Application of Bioimage Informatics to Quantification of Focal Adhesions and Invadopodia

The development of the ability to fluorescently label functional proteins and visualize their subcellular localization using microscopy in living cells, has made it possible to study a wide range of single cell phenomena. To understand the results of imaging assays, cell biologists have used manual methods for determining the quantitative properties of the cellular structures visualized fluorescent microscopy. As the quantity and complexity of the images that can be collected using fluorescence microscopy has increased, a new subfield of Bioinformatics has developed, named Bioimage Informatics, which specializes in adapting and developing new methods to quantify the image sets resulting from biological assays. In this thesis, I describe the application and development of Bioimage Informatic methods to the analysis of Focal Adhesions and Invadopodia. Focal Adhesions are subcellular protein complexes, whose role include acting as the points of contact for cellular motility and sensing the outside environment. Focal Adhesions have traditionally been analyzed using manual methods, which has limited the number of Focal Adhesions that could be analyzed and the depth of properties that could be collected. I have developed a set of methods which can identify, track and quantify Focal Adhesion properties from live cell image sets. This Focal Adhesion analysis framework has been expanded to include spatial and global methods for describing Focal Adhesion morphology. I have also developed a system for quantifying Invadopodia properties. Invadopodia are subcellular protein complexes present in metastatic cancer cells, which actively degrade the extracellular matrix, allowing migration of cancer cells away from primary tumors. This analysis system has two parts, one which can follow single Invadopodia and assess their properties and a complementary component which assesses degradation behavior in cell populations.Doctor of Philosoph

The Dark Kinase Knowledgebase: An online compendium of knowledge and experimental results of understudied kinases

Kinases form the backbone of numerous cell signaling pathways, with their dysfunction similarly implicated in multiple pathologies. Further facilitated by their druggability, kinases are a major focus of therapeutic development efforts in diseases such as cancer, infectious disease and autoimmune disorders. While their importance is clear, the role or biological function of nearly one-third of kinases is largely unknown. Here, we describe a data resource, the Dark Kinase Knowledgebase (DKK; https://darkkinome.org), that is specifically focused on providing data and reagents for these understudied kinases to the broader research community. Supported through NIH\u27s Illuminating the Druggable Genome (IDG) Program, the DKK is focused on data and knowledge generation for 162 poorly studied or \u27dark\u27 kinases. Types of data provided through the DKK include parallel reaction monitoring (PRM) peptides for quantitative proteomics, protein interactions, NanoBRET reagents, and kinase-specific compounds. Higher-level data is similarly being generated and consolidated such as tissue gene expression profiles and, longer-term, functional relationships derived through perturbation studies. Associated web tools that help investigators interrogate both internal and external data are also provided through the site. As an evolving resource, the DKK seeks to continually support and enhance knowledge on these potentially high-impact druggable targets

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

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

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

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

LKB1 loss in melanoma disrupts directional migration toward extracellular matrix cues

Somatic inactivation of the serine/threonine kinase gene STK11/LKB1/PAR-4 occurs in a variety of cancers, including ∼10% of melanoma. However, how the loss of LKB1 activity facilitates melanoma invasion and metastasis remains poorly understood. In LKB1-null cells derived from an autochthonous murine model of melanoma with activated Kras and Lkb1 loss and matched reconstituted controls, we have investigated the mechanism by which LKB1 loss increases melanoma invasive motility. Using a microfluidic gradient chamber system and time-lapse microscopy, in this paper, we uncover a new function for LKB1 as a directional migration sensor of gradients of extracellular matrix (haptotaxis) but not soluble growth factor cues (chemotaxis). Systematic perturbation of known LKB1 effectors demonstrated that this response does not require canonical adenosine monophosphate–activated protein kinase (AMPK) activity but instead requires the activity of the AMPK-related microtubule affinity-regulating kinase (MARK)/PAR-1 family kinases. Inhibition of the LKB1–MARK pathway facilitated invasive motility, suggesting that loss of the ability to sense inhibitory matrix cues may promote melanoma invasion