CHAPTER 3: CELL MIGRATION IN 3D ENVIRONMENTS: IMPLICATIONS FOR
3.3 Cellular Factors Affecting 3D Migration
Interstitial migration is a coordinated dance of adhesion, cytoskeletal dynamics, deformation of the cell body and its intracellular constituents, and/or matrix remodeling. The mode of migration is primarily dependent on the cell type. Leukocytes are ellipsoid, deformable, and move rapidly in an ‘amoeboid’ mode, which is characterized by transient adhesion and low contractility (Friedl et al. 2010). In contrast, spindle-shaped fibroblasts move in a slower ‘mesenchymal’ mode, which involves the formation of focal adhesions and contractile stress fibers. Single-cell migration is also different from collective migration, where cellular aggregates move in a coordinated stream or sheet, relying in part on their adhesion to one another to influence mobility (Friedl et al. 2010). Although the rest of this chapter will focus on single-cell ‘mesenchymal’ migration, the basic principles covered apply to all migrating cells.
3.3.1 Cell Adhesion and Mechanotransduction
Cell-matrix adhesion occurs when transmembrane receptors known as integrins engage with ECM components. Integrins are heterodimeric proteins that consist of α and β subunits, which bind to various ligands via specific peptide sequences (Walters et al. 2015). When integrins bind to their respective ligands, both structural and signaling molecules are recruited to the cell membrane to form focal adhesions that join with actin filaments to mechanically link the ECM and cytoskeleton. Focal adhesions anchor the cell to its substrate, enabling transmission of mechanical information as the actomyosin machinery contracts via the sliding of nonmuscle myosin II and actin filament stress fibers (Figure3-1A). This increase in cytoskeletal tension is transmitted back to the ECM to pull the cell forward. Force sensation by stress fibers can also feed back to alter the focal adhesions themselves, activating the RhoA/ROCK (Rho associated protein kinase) pathway to increase contractility (Petrie et al. 2012). Blocking integrin-mediated adhesion,
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as well as ROCK phosphorylation of its downstream effector, myosin light chain (MLC), reduces migration speed in a dose-dependent manner (Wolf et al. 2013) (Figure 3-1B and Figure3-1C).
Figure 3-1: Cell adhesion and contractility are required for 3D migration. (A) Mechanosensing matrix rigidity via the RhoA/ROCK/myosin II axis. Adapted from (Petrie et al. 2012). (B) Blocking cell adhesion via an anti-β1 integrin antibody (4B4, top) and contractility via the ROCK inhibitor Y-27632 (bottom) reduces cell migration speed in collagen gels. Cell-produced MMPs are inhibited in the GM6001 group. Adapted from (Wolf et al. 2013).
Several studies report that migration occurs most rapidly at an intermediate adhesive ligand density (Lautscham et al. 2015, Singh et al. 2014), similar to migration in 2D (Palecek et al. 1997). Interestingly, focal adhesion proteins that normally form aggregates on 2D surfaces (e.g., vinculin, paxillin, talin, α‑actinin) remain diffuse in cells moving within 3D environments. Fraley and colleagues found that, rather than producing a single prolonged protrusion, focal adhesions in 3D were associated with increased protrusive
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activity and matrix deformation, suggesting that cells actively engage with their environment before selecting a route for migration (Fraley et al. 2010).
3.3.2 Nuclear Mechanics
In confined passages, cells must physically deform to move forward (Figure 3-2A). The nucleus is considered the rate-limiting organelle in migration due to its large size and stiffness, which is 2
–
4 times higher than the surrounding cytoplasm (Guilak et al. 2000). When the nuclear cross-sectional area is >4 times the area of the constriction, cells stall at the channel entrance and migration speed significantly declines as they enter (Friedl et al. 2011, Lautscham et al. 2015) (Figure 3-2B). As the nucleus exits the constriction, its velocity increases due to the dissipation of stored elastic energy. Indeed, nuclear deformation of most cells is limited to a diameter of 3–4 μm (Wolf et al. 2013). This limitation is partly a function of the chromatin structure, such that higher degrees of chromatin condensation reduce nuclear plasticity and increase stiffness (Friedl et al. 2011, Pajerowski et al. 2007). Of equal importance are type V intermediate filament proteins called lamins that provide structure and stability to the nuclear envelope, with lamins A and C (lamin A/C) being the major contributors to nuclear mechanics (Lammerding et al. 2006). While cells with stiff nuclei display limited migratory capacity inside dense collagen gels, cells with compliant nuclei that lack lamin A/C, such as leukocytes and certain cancer cells, remain highly mobile (Friedl et al. 2011, Rowat et al. 2013, Wolf et al. 2013). Restoring nuclear plasticity by reducing lamin A/C enhances 3D migration through small pores (Greiner et al. 2014, Harada et al. 2014), whereas overexpressing lamin A reduces migratory capacity (Booth-Gauthier et al. 2013, Lautscham et al. 2015, Rowat et al. 2013) (Figure 3-2C–E). Since nuclear deformation also relies on actomyosin-generated contractility and nuclear-cytoskeletal force transmission (Driscoll et al. 2015), disrupting34
linkers of the nucleoskeleton to the cytoskeleton likewise reduces migration (Khatau et al. 2012). Alternatively, the nucleus may also act as a piston that physically compartmentalizes the cytoplasm, such that increasing hydrostatic pressure between the nucleus and the forward elements of the cell can act to push the cell forward (Petrie et al. 2014). It should be recognized that while the nucleus is a steric barrier to migration, depletion of lamin beyond certain levels can make them susceptible to stress-induced cell death, where the act of squeezing the nucleus through a small pore results in nuclear fragmentation (Harada et al. 2014).
Figure 3-2: Nuclear stiffness is a physical impediment to interstitial cell migration. (A) Schematic showing a cell migrating through a fibrous matrix, deforming the cell body and nucleus as it passes through a constriction. (B) Phases of nuclear deformation: (1) resistance, (2) local prolapse, (3) compression and gliding, and (4) rear release. Plot illustrating nuclear velocity over time for the phases above. Scale = 5 µm. Adapted from (Friedl et al. 2011). (C) Plot illustrating cell migration speed as a function of lamin A expression (Knockout (KO), Wild Type (WT), Overexpression (OE)). Migration efficiency is highest with a moderate level of lamin A KO. Adapted from (Swift et al. 2014). (D) Lamin A KO enhances cell migration through porous barriers. Adapted from (Greiner et al. 2013). (E) Lamin A OE reduces cell migration through microporous membranes. Adapted from (Rowat et al. 2013).
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3.3.3 Cell-Mediated Matrix Degradation
Interstitial migration through dense or impenetrable matrices is often aided by cell- produced matrix metalloproteinases (MMPs), which cleave ECM molecules at specific peptide sequences to generate a gap wide enough to allow for cell passage (Lu et al. 2011) (Figure 3-3). MMP expression is also mechanosensitive: it is enhanced on stiff substrates (Haage et al. 2014, Nukuda et al. 2015) via increased focal adhesion kinase (FAK) activity that is triggered by integrin clustering (Wu et al. 2005, Zhao et al. 2015). Cells can likewise remodel the matrix by contact-dependent, membrane-bound MMPs or by secretion of MMPs into the pericellular space (Friedl et al. 2000a). Cell surface binding of MMPs, which includes MMP-2, MMP-9, and membrane-type (MT)-MMPs, localizes proteolysis to the cell periphery, such that tube-like trails are generated behind the migrating cell (Friedl et al. 2009). Normal fibroblasts use this method, as do malignant neoplastic cells during metastatic invasion (Friedl et al. 2000a). In contrast, protease secretion results in a diffuse proteolysis that reduces biophysical matrix resistance at distances further than the cell membrane, acting to soften the tissue around pre-existing gaps in the ECM so as to make it easier to deform during passage. This method is commonly used in large-scale tissue remodeling events, such as morphogenesis and wound healing, although deregulated expression of matrix-degrading enzymes can result in collective tumor invasion (Lu et al. 2011). If cell-produced MMPs are inhibited, cells migrate (where they can) by adapting their shape to squeeze through pre-existing pores and by locally deforming the ECM network (Friedl et al. 2010), significantly reducing migration speed (Raeber et al. 2007, Wolf et al. 2013, Zaman et al. 2006). However, when pore dimensions are below the lower limit of nuclear deformation, cells become immobilized (Wolf et al. 2013).
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Figure 3-3: Stages of interstitial migration with or without matrix remodeling. (1) Initial cell polarization with rotation of the nucleus, (2) cellular adhesion of integrins, (3) cytoskeletal contraction of stress fibers (red), (4a) surface localization and secretion of matrix-degrading enzymes (green) to enlarge the matrix pores allowing for cell passage without deformation, and (4b) non-proteolytic migration requiring deformation of the cell and nucleus. Adapted from (Friedl et al. 2011).