Protrusion Fluctuations Direct Cell Motion

Many physiological phenomena involve directional cell migration. It is usually attributed to chemical gradients in vivo. Recently, other cues have been shown to guide cells in vitro, including stiffness/adhesion gradients or micropatterned adhesive motifs. However, the cellular mechanism leading to these biased migrations remains unknown, and, often, even the direction of motion is unpredictable. In this study, we show the key role of fluctuating protrusions on ratchet-like structures in driving NIH3T3 cell migration. We identified the concept of efficient protrusion and an associated direction index. Our analysis of the protrusion statistics facilitated the quantitative prediction of cell trajectories in all investigated conditions. We varied the external cues by changing the adhesive patterns. We also modified the internal cues using drug treatments, which modified the protrusion activity. Stochasticity affects the short- and long-term steps. We developed a theoretical model showing that an asymmetry in the protrusion fluctuations is sufficient for predicting all measures associated with the long-term motion, which can be described as a biased persistent random walk.     

Protrusion Fluctuations Direct Cell Motion

Many physiological phenomena involve directional cell migration. It is usually attributed to chemical gradients in vivo. Recently, other cues have been shown to guide cells in vitro, including stiffness/adhesion gradients or micropatterned adhesive motifs. However, the cellular mechanism leading to these biased migrations remains unknown, and, often, even the direction of motion is unpredictable. In this study, we show the key role of fluctuating protrusions on ratchet-like structures in driving NIH3T3 cell migration. We identified the concept of efficient protrusion and an associated direction index. Our analysis of the protrusion statistics facilitated the quantitative prediction of cell trajectories in all investigated conditions. We varied the external cues by changing the adhesive patterns. We also modified the internal cues using drug treatments, which modified the protrusion activity. Stochasticity affects the short- and long-term steps. We developed a theoretical model showing that an asymmetry in the protrusion fluctuations is sufficient for predicting all measures associated with the long-term motion, which can be described as a biased persistent random walk.     

Netrin, Slit and Wnt receptors allow axons to choose the axis of migration

One of the challenges to understanding nervous system development has been to establish how a fairly limited number of axon guidance cues can set up the patterning of very complex nervous systems. Studies on organisms with relatively simple nervous systems such as Drosophila melanogaster and C. elegans have provided many insights into axon guidance mechanisms. The axons of many neurons migrate along both the dorsal-ventral (DV) and the anterior-posterior (AP) axes at different phases of development, and in addition they may also cross the midline. Axon migration in the dorsal-ventral (DV) direction is mainly controlled by Netrins with their receptors; UNC-40/DCC and UNC-5, and the Slits with their receptors; Robo/SAX-3. Axon guidance in the anterior-posterior (AP) axis is mainly controlled by Wnts with their receptors; the Frizzleds/Fz. An individual axon may be subjected to opposing attractive and repulsive forces coming from opposite sides in the same axis but there may also be opposing cues in the other axis of migration. All the information from the cues has to be integrated within the growth cone at the leading edge of the migrating axon to elicit a response. Recent studies have provided insight into how this is achieved.Evidence suggests that the axis of axon migration is determined by the manner in which Netrin, Slit and Wnt receptors are polarized (localized) within the neuron prior to axon outgrowth. The same molecules are involved in both axon outgrowth and axon guidance, for at least some neurons in C. elegans, whether the cue is the attractive cue UNC-6/Netrin working though UNC-40/DCC or the repulsive cue SLT-1/Slit working though the receptor SAX-3/Robo (Adler et al., 2006, Chang et al., 2006, Quinn et al., 2006, 2008). The molecules involved in cell signaling in this case are polarized within the cell body of the neuron before process outgrowth and direct the axon outgrowth. Expression of the Netrin receptor UNC-40/DCC or the Slit receptor SAX-3/Robo in axons that normally migrate in the AP direction causes neuronal polarity reversal in a Netrin and Slit independent manner (Levy-Strumpf and Culotti 2007, Watari-Goshima et al., 2007). Localization of the receptors in this case is caused by the kinesin-related VAB-8L which appears to govern the site of axon outgrowth in these neurons by causing receptor localization. Therefore, asymmetric localization of axon guidance receptors is followed by axon outgrowth in vivo using the receptor's normal cue, either attractive, repulsive or unknown cues.     

Plexins, semaphorins, and scatter factor receptors: a common root for cell guidance signals?

Semaphorins, the plexin family of semaphorin receptors, and scatter factor receptors share evolutionarily conserved protein modules, such as the semaphorin domain and Met Related Sequences (MRS). All these proteins also have in common a role in mediating cell guidance cues. During development, scatter factor receptors control cell migration, epithelial tubulogenesis, and neurite extension. Semaphorins and their receptors are known signals for axon guidance; they are also suspected to regulate developmental processes involving cell migration and morphogenesis, and have been implicated in immune function and tumor progression. Scatter factors and secreted semaphorins are diffusible ligands, whereas membrane-bound semaphorins signal by cell-cell interaction. Cell guidance control by semaphorins requires plexins, alone or in a receptor complex with neuropilins. Semaphorins, besides their role in axon guidance, are expected to have multiple functions in morphogenesis and tissue remodeling by mediating cell-repelling cues through plexin receptors.     

Substrate-cytoskeletal coupling as a mechanism for the regulation of growth cone motility and guidance

Growth cones are highly motile structures at the end of neuronal processes, capable of receiving multiple types of guidance cues and transducing them into directed axonal growth. Thus, to guide the axon toward the appropriate target cell, the growth cone carries out different functions: it acts as a sensor, signal transducer, and motility device. An increasing number of molecular components that mediate axon guidance have been characterized over the past years. The vast majority of these molecules include proteins that act as guidance cues and their respective receptors. In addition, more and more signaling and cytoskeleton-associated proteins have been localized to the growth cone. Furthermore, it has become evident that growth cone motility and guidance depends on a dynamic cytoskeleton that is regulated by incoming guidance information. Current and future research in the growth cone field will be focussed on how different guidance cues transmit their signals to the cytoskeleton and change its dynamic properties to affect the rate and direction of growth cone movement. In this review, we discuss recent evidence that cell adhesion molecules can regulate growth cone motility and guidance by a mechanism of substrate-cytoskeletal coupling.     

Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho

Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance [1-3]. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance [4-7]. Using yeast two-hybrid screening and in vitro interaction assays, we show that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induced similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. Our findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration.     

Regulation of chemotactic networks by 'atypical' receptors

Directed cell migration is a fundamental component of numerous biological systems and is critical to the pathology of many diseases. Although the importance of secreted chemoattractant factors in providing navigational cues to migrating cells bearing specific chemoattractant receptors is now well-established, how the function of these factors is regulated is not so well understood and may be of key importance to the design of new therapeutics for numerous human diseases. While regulation of migration clearly takes place on a number of different levels, it is becoming clear that so-called 'atypical' receptors play a role in scavenging, or altering the localisation of, chemoattractant molecules such as chemokines and complement components. These receptors do this through binding and/or internalising their chemoattractant ligands without activating signal transduction cascades leading to cell migration. The atypical chemokine receptor family currently comprises the receptors D6, DARC and CCX-CKR. In this review, we discuss the evidence from in vitro and in vivo studies that these receptors play a role in regulating cell migration, and speculate that other orphan receptors may also belong to this family. Furthermore, with the advent of gene therapy on the horizon, the therapeutic potential of these receptors in human disease is also considered.     

Localized Lipid Packing of Transmembrane Domains Impedes Integrin Clustering

Integrin clustering plays a pivotal role in a host of cell functions. Hetero-dimeric integrin adhesion receptors regulate cell migration, survival, and differentiation by communicating signals bidirectionally across the plasma membrane. Thus far, crystallographic structures of integrin components are solved only separately, and for some integrin types. Also, the sequence of interactions that leads to signal transduction remains ambiguous. Particularly, it remains controversial whether the homo-dimerization of integrin transmembrane domains occurs following the integrin activation (i.e. when integrin ectodomain is stretched out) or if it regulates integrin clustering. This study employs molecular dynamics modeling approaches to address these questions in molecular details and sheds light on the crucial effect of the plasma membrane. Conducting a normal mode analysis of the intact αllbβ3 integrin, it is demonstrated that the ectodomain and transmembrane-cytoplasmic domains are connected via a membrane-proximal hinge region, thus merely transmembrane-cytoplasmic domains are modeled. By measuring the free energy change and force required to form integrin homo-oligomers, this study suggests that the β-subunit homo-oligomerization potentially regulates integrin clustering, as opposed to α-subunit, which appears to be a poor regulator for the clustering process. If α-subunits are to regulate the clustering they should overcome a high-energy barrier formed by a stable lipid pack around them. Finally, an outside-in activation-clustering scenario is speculated, explaining how further loading the already-active integrin affects its homo-oligomerization so that focal adhesions grow in size.     

Preimplantation expression of the somatic form of Dnmt1 suggests a role in the inheritance of genomic imprints

Background

Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors.

Results

We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally.

Conclusion

In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.     

Biophysical Induction of Vascular Smooth Muscle Cell Podosomes

Vascular smooth muscle cell (VSMC) migration and matrix degradation occurs with intimal hyperplasia associated with atherosclerosis, vascular injury, and restenosis. One proposed mechanism by which VSMCs degrade matrix is through the use of podosomes, transient actin-based structures that are thought to play a role in extracellular matrix degradation by creating localized sites of matrix metalloproteinase (MMP) secretion. To date, podosomes in VSMCs have largely been studied by stimulating cells with phorbol esters, such as phorbol 12,13-dibutyrate (PDBu), however little is known about the physiological cues that drive podosome formation. We present the first evidence that physiological, physical stimuli mimicking cues present within the microenvironment of diseased arteries can induce podosome formation in VSMCs. Both microtopographical cues and imposed pressure mimicking stage II hypertension induce podosome formation in A7R5 rat aortic smooth muscle cells. Moreover, wounding using a scratch assay induces podosomes at the leading edge of VSMCs. Notably the effect of each of these biophysical stimuli on podosome stimulation can be inhibited using a Src inhibitor. Together, these data indicate that physical cues can induce podosome formation in VSMCs.     

A calcium dependent de-adhesion mechanism regulates the direction and rate of cell migration: a mathematical model

Cell migration has long been studied by a variety of techniques and many proteins have been implicated in its regulation. Integrins, key proteins that link the cell to the extracellular matrix, are central to adhesion complexes whose turnover defines the rate of cell locomotion. The formation and disassembly of these adhesions is regulated by both intracellular and extracellular factors. In this study we have focused on the Ca2+-dependent protein network (module) that disassembles the adhesion complexes. We have developed a mathematical model that includes the Ca2+-dependent enzymes micro-calpain and phospholipase C (PLC) as well as IP3 receptors and stretch activated Ca2+ channels, all of which have been reported to regulate migration. The model also considers the spatial effects of Ca2+ propagation into lamella. Our model predicts differential activation of calpain at the leading and trailing edges of the cell. Since disassembly of integrin adhesive contacts is proportional to the degree of calpain activation, this leads to cell migration in a preferred direction. We show how the dynamics of Ca2+ spiking affects calpain activation and thus changes the disassembly rate of adhesions. The spiking is controlled by PLC activity and currents through stretch-activated Ca2+ channels. Our model thus combines the effects of various molecular factors and leads to a consistent explanation of the regulation of the rate and direction of cell migration.     

Characterization of integrin-tetraspanin adhesion complexes: role of tetraspanins in integrin signaling.

Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of alpha3beta1-TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of alpha3beta1-TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B alpha3beta1-TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility.     

Relationships between glycosaminoglycan and receptor binding sites in chemokines-the CXCL12 example

Chemokines are small proteins, promoting directional migration and activation of different cells through binding to specific receptors. Most chemokines also bind to heparan sulfate (HS), a family of complex and highly sulfated glycosaminoglycan (GAG) found at the cell surface and in the extracellular matrix. This class of molecules has recently emerged as critical regulators of many events involving cell response to the external environment. Binding to HS is thought to be functionally important. Current models suggested that HS ensures the correct positioning of chemokines within tissues and maintains haptotactic gradients of the proteins along cell surfaces, thus providing directional cues for migrating cells. On the chemokine surface, the GAG binding epitopes can be displayed on different areas, some of which overlap the receptor binding domain, while others are clearly separated. We review here some structural aspects of the interaction between GAGs or receptors and chemokines. In particular, we will address the case of CXCL12, a chemokine whose receptor binding site is distinct from the GAG binding site and whose different isoforms display different GAG binding abilities. This chemokine system thus offers an unprecedented opportunity to ascertain the importance of chemokine/GAG interaction in the regulation of cell migration.     

Orientation Cues for High-Flying Nocturnal Insect Migrants: Do Turbulence-Induced Temperature and Velocity Fluctuations Indicate the Mean Wind Flow?

Migratory insects flying at high altitude at night often show a degree of common alignment, sometimes with quite small angular dispersions around the mean. The observed orientation directions are often close to the downwind direction and this would seemingly be adaptive in that large insects could add their self-propelled speed to the wind speed, thus maximising their displacement in a given time. There are increasing indications that high-altitude orientation may be maintained by some intrinsic property of the wind rather than by visual perception of relative ground movement. Therefore, we first examined whether migrating insects could deduce the mean wind direction from the turbulent fluctuations in temperature. Within the atmospheric boundary-layer, temperature records show characteristic ramp-cliff structures, and insects flying downwind would move through these ramps whilst those flying crosswind would not. However, analysis of vertical-looking radar data on the common orientations of nocturnally migrating insects in the UK produced no evidence that the migrants actually use temperature ramps as orientation cues. This suggests that insects rely on turbulent velocity and acceleration cues, and refocuses attention on how these can be detected, especially as small-scale turbulence is usually held to be directionally invariant (isotropic). In the second part of the paper we present a theoretical analysis and simulations showing that velocity fluctuations and accelerations felt by an insect are predicted to be anisotropic even when the small-scale turbulence (measured at a fixed point or along the trajectory of a fluid-particle) is isotropic. Our results thus provide further evidence that insects do indeed use turbulent velocity and acceleration cues as indicators of the mean wind direction.     

Cross-talk between calcium and protein kinase A in the regulation of cell migration

Calcium (Ca(2+)) and the cAMP-dependent protein kinase (PKA) are pleiotropic cellular regulators and both exert powerful, diverse effects on cytoskeletal dynamics, cell adhesion, and cell migration. Localization, by A-kinase-anchoring proteins (AKAPs), of PKA activity to the protrusive leading edge, integrins, and other regulators of cytoskeletal dynamics has emerged as an important facet of its role in cell migration. Additional recent work has firmly established the importance of Ca(2+) influx through mechanosensitive transient receptor potential (TRP) channels and through store-operated Ca(2+) entry (SOCE) in cell migration. Finally, there is considerable evidence showing that these mechanisms of Ca(2+) influx and PKA regulation intersect--and often interact--and thus may work in concert to translate complex extracellular cues into the intracellular biochemical anisotropy required for directional cell migration.     

Cooperativity between integrin activation and mechanical stress leads to integrin clustering

Integrins are transmembrane receptors involved in crucial cellular biological functions such as migration, adhesion, and spreading. Upon the modulation of integrin affinity toward their extracellular ligands by cytoplasmic proteins (inside-out signaling) these receptors bind to their ligands and cluster into nascent adhesions. This clustering results in the increase in the mechanical linkage among the cell and substratum, cytoskeleton rearrangements, and further outside-in signaling. Based on experimental observations of the distribution of focal adhesions in cells attached to micropatterned surfaces, we introduce a physical model relying on experimental numerical constants determined in the literature. In this model, allosteric integrin activation works in synergy with the stress build by adhesion and the membrane rigidity to allow the clustering to nascent adhesions independently of actin but dependent on the integrin diffusion onto adhesive surfaces. The initial clustering could provide a template to the mature adhesive structures. Predictions of our model for the organization of focal adhesions are discussed in comparison with experiments using adhesive protein microarrays.     

Cells on the move: a dialogue between polarization and motility

Throughout evolution, both prokaryotic and eukaryotic cells have developed a variety of biochemical mechanisms to define the direction and proximity of extracellular stimuli. This process is essential for the cell to reply properly to the environmental cues that determine cell migration, proliferation, and differentiation. Chemotaxis is the cellular response to chemical attractants that direct cell migration, a process that plays a central role in many physiological situations, such as host immune responses, angiogenesis, wound healing, embryogenesis, and neuronal patterning, among others. In addition, cell migration takes part in pathological states, including inflammation and tumor metastasis. Indeed, tumor progression to invasion and metastasis depends on the active motility of the invading cancer cells and the endothelial cell bed during tumor neovascularization. Cell migration switches "off" and "on," based on quantitative differences in molecular components such as adhesion receptors, cytoskeletal linking proteins, and extracellular matrix ligands, and by regulating the affinity of membrane-bound chemoattractant receptors. A clear understanding of how cells sense chemoattractants is, therefore, of pivotal importance in the biology of the normal cell as well as in prevention of malignant cell invasion. Here we offer a perspective on cell migration that emphasizes the relationship between cell polarization and cell movement and the importance of the equilibrium between the signals that drive each process for the control of tumor cell invasion.     

The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis

The cues and signaling systems that guide the formation of embryonic blood vessels in tissues and organs are poorly understood. Members of the Eph family of receptor tyrosine kinases and their cell membrane-anchored ligands, the ephrins, have been assigned important roles in the control of cell migration during embryogenesis, particularly in axon guidance and neural crest migration. Here we investigated the role of EphB receptors and their ligands during embryonic blood vessel development in Xenopus laevis. In a survey of tadpole-stage Xenopus embryos for EphB receptor expression, we detected expression of EphB4 receptors in the posterior cardinal veins and their derivatives, the intersomitic veins. Vascular expression of other EphB receptors, including EphB1, EphB2 or EphB3, could however not be observed, suggesting that EphB4 is the principal EphB receptor of the early embryonic vasculature of Xenopus. Furthermore, we found that ephrin-B ligands are expressed complementary to EphB4 in the somites adjacent to the migratory pathways taken by intersomitic veins during angiogenic growth. We performed RNA injection experiments to study the function of EphB4 and its ligands in intersomitic vein development. Disruption of EphB4 signaling by dominant negative EphB4 receptors or misexpression of ephrin-B ligands in Xenopus embryos resulted in intersomitic veins growing abnormally into the adjacent somitic tissue. Our findings demonstrate that EphB4 and B-class ephrins act as regulators of angiogenesis possibly by mediating repulsive guidance cues to migrating endothelial cells.     

EphB/ephrinB Signaling in Cell Adhesion and Migration

Eph receptors and their ligands, ephrins, represent the largest group of the receptor tyrosine kinase (RTK) family, and they mediate numerous developmental processes in a variety of organisms. Ephrins are membrane-bound proteins that are mainly divided into two classes: A class ephrins, which are linked to the membrane by a glycosylphosphatidylinositol (GPI) linkage, and B class ephrins, which are transmembrane ligands. Based on their domain structures and affinities for ligand binding, the Eph receptors are also divided into two groups. Trans-dimerization of Eph receptors with their membrane-tethered ligands regulates cell-cell interactions and initiates bidirectional signaling pathways. These pathways are intimately involved in regulating cytoskeleton dynamics, cell migration, and alterations in cellular dynamics and shapes. The EphBs and ephrinBs are specifically localized and modified to promote higher-order clustering and initiate of bidirectional signaling. In this review, we present an in-depth overview of the structure, mechanisms, cell signaling, and functions of EphB/ephrinB in cell adhesion and migration.     

Cell Migration and Cell-Cell Interaction in the Presence of Mechano-Chemo-Thermotaxis

Although there are several computational models that explain the trajectory that cells take during migration, till now little attention has been paid to the integration of the cell migration in a multi-signaling system. With that aim, a generalized model of cell migration and cell-cell interaction under multisignal environments is presented herein. In this work we investigate the spatio-temporal cell-cell interaction problem induced by mechano-chemo-thermotactic cues. It is assumed that formation of a new focal adhesion generates traction forces proportional to the stresses transmitted by the cell to the extracellular matrix. The cell velocity and polarization direction are calculated based on the equilibrium of the effective forces associated to cell motility. It is also assumed that, in addition to mechanotaxis signals, chemotactic and thermotactic cues control the direction of the resultant traction force. This model enables predicting the trajectory of migrating cells as well as the spatial and temporal distributions of the net traction force and cell velocity. Results indicate that the tendency of the cells is firstly to reach each other and then migrate towards an imaginary equilibrium plane located near the source of the signal. The position of this plane is sensitive to the gradient slope and the corresponding efficient factors. The cells come into contact and separate several times during migration. Adding other cues to the substrate (such as chemotaxis and/or thermotaxis) delays that primary contact. Moreover, in all states, the average local velocity and the net traction force of the cells decrease while the cells approach the cues source. Our findings are qualitatively consistent with experimental observations reported in the related literature.