Cell Migration——An Overview

vol.294 Guan J.-L. (ed.) Cell Migration-Developmental Methods and Protocols Chapter 1

Summary

Cell migration is an essential process for normal development and homeostasis thatcan also contribute to important pathologies. Not surprisingly, there is considerableinterest in understanding migration on a molecular level, but this is a difficult task. However,technologies are rapidly emerging to address the major intellectual challengesassociated with migration. In this chapter, we outline the basics of cell migration with anemphasis on the diverse systems, methodologies, and techniques described in this book.

From the contributions presented, it is apparent that the next few years should producemajor advances in our understanding of cell migration.

Key Words: Cell migration; adhesion; signaling; protrusion; development.

1. Introduction

Cell migration is a complex process that is essential for embryonic developmentand homeostasis (1,2). In gastrulation, migration is particularly robust,where essentially all cells migrate as sheets to form the three layers, includingendoderm, ectoderm, and mesoderm that comprise the resulting embryo. Cellswithin these layers migrate to target locations throughout the developingembryo, where they differentiate and form various tissues and organs. Themigration of cells from epithelial layers to their targets is a general phenomenonthat occurs throughout development. In the developing cerebellum, neuronalprecursor cells migrate from the epithelium to their residences in distinctlayers. One special form of migration during development is the extension ofneurites. The tip of a developing neurite, the growth cone, shares many similaritieswith a migrating cell. The precise guidance and target recognition of growth cones are central to the establishment of the neuronal network and thuscognitive functions.

Migration is not limited to development, but occurs in the adult, where it iscentral to both normal and pathological states. For example, the migration ofprecursor cells from the basal layer to the epidermis functions to continuouslyrenew skin. Other homeostatic processes, including wound repair and mountingan effective immune response, also require migration. Leukocyte migrationfrom the circulation into the surrounding tissue, where they ingestbacteria, is important for mounting an immune response. Migration also cancontribute to some pathological processes, such as vascular disease, chronicinflammatory diseases, and tumor formation and metastasis. For example, tumorformation is accompanied by the construction of a new vascular network,which involves migration of the endothelial cells from pre-existing blood vesselsinto the tumor, where they proliferate and form the new vessels (angiogenesis).

Migration also occurs during metastasis when some tumor cellsmigrate out of the initial tumor into the circulation and move to new locations,where they form a secondary tumor. Because the invasion of tumor cells fromthe primary site into the surrounding area and angiogenesis is essential fortumor development, assays have been developed to study these processes. InChapter 9, Shaw outlines an assay for examining the invasion of tumor cellsthrough Matrigel. Assays to dissect the signaling events involved inendothelial cell migration and model systems for angiogenesis are describedin Chapters 10, 11, and 19.

Although many studies have examined cell migration in vertebrates, migrationis equally important in invertebrates, plants, and some single-cell organisms.

For example, during development of Caenorhabditis elegans, cellsmigrate within the embryo along defined trajectories. Each cell stops to divide,and the daughter cells continue to migrate. Because of the small size and thetransparency of C. elegans, individual cells can be followed as they migrate inliving embryos, making it a simple system for studying migration. In Chapter13, Shakir and Lundquist describe the methods for analyzing migration inC. elegans. Another invertebrate model system is the fruit fly, Drosophilamelanogaster. Drosophila has a more complicated body plan and therefore hasincreased complexity in its migration patterns during development and adultlife. One example is primordial germ cells, which migrate through the midgutepithelium and attach to the mesoderm, where they associate with the gonadalprecursors and eventually form a gonad on either side of the embryo. Chapters14 and 15 outline protocols for studying migration of different cell types inDrosophila and live imaging of Drosophila embryos. The information gainedfrom studying invertebrate cell migration can be very useful for understandingmigration in more complex organisms, as confirmed by the high degree of homology between many invertebrate and vertebrate gene products that areinvolved in migration.

2. The Migratory Cycle

Migration can be thought of as a cyclical process. It begins when a cellresponds to an external signal by polarizing and extending a protrusion in thedirection of movement. The formation of adhesion complexes functions tostabilize the protrusion by attaching it to the substratum on which the cell ismigrating. These adhesions, which serve as traction points for migration, initiatesignals that regulate adhesion dynamics and protrusion formation (3).

Contraction then moves the cell body forward and release of the attachmentsat the rear, as the cell retracts, completes the cycle. Slow-moving cells, suchas fibroblasts, show these distinct steps of migration, but they are less obviousin other cell types. For example, rapidly migrating cells, such as keratocytesand leukocytes, glide over the substratum by protruding and retractingsmoothly without forming obvious attachments.

2.1. Polarization

Many different molecules serve as external agents that initiate and promotemigration. For example, some molecules initiate a migratory phenotype(chemokinetic) whereas others reside in soluble (chemotactic)- or substrate(haptotactic)-associated gradients and lead to directed movement. These moleculesand their receptors are well studied in leukocytes. These cells can sensethe presence of even a shallow gradient, in which they polarize and migratepersistently in one direction (4). Their persistent polarity is apparent whenthe cells sense changing chemotactic gradients and the entire cell turns ratherthan extending a new protrusion from another region. In contrast, fibroblastsare more plastic and can extend protrusions from any position in the cell asthey change directions. Analysis of directional cell migration using theBoyden chamber and the Dunn chemotaxis chamber are discussed in Chapters2 and 4. In addition, Chapter 24 outlines a method for generating gradientsfor studying directional cell migration.

2.2. Adhesion Complexes

Adhesion complexes, which are sites of attachment between the cell and theextracellular matrix (ECM), are composed of a number of proteins, includingthe integrin family of transmembrane receptors, kinases, adaptor and structuralmolecules (3). Integrins serve as the functional connection between the ECMand the actin cytoskeleton. The small GTPase, Rac, induces the formation ofsmall adhesions at the leading edge (5–7). These adhesions serve as traction points and transmit strong propulsive forces that move the cell body forward(8). The maturation of these small adhesions into larger, more organized structuresactually inhibits migration (3,9). In Chapter 5, Kucik and Wu presentprotocols for analyzing cell adhesion under static conditions and shear stress,and in Chapter 6, Berrier and LaFlamme provide a method to quantify cellspreading, which is an important aspect of cell adhesion. Finally, in Chapter21, Drees et al. describe methods for analyzing protein complexes at the cellmembrane after cell–cell and cell–ECM adhesion.

Because tyrosine phosphorylation of adhesion components is thought toregulate their dynamics, there is great interest in studying this process. Thiscan be accomplished with a “phosphotyrosine reporter” in which yellow fluorescentprotein (YFP) is fused to two phosphotyrosine-binding Src-homology2-domains derived from c-Src (10). Quantitative fluorescent microscopywith this reporter has been used to study the kinetics of tyrosine phosphorylationin adhesions. Details of this method are provided in Chapter 20.

2.3. Moving Forward and Trailing Behind

Actin–myosin contractility at the front of the cell serves to pull the cell bodyforward in the direction of movement. Release of adhesions at the cell rear andretraction of the tail are also mediated by myosin. Spatial and temporal regulationof Rho GTPases controls these processes through effectors, such as Rhokinase, that regulate actomyosin contractility. Rho kinase has been implicatedin release of adhesions at the cell rear through regulation of myosin II (11).

Other molecules implicated in the release of adhesions include the proteasecalpain and a phosphatase, calcineurin (12,13). Microtubules also function inthe regulation of adhesion disassembly, probably through the modulation ofRac activity (14).

3. Modes of Migration

3.1. Single-Cell Migration

As discussed previously, the migration of individual cells requires theasymmetrical organization of cellular activities. In culture, many differentcell types can become polarized with a front and rear asymmetry, but this isusually transient and results in random migration. The stabilized, directionalmovement of cells requires external cues. In tissue, these external cues aretypically provided by the surrounding environment. The external cues activateintracellular signaling pathways that control polarization and directedcell movement. The small GTPases, Rac, and Cdc42, play a prominent rolein regulating this process (5,6).

3.2. Monolayer Cell Migration

During embryogenesis, many cells do not migrate as single cells but rather assheets or loosely associated clusters. In vitro, scratching or wounding a cellmonolayer induces the synchronized movement of sheets of cells. In Chapter 3,Rodriguez et al. outline a protocol for performing a wound assay to study directionalmigration. As with single cells, the migrating sheets detect the direction ofmigration and polarize with protrusive activity constrained to the front. Interactionswith their neighbors can provide additional directional cues to cells in amonolayer. Like single cell movement, the migration of cell monolayers is regulatedby the Rho GTPases (15). Rac and CDC42 are essential for the polarizationand migration of the cell monolayers.

Some tumor cells also adopt this mode of migration. Primary melanomaexplants migrate in collagen gels as multicellular clusters that are polarizedwith a clearly defined leading edge at the front of the clusters and a trailingedge at the rear (16). The invasive movement of the multicellular clusters, likesingle cell migration, is integrin dependent. Inhibition of β1 integrin functionproduces dramatic phenotypic changes in the migration, such as dispersion ofthe clusters. The cells respond to this by converting to a single cell mode ofmigration. The dispersion of clusters of cells can be measured with a scatterassay. In Chapter 7, Chen describes an assay for studying the scatter responseof epithelial cells to stimulation with growth factors.

4. Cell Migration In Vivo

Much of our knowledge regarding migration has been obtained from cellsgrowing on flat surfaces, such as cover slips or tissue culture dishes. Onlyrecently, have studies begun to examine migration in environments, whichmore closely mimic that observed in vivo, such as three-dimensional (3-D)matrices and slice cultures. Migration modes and cell morphologies in 3-Denvironments can differ significantly from those observed with dissociatedcells migrating on planar substrates (17,18). One approach is to put culturedcells between two layers of flexible polyacrylamide substrata, which createsan environment that more closely resembles tissue (19). In another approach,when fibroblasts are placed in 3-D matrices derived from tissues or cell culture,enhanced adhesion and migration are observed compared with cellsplated on 2-D substrates (17). In the 3-D matrix, adhesions are very long andslender in contrast to the oval-shaped focal adhesions found in cultured cells.

Chapter 8 by Cukierman describes a method for measuring rates and directionalityof fibroblasts in 3-D matrices. In slice culture, myogenic precursorcells extend long, highly polarized, persistent protrusions that are not usuallyseen in fibroblasts migrating on flat, rigid substrates (18). However, like cultured cells, the formation of these exaggerated protrusions is regulated bylocal activation of the small GTPase, Rac.

Neural crest cells provide another system for studying migration in vivo.

Chapter 18 outlines methods for studying neural crest migration in chickembryos. During development, the neural tube forms by rolling up and pinchingoff from the future epidermis. Along the “crest” where the neural tubepinches off, a number of cells break loose and migrate individually alongdefined pathways. These neural crest cells eventually form most of the peripheralnervous system as well as other cells and organs. As neural crest cellsbreak away from the neural tube, they undergo an epithelial-mesenchymaltransition. This process is similar to what occurs when cancer cells escapefrom the primary tumor and enter the circulation. Therefore, understandingthis process in vivo can provide tremendous insight into the mechanisms ofcancer metastasis.

In rodents, the migration of neuronal precursor cells from the subventricularzone (SVZ) to the olfactory bulb is particularly robust and directional.

This restricted pathway is known as the rostral migratory stream(RMS). The RMS provides a model system for studying neuronal migrationusing explants from the forebrain of postnatal rats. Detailed methods for theSVZ migration assay in 3-D gels are provided in Chapter 12 by Ward andRao. They also provide methods for introducing cDNAs into SVZ neuronsand live-cell imaging of neuronal migration. Using this system, Rao and colleaguesfound that the migration of these neuronal precursors was repelledby the secreted protein Slit and attracted by a novel chemoattractant (20,21).

In another study using slice cultures from the forebrain, the migrating neuronalprecursors, which were visualized with DiI crystals, were observed toextend a single, long, persistent leading protrusion in the direction of movementand migrate along a defined pathway (22). Both integrins and the netrin-1 receptor, deleted in colorectal carcinoma (DCC), play a role in RMSmigration. DCC is involved in directional migration of the neural precursorswhereas integrin ligation is essential for any movement of these cells.

Finally, migration can be studied in vivo using zebrafish embryos. Duringgastrulation, cell migration is more robust than at any other stages of life.

The zebrafish is an ideal system for studying this process because of the transparencyof the embryos, the availability of the mutants and the ability of theembryos to develop externally. In Chapter 16, Sepich and Solnica-Krezelprovide some background and protocols for the analyses of migration duringgastrulation. They also present data collected from wild-type and mutantsshowing morphogenetic defects. Gastrulation cell movements can be similarlystudied in Xenopus embryos. In Chapter 17, DeSimone et al. describemethods for examining cell migration in Xenopus embryos.

5. Imaging Migrating Cells

Cell polarization and migration require the coordinated regulation of signalingand adaptor molecules at specific locations within the cell. This requires thedevelopment of probes to measure the activation of these key regulatory moleculesin living cells with high temporal and spatial resolution. GFP-taggedsensors that bind to the activated molecules can be used to reveal their locationsand monitor changes in their activation over time (23). However, the useof these GFP-tagged sensors is limited because of high background levels andlow affinities for the activated molecules. Therefore, probes have been generatedto monitor the direct interaction between a regulatory molecule and abinding domain from one of its effectors by fluorescence energy transfer(FRET; ref. 24). In Chapter 22, Ballestrem and Geiger outline the use of FRETfor studying dynamic interactions in focal adhesions and provide a protocolfor FRET measurements in living cells. To generate an effective FRET signal,the protein levels of the regulatory molecule and its effector need to becomparable. To circumvent this problem, biosensors, in which the fluorescencederivatives of the regulatory molecule and binding domain are encodedon a single cDNA, have been developed.

Recent technical advances have allowed investigators to monitor cellbehavior in living animals. Multiphoton microscopy is particularly wellsuitedfor in vivo studies because it provides a way of imaging deeply intoliving tissues without the contribution of out-of-focus light. In Chapter 23,Flesken-Nikitin et al. describe the use of multiphoton microscopy to studycell migration in living mice. They track the migration of individual cells bylabeling them with enhanced GFP. This provides a powerful method thatmight eventually be used to study migration in vivo on a molecular level.

6. Conclusions

Cell migration is an important process that takes place throughout life.

Because of its central importance, migration has become the focus of muchresearch. However, since migration requires the coordinated activity of severalindividual component processes, the mechanisms by which these componentprocesses are integrated present a major challenge. Fortunately,technologies and methodologies are emerging that allow us to study the spatialand temporal regulation that generates the coordination of these processessome of which are described in this book. For example, FRET biosensors,which allow spatially resolved assays of signaling events in real time in livingcells, and the development of 3-D systems that allow imaging of cellularand molecular dynamics under in vivo-like conditions. A rapidly emergingtheme is that migration in vivo differs from that studied in vitro, which may reflect different signaling mechanisms or cellular mechanics. With the technologiesand methodologies in place the next few years should provide awealth of information in the area of migration.

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