This review discusses recent advances in our understanding of the role filopodia and filopodia-like structures in cell adhesion and three dimensional (3D) cell migration both in vitro and in vivo. In particular, we focus on recent advances demonstrating that filopodia are involved in substrate tethering and environment sensing in vivo. We further discuss the emerging role of filopodia and filopodial proteins in tumor dissemination as mounting in vitro, in vivo and clinical evidence suggest that filopodia drive cancer cell invasion and highlight filopodia proteins as attractive therapeutic targets. Finally, we outline outstanding questions that remain to be addressed to elucidate the role of filopodia during 3D cell migration.
Guillaume Jacquemet, Hellyeh Hamidi & Johanna Ivaska
2D surfaces offer simple analysis of cells in culture, yet these often
yield different cell morphologies and responses from those observed in vivo.
Considerable effort has therefore been expended on the generation of
more tissue-like environments for the study of cell behavior in vitro. Purified matrix proteins provide a 3D scaffold that better mimics the in vivo situation; however, these are far removed from the complex tissue composition seen in vivo. Cell-derived matrices (CDMs) offer a more physiologically relevant alternative for studying in vivo-like cell behavior in vitro.
In the protocol described here, fibroblasts cultured on gelatin-coated
surfaces are maintained in the presence of ascorbic acid to strengthen
matrix deposition over 1–3 weeks. The resulting fibrillar CDMs are
denuded of cells, and other cells are subsequently cultured on them,
after which their behavior is monitored. We also demonstrate how to use
CDMs as an in vivo-relevant reductionist model for studying tumor-stroma-induced changes in carcinoma cell proliferation and migration.
Riina Kaukonen, Guillaume Jacquemet, Hellyeh Hamidi & Johanna Ivaska
Defective filopodia formation is linked to pathologies such as cancer, wherein actively protruding filopodia, at the invasive front, accompany cancer cell dissemination. Despite wide biological significance, delineating filopodia function in complex systems remains challenging and is particularly hindered by lack of compatible methods to quantify filopodia properties. Here, we present FiloQuant, a freely available ImageJ plugin, to detect filopodia-like protrusions in both fixed- and live-cell microscopy data. We demonstrate that FiloQuant can extract quantifiable information, including protrusion dynamics, density, and length, from multiple cell types and in a range of microenvironments. In cellular models of breast ductal carcinoma in situ, we reveal a link between filopodia formation at the cell–matrix interface, in collectively invading cells and 3D tumor spheroids, and the in vitro invasive capacity of the carcinoma. Finally, using intravital microscopy, we observe that tumor spheroids display filopodia in vivo, supporting a potential role for these protrusions during tumorigenesis.
Guillaume Jacquemet , Ilkka Paatero, Alexandre F. Carisey, Artur Padzik, Jordan S. Orange, Hellyeh Hamidi & Johanna Ivaska
Filopodia are fingerlike membrane protrusions that are extended by cells in vitro and in vivo. Due to important roles in sensing the extracellular microenvironment, filopodia and filopodia-like protrusions have been implicated in numerous biological processes including epithelial sheet zippering in development and wound healing and in cancer progression. Recently, there has been an explosion in the number of software available to analyze specific features of cell protrusions with the aim of gaining mechanistic insights into the action of filopodia and filopodia-like structures. In this methods chapter, we highlight an open-access software called FiloQuant that has been developed to specifically quantify the length, density, and dynamics of filopodia and filopodia-like structures from in vitro and in vivo generated samples. We provide step-by-step protocols on (i) how to install FiloQuant in the ImageJ platform (Fiji), (ii) how to quantify filopodia and filopodia-like protrusions from single images using FiloQuant, and (iii) how to track filopodial protrusions from live-cell imaging experiments using FiloQuant and TrackMate.
Guillaume Jacquemet, Hellyeh Hamidi & Johanna Ivaska
Mounting in vitro, in vivo and clinical evidence suggest an important role for filopodia in driving cancer cell invasion. Using a high-throughput microscopic-based drug screen, we identify FDA-approved calcium channel blockers (CCBs) as potent inhibitors of filopodia formation in cancer cells. Unexpectedly, we discover that L-type calcium channels are functional and frequently expressed in cancer cells suggesting a previously unappreciated role for these channels during tumorigenesis. We further demonstrate that, at filopodia, L-type calcium channels are activated by integrin inside-out signalling, integrin activation and Src. Moreover, L-type calcium channels promote filopodia stability and maturation into talin-rich adhesions through the spatially restricted regulation of calcium entry and subsequent activation of the protease calpain-1. Altogether we uncover a novel and clinically relevant signalling pathway that regulates filopodia formation in cancer cells and propose that cycles of filopodia stabilization, followed by maturation into focal adhesions, directs cancer cell migration and invasion.
Guillaume Jacquemet, Habib Baghirov, Maria Georgiadou, Harri Sihto, Emilia Peuhu, Pierre Cettour-Janet, Tao He, Merja Perälä, Pauliina Kronqvist, Heikki Joensuu & Johanna Ivaska
Filopodia are adhesive cellular protrusions specialized in the detection of extracellular matrix (ECM)-derived cues. Although ECM engagement at focal adhesions is known to trigger the recruitment of hundreds of proteins (“adhesome”) to fine-tune cellular behavior, the components of the filopodia adhesions remain undefined. Here, we performed a structured-illumination-microscopy-based screen to map the localization of 80 target proteins, linked to cell adhesion and migration, within myosin-X-induced filopodia. We demonstrate preferential enrichment of several adhesion proteins to either filopodia tips, filopodia shafts, or shaft subdomains, suggesting divergent, spatially restricted functions for these proteins. Moreover, proteins with phosphoinositide (PI) binding sites are particularly enriched in filopodia. This, together with the strong localization of PI(3,4)P2 in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions, and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. We demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its C-terminal Cas family homology domain (CCHD) and acts as a mechanosensitive regulator of filopodia stability. Finally, we demonstrate that our map based on myosin-X-induced filopodia can be translated to endogenous filopodia and fascin- and IRSp53-mediated filopodia.
Guillaume Jacquemet, Aki Stubb, Rafael Saup, Mitro Miihkinen, Elena Kremneva, Hellyeh Hamidi & Johanna Ivaska
The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance and wound healing. In order for cells to migrate, they must interact with their environment using adhesion receptors, such as integrins, and form specialized adhesion complexes that mediate responses to different extracellular cues. In this review, we discuss the role of integrin adhesion complexes (IACs) in cell migration, highlighting the layers of regulation that are involved, including intracellular signalling cascades, mechanosensing and reciprocal feedback to the extracellular environment. We also discuss the role of IACs in extracellular matrix remodeling and how they impact upon cell migration.