpin (1061–1066) selectively impairs actin bundling in vitro. Moreover, expression of vinculin lacking the CT hairpin in vinculin knock-out murine embryonic fibroblasts affects the number of focal adhesions formed, cell spreading as well as cellular stiffening in response to mechanical force.

The Focal Adhesion Analysis Server: a web tool for analyzing focal adhesion dynamics

The Focal Adhesion Analysis Server (FAAS) is a web-based implementation of a set of computer vision algorithms designed to quantify the behavior of focal adhesions in cells imaged in 2D cultures. The input consists of one or more images of a labeled focal adhesion protein. The outputs of the system include a range of static and dynamic measurements for the adhesions present in each image as well as how these properties change over time. The user is able to adjust several parameters important for proper focal adhesion identification. This system provides a straightforward tool for the global, unbiased assessment of focal adhesion behavior common in optical microscopy studies. The webserver is available at: http://faas.bme.unc.edu/.

Dissecting motility signaling through activation of specific Src-effector complexes

We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells, and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed RapRTAP (rapamycin regulated targeted activation of pathways) was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases.

Intravital imaging of donor allogeneic effector and regulatory T cells with host dendritic cells during GVHD

Graft-versus-host disease (GVHD) is a systemic inflammatory response due to the recognition of major histocompatibility complex disparity between donor and recipient after hematopoietic stem cell transplantation (HSCT). T-cell activation is critical to the induction of GVHD, and data from our group and others have shown that regulatory T cells (Tregs) prevent GVHD when given at the time of HSCT. Using multiphoton laser scanning microscopy, we examined the single cell dynamics of donor T cells and dendritic cells (DCs) with or without Tregs postallogeneic transplantation. We found that donor conventional T cells (Tcons) spent very little time screening host DCs. Tcons formed stable contacts with DCs very early after transplantation and only increased velocity in the lymph node at 20 hours after transplant. We also observed that Tregs reduced the interaction time between Tcons and DCs, which was dependent on the generation of interleukin 10 by Tregs. Imaging using inducible Tregs showed similar disruption of Tcon-DC contact. Additionally, we found that donor Tregs induce host DC death and down-regulate surface proteins required for donor T-cell activation. These data indicate that Tregs use multiple mechanisms that affect host DC numbers and function to mitigate acute GVHD.

Nck enables directional cell migration through the coordination of polarized membrane protrusion with adhesion dynamics.

Summary Directional migration requires the coordination of cytoskeletal changes essential for cell polarization and adhesion turnover. Extracellular signals that alter tyrosine phosphorylation drive directional migration by inducing reorganization of the actin cytoskeleton. It is recognized that Nck is an important link between tyrosine phosphorylation and actin dynamics; however, the role of Nck in cytoskeletal remodeling during directional migration and the underlying molecular mechanisms remain largely undetermined. In this study, a combination of molecular genetics and quantitative live cell microscopy was used to show that Nck is essential in the establishment of front–back polarity and directional migration of endothelial cells. Time-lapse differential interference contrast and total internal reflection fluorescence microscopy showed that Nck couples the formation of polarized membrane protrusions with their stabilization through the assembly and maturation of cell–substratum adhesions. Measurements by atomic force microscopy showed that Nck also modulates integrin &agr;5&bgr;1-fibronectin adhesion force and cell stiffness. Fluorescence resonance energy transfer imaging revealed that Nck depletion results in delocalized and increased activity of Cdc42 and Rac. By contrast, the activity of RhoA and myosin II phosphorylation were reduced by Nck knockdown. Thus, this study identifies Nck as a key coordinator of cytoskeletal changes that enable cell polarization and directional migration, which are crucial processes in development and disease.

Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms

Significance Src family kinases (SFKs), critical in many aspects of homeostasis and disease, occur as multiple isoforms. It has been difficult to dissect the unique function of each isoform because their structures are so similar. Here we specifically activated each SFK isoform through insertion of an engineered domain. The domain caused the kinases to be catalytically inactive until they were reactivated by the small molecule rapamycin. Computational methods for quantifying dynamic changes in cell shape revealed that activation of each isoform produced dramatically different cell behaviors. Quantitative analysis showed that these behaviors correlated with specific patterns of subcellular trafficking, and depended on isoform acylation. The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases’ homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.

Gleevec, an Abl family inhibitor, produces a profound change in cell shape and migration

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.

Automated analysis of invadopodia dynamics in live cells

Multiple cell types form specialized protein complexes that are used by the cell to actively degrade the surrounding extracellular matrix. These structures are called podosomes or invadopodia and collectively referred to as invadosomes. Due to their potential importance in both healthy physiology as well as in pathological conditions such as cancer, the characterization of these structures has been of increasing interest. Following early descriptions of invadopodia, assays were developed which labelled the matrix underneath metastatic cancer cells allowing for the assessment of invadopodia activity in motile cells. However, characterization of invadopodia using these methods has traditionally been done manually with time-consuming and potentially biased quantification methods, limiting the number of experiments and the quantity of data that can be analysed. We have developed a system to automate the segmentation, tracking and quantification of invadopodia in time-lapse fluorescence image sets at both the single invadopodia level and whole cell level. We rigorously tested the ability of the method to detect changes in invadopodia formation and dynamics through the use of well-characterized small molecule inhibitors, with known effects on invadopodia. Our results demonstrate the ability of this analysis method to quantify changes in invadopodia formation from live cell imaging data in a high throughput, automated manner.

Abstract C14: Loss of haptotaxis facilitates invasion in LKB1-deficient melanoma

Germline mutations in the serine/threonine kinase STK11/LKB1 are associated with Peutz-Jehgers Syndrome, which is characterized by hyperpigmentation of the oral mucosa. Inactivating somatic mutations occur in approximately 10-20% of melanomas; however, how the loss of LKB1 facilitates melanoma invasion remains poorly understood. Using cell lines derived from simultaneous activation of KRas and inactivation of LKB1 in melanocytes, we have investigated melanoma migration upon reconstitution with LKB1. Reexpression of LKB1 diminishes migration during wound healing, spheroid outgrowth into 3D collagen, and overall single cell speed in random motility assays. Furthermore, the formation of invadopodia is independent of LKB1 status in both human and mouse melanomas. Interestingly, using microfluidic devices we have found that loss of LKB1 abrogates the ability of cells to respond to gradients of extracellular matrix (haptotaxis) but does not impair their ability to chemotax to EGF. We have also recently developed a model of orthotopic implantation of multicellular tumor spheroids into the dermis of the mouse ear skin and have validated this approach by recapitulating the finding that LKB1 limits tumorigenesis. We are using this model to image local invasion in vivo by multiphoton microscopy and are currently examining the intriguing hypothesis that loss of extracellular matrix sensing is one aspect that contributes to metastatic migration. Citation Format: Keefe T. Chan, Sreeja B. Asokan, Tao Bo, Matthew E. Berginski, Wenjin Liu, Shelly D. Cochran, Norman E. Sharpless, James E. Bear. Loss of haptotaxis facilitates invasion in LKB1-deficient melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C14.