Complexes were washed, denatured, and resolved by SDS-PAGE. confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion. Graphical Abstract Open in a separate window Introduction Fascin is an actin-binding protein that is known to regulate the parallel bundling of actin filaments (Vignjevic et?al., 2006), stabilize filopodia and invadopodia (Jayo et?al., 2012, Li et?al., 2010), and regulate adhesion dynamics in migrating cells (Elkhatib et?al., 2014). Fascin has received considerable attention in recent years as its expression is very low or absent in normal adult epithelia, but it is dramatically upregulated at both transcript and protein levels in all forms of human carcinomas studied to date (Hashimoto et?al., 2005). Thus, fascin is emerging as an excellent prognostic marker and a potential therapeutic target for metastatic disease (Tan et?al., 2013, Adams, 2015). Despite this recognized clinical importance, there is still very little molecular detail available defining the mechanisms underpinning fascin-dependent cell invasion, thus significantly limiting strategic approaches for therapeutic design. It is also unclear whether these defined roles for fascin in tumorigenesis rely upon the classical F-actin-bundling function or whether other roles may exist that coordinate cell invasion. Fascin comprises four tandem -trefoil domains that form a bilobed structure, with -hairpin triplets located symmetrically on opposite sides of each lobe that are proposed APC to act as the actin-binding domains (Sedeh et?al., 2010). These actin bundles, whether in the form of filopodia extending beyond the cell edge or microspikes within lamellae of migrating cells or neuronal growth cones, are involved in controlling cell migration in?vitro (Adams, 2004) and embryonic development (24S)-MC 976 in?vivo (Wood and Martin, 2002, Mattila and Lappalainen, 2008, Hashimoto et?al., 2011). Invasion of carcinoma cells is a highly coordinated process that depends largely on alterations to cell-cell and cell-extracellular matrix (ECM) adhesion and organization of the actin cytoskeleton (Guo and Giancotti, 2004). Carcinoma cells migrating in 3D ECM and in living tissues assemble membrane protrusions and specialized ECM-degrading adhesions termed invadopodia to enable tunneling through the matrix (Friedl and Wolf, 2003, Condeelis et?al., 2005, Li et?al., 2010). We and other groups have shown that loss of fascin function in a range of cell types results in reduced assembly of actin protrusions, more stable adhesions, and reduced migration and invasion in?vivo (Hashimoto et?al., 2007, Kim et?al., 2009, Chen et?al., 2010, Jayo et?al., 2012, Zanet et?al., 2012). However, it remains unclear whether these reported functions for fascin depend upon actin bundling within filopodia (24S)-MC 976 alone, or whether other roles for fascin exist within normal and metastatic cells that promote motility. Physicochemical properties of the ECM play an important role in the regulation of cell migration (Charras and Sahai, 2014, Friedl and Alexander, 2011) and cancer cells have been shown to have great plasticity, enabling them to adapt their migratory strategies to external cues (Sanz-Moreno et?al., 2008, Wolf et?al., 2003, Balzer et?al., 2012). Several studies have demonstrated that nuclear size and deformation act as limiting factors of cell migration in physically confined environments (Wolf et?al., 2013, Rowat (24S)-MC 976 et?al., 2013, Davidson et?al., 2014). Contractile force generation, cytoskeleton-driven force transmission to the nucleus, and nuclear stiffness (Harada et?al., 2014, Lammermann et?al., 2008, Lombardi et?al., 2011, Alam et?al., 2015) can together create a migratory threshold (Isermann and Lammerding, 2013, Swift and Discher, 2014). The linker of the nucleus and cytoskeleton (LINC) complex connects the cytoskeleton to the nuclear inner.