Proteolytic interstitial cell migration: a five-step process
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- Friedl, P. & Wolf, K. Cancer Metastasis Rev (2009) 28: 129. doi:10.1007/s10555-008-9174-3
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Cell migration is a multi-step process that leads to the actin-driven translocation of cells on or through tissue substrate. Basic steps involved in cell migration have been defined for two-dimensional haptokinetic migration which, however, does not provide physical constraints imposed by three-dimensional interstitial tissues. We here describe the process of pericellular proteolysis that leads to extracellular matrix (ECM) degradation and realignment during cell movement and integrate it into established steps of cell migration. After actin-driven leading edge protrusion (step I) and anterior formation of integrin-mediated focal interactions to the substrate (step II), ECM breakdown is focalized towards physical ECM barriers several micrometer rearward of the leading edge (step III). Actomyosin-mediated cell contraction (step IV) then leads to rear-end retraction and forward sliding of cell body and nucleus so that a small tube-like matrix defect bordered by realigned ECM fibers becomes apparent (step V). Pericellular proteolysis is thus integral to the migration cycle and serves to widen ECM gaps and thereby lowers physical stress upon the cell body, which ultimately leads to aligned higher-oder ECM patterns.
KeywordsCancer metastasis Cell migration Proteases Integrins
Cell migration is fundamental to cell and tissue dynamics in morphogenesis, immune defence, repair as well as cancer invasion and metastasis. Basic concepts on cell migration as a physiochemical process have been established using isolated cells during their movement across ECM-coated two-dimensional (2D) surfaces . In the multicellular organism, cell migration across flat ECM substrate is present in epithelial migration i.e. during regeneration, such as epidermal wound healing , and the lateral movement of gut epithelium along the basement membrane . Most cells in the mammalian body, however, migrate within three-dimensional (3D) tissues, such as interstitial connective or neuronal tissue. 3D migration is carried out by different cell types using different shapes, kinetics and stringency of cell-cell interactions and, thus, shows significant diversity . In difference to migration across 2D substrate, flat and spread-out cell morphologies are prevented in 3D tissue whereas spherical shapes of varying length, ranging from short ellipsoid to elongated and spindle-shaped are maintained . Rather than forming a flat continuous leading lamella, the leading edge protruding into 3D tissue contains one or few cylindrical pseudopodia with terminal filopodia that dynamically engage with ECM fibers [6, 7, 8]. Whereas cells moving across 2D surfaces contain stress fibres that span between focal adhesions, cells migrating in 3D tissues lack stress fibres but form a cortical cytoskeleton. Thus, dependent on whether cell migration occurs in 2D or 3D environments, cell morphology and actin-rich protrusions that engage with the ECM vary considerably.
2 Physics of 3D cell migration and proteolytic ECM fiber cleavage
Whereas the basis of actmyosin-mediated protrusion and contractility is common to all migration types, their biophysical principles diverge. The prototypic mode of stromal cell migration is derived from the mesenchymal migration of fibroblasts or fibroblast-like cells . Hallmarks of mesenchymal migration in 3D ECM are spindle-shaped morphology and relatively slow migration (0.1 to 1 µm/min) as well as focalized cell-matrix interactions towards the ECM scaffold that mediate high traction forces and the capability to proteolytically remodel the ECM structure during migration [5, 6, 7]. Besides in fibroblasts, mesenchymal migration is found in smooth muscle cells, pericytes, activated macrophages, neural crest cells, and many dedifferentiated cancer cells, including fibrosarcoma or breast cancer cells.
3 Integrating proteolysis into the migration cycle—the 5-step model
4 Spatial control of pericellular proteolysis at the leading edge
Adhesion and proteolysis are principally countercurrent processes because uncontrolled ECM degradation at or very nearby adhesion sites would compromise force generation due to the premature loss of ECM scaffold. As consequence, the cell would be surrounded by ECM fragments rather than a solid tissue structure, detach, and float in a “sea” of soluble ligand. This worst-case scenario is prevented by separating the anterior tip region from the proteolytic action, which in turn is apparent further rearward where litte or no traction force needs to be generated. Thus, adhesion and force generation, and pericellular proteolysis are executed in different subcellular regions, thereby sparing the leading edge from premature loss of substrate (Fig. 3B). The mechanisms that segregate these two zones are unknown. Likely they reside in local signalling control of proteolytic action but not in alterations of the composition of the lytic adhesion structure, because the main structural components F-actin, ß1 integrins and surface-localized MMPs are equally focalized to substrate contact points in front, mid and rear regions of the cell [9, 16]. Further, outside pressure towards the cell body, the duration of fiber contact to the cell surface and the amount of active protease present in ECM-binding regions may be rate-limiting. Thus, fibers that either press against the cell body and/ or fibers that remain long enough in contact to the cell surface are most likely those that undergo proteolytic degradation.
5 Temporal control of pericellular proteolysis
The proteolytic action at the cell surface during migration is a stereotypic multi-step process (Fig. 3C). First, ECM polymer needs to physically touch the cell surface, either loosely or as compression zone which causes inward pressure upon the cell body (Fig. 3C, step 1). These very specific regions then undergo proteolytic action resulting ultimately in the focal cleavage of ECM fibers and the generation of two loose ends (Fig. 3C, step 2). As the cell moves, blunt fiber ends that continue to touch the cell surface become transported and aligned along the length axis of the cell (Fig. 3C, step 3). Possibly by secondary remodeling, such as secretion of additional ECM components, realigned fibers remain stable in their new position lateral to the cell body which becomes apparent as the rear end has released the newly formed matrix defect (Fig. 3C, step 4). The ECM defect then consists of a hollow tube the size of which approximately corresponds to the diameter of the cell. Thus, migration-associated pericellular proteolysis consists of individual biochemical and biophysical steps that ultimately lead to an oriented, compacted ECM scaffold that borders a quite linear track.
6 Consequences of proteolytic ECM remodeling
7 Deviations from the rule
This five-step model of proteolytic migration forms an integrated program of slow migration designed to remodel tissue structure, and only mesenchymal cells are known to execute the complete 5 step-cycle. Cell migration, however, is a diverse process and different cell types employ different mechanisms, rendering the proteolytic step as optional, rather than indispensable. Cell types that do not execute step III are either cells that constitutively move without protease function, or cells after pharmacological abrogation of protease activity [18, 23].
In contrast to the here described mesenchymal migration mode, leukocytes are rapidly moving cells that employ amoeboid movement characterized by a short, ellipsoid shape. They preferentially travel through loose interstitial tissue at 10 to 40 fold higher speed than mesenchymal cells, lack focalized adhesion structures to the substrate and do not execute pericellular proteolysis while migrating [19, 20]. Instead of removing ECM barriers by proteolytic cleavage, they glide through narrow regions by flow of the cytoplasm and squeezing of the nucleus which is supported by actomyosin contraction in the cell rear [21, 22]. The zone of usually strong cell deformation through narrow tissue is a hallmark of amoeboid movement, termed constriction ring or compression zones [20, 23].
In reminiscence of leukocytes, mesenchymal cells that loose their ability to cleave ECM, can convert to non-proteolytic migration and overcome matrix barriers by squeezing through rather than degrading ECM. After treatment with protease inhibitors or RNA knockdown of MT1-MMP/MMP-14, the rate-limiting collagenase towards degradation of fibrillar collagen, invading fibrosarcoma or breast cancer cells continue to move by an amoeboid mechanism and squeeze through ECM gaps and trails, similar to leukocytes [9, 23]. This process, termed mesenchymal-amoeboid transition, secures migration without the proteolytic step. Thus, pericellular proteolysis and ECM remodeling are secondary to cell migration and facilitate forward gliding in a persistent manner along linear tracks, yet alternative, nonproteolytic shape change and amoeboid squeezing secure a less peristent migration mode that follows preformed rather than newly remodeled tissue structures.
The proteolytic step is facultative in the migration cycle, dependent on the type of cell and ECM substrate. When present in a regulated manner, proteolysis prevents the cell from physical stress, retains the nucleus in round to ellipsoid state and provides a physical mechanism to align tissue structures. It further serves to establish trails of least resistance that are used by other cells for track guidance. Proteolysis is therefore a step that adds complexity to the cell migration process enabling cells to shape tissues by introducing ordered structure. On the other hand, cells that lack step III either transmigrate narrow ECM gaps by non-proteolytic strategies, such as by changing the cell morphology, or circummigrate this region by retracting the leading edge and deviating from the previous path. In conclusion, despite their clear functions in track generation and tissue remodeling, proteases can be dispensible for migration if the cells passage through gaps and pores that are large enough to accommodate the nucleus and as long as the nucleus is able to deform. It will be important to dissect different cell types and ECM environments for the presence, necessity and consequences of proteolytic tissue remodeling.
We gratefully acknowledge Dr. H. Lü for sample preparation and image acquisition. The work was supported by DFG grant FR1155/8-2 and EU a grant within the European Network of Excellence of Molecular Imaging Laboratories (EMIL–LSHC-CT–2004-503569) to PF.
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