Pdk1^-/- EBs (right) do not develop into networks of ECs.

The phosphatidylinositol 3-kinase (PI3K) signalling pathway and its downstream effectors phosphoinositide-dependent kinase-1 (PDK1) and AKT/PKB are known to regulate cell motility. But the way in which these components interact has yet to be elucidated. It is thought that phosphatidylinositol 3,4,5-trisphosphate (PIP3), the product of PI3K, might be involved in the PDK1-AKT/PKB interaction as both kinases contain a PH domain that binds to PIP3. Now, in the Journal of Cell Biology, Primo et al. report for the first time that PDK1 regulates cell motility by activating AKT/PKB through its interaction with PIP3.

Pdk1^-/- mice lack normal vascularisation, indicating that PDK1 might be required for endothelial cell (EC) migration during vessel formation. The authors found that Pdk1^-/- embryoid bodies (EBs) did not develop into networks of ECs. Pdk1^-/- EC migration was defective and disruption of the PDK1 PH domain led to impaired migration and vessel formation. These observations confirm that PDK1 activity is required for cell migration in ECs.

Overexpression of PDK1 in ECs resulted in an increase in migration that followed the gradient of vascular endothelial growth factor (VEGF). Enhanced migration was associated with increased phosphorylation of AKT/PKB, but not of PDK1, suggesting that AKT/PKB might be responsible for the enhanced migration. Kinase-deficient PDK1 did not induce migration of ECs, indicating that PDK1 is responsible for AKT/PKB phosphorylation. Upon inhibition of PI3K, VEGF-mediated EC migration was reduced. This observation points to a link between PI3K action, VEGF receptor signalling and PDK1 activity.

The authors postulated that PDK1 might activate AKT/PKB via docking to membrane-bound PIP3. This hypothesis was corroborated by the discovery that, in contrast to control PDK1, PDK1 which lacked the PH domain did not relocate to the leading edge. In addition, phosphorylation of AKT/PKB increased at the edge and AKT/PKB colocalised with PDK1 at the plasma membrane.

Based on their results, Primo et al. propose that after exposure to a stimulus such as VEGF, PIP3 synthesis leads to the translocation of PDK1 and AKT/PKB to the EC cell membrane. PDK1 then activates AKT/PKB, which then guides directional cell movement akin to the chemotactic cell motility observed in Dictyostelium discoideum. The results of Primo et al. offer the first evidence that the PI3K signalling pathway is involved in tyrosine kinase receptor-mediated cell migration.

Author: Mirko von Elstermann

Integrin (red) associates with Type I PKA (green).

A-kinase anchoring proteins (AKAPs) aid the docking of cAMP-dependent protein kinase (PKA) to specific cellular regions during various signalling processes. There are two types of PKA that each differ in their regulatory (PKA-RI and II) and catalytic (PKA-CI and II) subunit makeup. Different AKAPs are either specific for only type II PKA or exhibit dual specificity for both types of PKA. Type I PKA is enriched at the front of migrating cells, where it phosphorylates 4 integrins. Although this locally restricted phosphorylation provides spatial cues governing cell motility, the details of the 4 integrin–PKA interaction are still poorly understood. Mark Ginsberg and colleagues now report in Nature Cell Biology that the cytoplasmic domain of 4 integrin is a novel form of type I PKA-specific AKAP.

The authors showed that PKA-RI colocalises with 4 integrin in migrating fibroblast protrusions. The cytoplasmic domain of 4 integrin, but not of other integrins, specifically interacts with the PKA holoenzyme-containing subunit PKA-RI, but not with PKA-RII. Importantly, the PKA-RI–4 integrin interaction differs from other AKAPs as it requires the presence of both the catalytic and regulatory subunits of PKA. The kinase activity, on the other hand, was required for phosphorylation of but not for binding to 4 integrin.

The authors investigated whether the type I PKA–4 integrin interaction influenced 41 integrin-dependent migration. They targeted either type I or type II PKA to the mitochondrion by transfecting Chinese Hamster Ovary (CHO) cells with modified PKA-R subunits (mitoAKB-RI or mitoAKB–RII). Phospho-4 integrin was detected at the leading edge of CHO cells expressing only mitoAKB–RII, indicating that leading-edge phosphorylation of 4 integrins requires the presence of type I PKA at the plasma membrane. Furthermore, mitoAKB RI-expressing cells exhibited defects in 4 integrin-mediated migration.

The anchoring of type I PKA by 4 integrin establishes unique roles for type I PKA in 4-mediated cell functions. 4 integrins cluster with antigen receptors to inhibit T-cell function, and 4 integrin phosphorylation leads to a reduction in the activation of tyrosine kinases that promote cell proliferation. Based on the results of this study, the authors postulate that both events involve the presence of type I PKA. Apart from the effects on signalling events, this study reveals a novel role for type I PKA in cell migration – type I PKA is recruited to the leading edge of migrating cells by its interaction with 4 integrins. This partitioning of type I PKA activity provides new insight into the maintenance of polarity during cell migration.

Author: Mirko von Elstermann

1 integrin uptake in GFP-EHD1 transfected cells.

Integrins are important regulators of cell-extracellular matrix (ECM) and cell-cell interactions. Upon activation, they cluster to form adhesion complexes and stimulate several intracellular signalling pathways that can lead to proliferation, apoptosis, cell survival and migration. The internalization and recycling of integrins represents an important mechanism for the regulation of integrin activity. In The Journal of Cell Science, Steve Caplan and colleagues shed light into the mechanisms of integrin transport, by identifying a new regulator, the C-terminal Eps15 homology domain-containing protein EHD1.

Most integrins are internalized in a clathrin-independent manner and proteins such as the small GTPase Arf6, protein kinase C- and dynamin have been shown to be involved in this process. More recently, several endocytic regulators of the Rab family were found to mediate the transport of integrins to the plasma membrane. Studies in Caenorhabditis elegans and RNA interference (RNAi)-mediated depletion of EHD1 in human cells have previously shown that EHD1 cooperates with Rab family members to regulate the recycling of membrane proteins that are internalized through both clathrin-dependent and -independent mechanisms (such as the transferrin receptor and the major histocompatibility complex class I, respectively). In this paper, the authors labelled surface 1 integrins and monitored their subcellular itinerary in human cancer cells and fibroblasts treated with EHD1-RNAi. Two hours after labelling, 1 integrin-containing vesicles accumulated in the perinuclear region of EHD1-depleted cells suggestive of a delay in integrin recycling. Flow cytometry-based recycling assays confirmed this observation.

Caplan and colleagues next assessed the effect of EHD1 loss in Ehd1-/- mouse embryonic fibroblasts (MEFs). These cells display lower overall levels of 1 integrin on the plasma membrane and an accumulation of 1 integrin in internal vesicles; however, their cell-ECM adhesions are more prominent. Increased levels of activated 1 integrin on the plasma membrane and a change in the localization of the adhesion-associated proteins focal adhesion kinase (FAK) and paxillin, which were found to preferentially localize to focal adhesions rather than the cytosol, are likely to account for this phenotype.

By monitoring adhesion dynamics using an immunofluorescence-based assay, the authors showed that focal adhesion disassembly is impaired in cells that lack EHD1. In agreement with these findings, Ehd1-/- MEFs migrate and spread significantly less than control cells when plated on fibronectin. Similar levels of colonization were observed for both Ehd1-/- MEFs and their wild-type counterparts on vitronectin — a ligand for v3 integrin — indicating that the effect of EHD1 on integrin trafficking might be subunit-specific.

Collectively, these data indicate that EHD1 contributes to 1 integrin recycling, positively regulating adhesion dynamics, cell spreading and migration. The mechanism by which EHD1 increases adhesion turnover and cooperates with Rab proteins to transport endocytosed 1 integrin to the plasma membrane awaits to be discovered.

Author: Monica Hoyos-Flight

Chemotaxis — the response to chemical stimuli by directed movement — is crucial for the development of germ layers during embryogenesis, cancer-cell spreading and immune-cell migration to sites of infection. However, the mechanisms that couple gradient sensing and movement in eukaryotic cells are still unclear. Most studies support the 'chemical compass model', which proposes that external chemotactic gradients activate a cascade of events that lead to actin polymerization and translocation towards or away from the chemotactic signal. However, in Nature Cell Biology, Natalie Andrew and Robert Insall report that pseudopod formation in Dictyostelium discoideum moving in a shallow gradient is controlled independently of chemotactic signalling.

In contrast to previous reports, the authors observed that neutrophils, mouse embryonic fibroblasts and amoeba generated most of their pseudopods by splitting of existing pseudopods, rather than de novo. The formation of 'lateral pseudopods', new protrusions that extend laterally from the leading edge and which are more accurately oriented towards the source of chemoattractant, were rarely observed. Furthermore, in D. discoideum the rate of pseudopod formation did not depend on orientation; new pseudopods were just as likely to be extended towards the source of attractant as away from it. These findings indicate that the rate of pseudopod formation is set internally and is independent of directional sensing.

The authors propose that instead of the preferential formation of protrusions towards a source of chemoattractant, it is actually the survival of pseudopods oriented towards the attractive signal that is important for directional migration. Using time-lapse microscopy, the authors show that there is a strong correlation between pseudopod position and survival. By maintaining pseudopods that sense the highest levels of chemoattractant and retracting the ones extended away from the attractive source Dictyostelium cells are able to migrate in the right direction.

Inhibition of phosphatidylinositol 3-kinase (PI3K), which is responsible for setting up an intracellular PIP3 gradient and positively regulating the actin cytoskeleton, greatly reduced the rate of pseudopod formation but did not affect the cells' ability to move towards chemoattractants. Therefore, it seems that phosphoinositide-mediated signalling regulates the rate of pseudopod extension in all directions, but not the actual direction of movement. In agreement with these results, two other recently published studies show that defective PIP3 signalling affects the motility but not chemotaxis of neutrophils.

In summary, this paper shows that pseudopod generation is a constitutive process and that pseudopod survival determines the direction of cell movement. Biased retention of randomly generated pseudopods has also been observed during bacterial chemotaxis indicating that these processes might have more in common than was previously thought. The role of PIP3 signalling in chemotaxis might have to be revisited as the proteins and signalling events that determine pseudopod lifetime could turn out to be key to gradient sensing.

Author: Monica Hoyos-Flight

The activation of integrins is crucial for regulating the attachment of a cell to the extracellular matrix (ECM) and modulating the transmission of forces and signals required for locomotion. Previous studies have established that the PTB-like F3 subdomain of the adaptor protein talin binds to integrin tails leading to integrin activation. Now in Cell, Iain Campbell and colleagues describe the structural basis of integrin activation by talin furthering our understanding of how this process might be manipulated.

The authors developed a chimeric peptide suitable for high-resolution studies that comprises the membrane proximal (MP) region of ₃ integrin and the integrin membrane distal (MD)-mimicking region of phosphatidylinositol phosphate kinase type I, which interact with the F3 domain of talin. The structure of this talin F3-peptide complex reveals new sites of contact between the MP region of ₃ integrins and talin. Phe727 and Phe730 of the ₃ tail make intimate contact with the F3 domain; mutating these residues to Ala results in an integrin mutant that is only weakly activated by talin.

Similarly, mutation of any of the MP contact residues in the talin F3 domain (Leu325Arg, Ser365Asp, Ser379Asp or Gln381Val) diminished its ability to activate IIb₃ integrin in transfected Chinese hamster ovary (CHO) cells. However, as binding to the MD region of the ₃ peptide was unchanged, the authors investigated whether these talin mutants could compete with endogenous talin and therefore, inhibit integrin activation. Indeed, all four mutants blocked the activation of a talin-dependent, constitutively active chimeric integrin expressed in CHO cells. Interestingly, this dominant-negative effect could be recapitulated with other PTB domain-containing proteins, such as DOK1, that are able to bind to the MD but not the MP region of the ₃ integrin tail. These findings are consistent with the idea that PTB domain-containing proteins compete with talin for binding to the MD site but do not interact with the MP region of ₃ which is critical for integrin activation.

The structures of the DOK1 and talin PTB domains differ in a short region between strands S1 and S2. It seems that the flexible loop that accepts the ₃ Phe727 and Phe730 in talin's PTB domain is responsible for its unique ability to activate integrins. Moreover, these results indicate that the affinity of other PTB-containing proteins for the MD region of ₃ could be important for regulating integrin activity in vivo.

Figuring out the precise mechanisms of integrin regulation is particularly interesting from a therapeutic point of view as integrins are involved in a wide variety of pathological events such as atherosclerosis and cancer. The structure of the integrin activation interface described in this paper could be an excellent starting point for the design of new drugs aimed at disrupting integrin activation.

Author: Monica Hoyos-Flight

Map of -actinin velocities in an MEF cell.

The linkage between the substratum and the actin cytoskeleton underlies a cell's ability to assemble and disassemble adhesions and thus, is crucial for regulating the formation of protrusions and cell migration. Paul Wiseman and colleagues now describe, in the Journal of Cell Science, the application of a novel image analysis tool to examine the movement of adhesion-related proteins and actin in migrating cells. Detailed protein velocity maps reveal differences in the efficiency of the linkage between integrin and actin among different cell types and indicate that the efficiency of linkage increases as actin and adhesions become more organized.

Integrins are key mediators of the linkage between the extracellular matrix (ECM) and actin. Previous studies indicate that the bond between the ECM and integrins is stronger than the link between integrins and the cytoskeleton as integrin-containing 'footprints' are often observed on the substratum after the rear region of a migrating cell retracts. Many other adhesion-related proteins such as talin, vinculin, focal adhesion kinase (FAK), Src and paxillin are also likely to be important regulators of the ECM-actin linkage, but the complexity of their interactions in both space and time has made it particularly difficult to asses their role in this linkage, until now.

By using spatio-temporal image correlation spectroscopy (STICS), the authors generated maps of molecular transport and quantified coupling between adhesion components and actin. STICS works by calculating the spatial-temporal correlation function from a series of images taken over time and directly reflects the movement of fluorescently-labelled macromolecules in live cells. An immobile filtering algorithm was developed to remove the contributions of large static structures, such as adhesions or actin filaments, and facilitate the visualization of proteins moving within or along these structures.

The authors first confirmed the validity of the technique by measuring the rates of mRFP-actin and -actinin-EGFP flow in mouse embryonic fibroblasts (MEFs). The values obtained, both in the absence and presence of retrograde flow inhibitors, were comparable to those measured by fluorescence speckle microscopy.

In CHO cells, under migration-promoting conditions, the movements of a-actinin and actin were highly coupled, but EGFP-fused paxillin, FAK, talin and vinculin had a much lower directional correlation with mRFP-actin. 5-integrin moved slowly and showed little directional correlation with the actin flow, supporting the notion of a strong interaction between 5-integrin and the ECM. In 3T3 cells and MEFs, which have highly organized actin filaments and larger adhesions, the movements of paxilin, talin and FAK were more tightly coupled to actin. The influence of the organization of actin filaments and adhesions on the linkage to the ECM was further corroborated when CHO cells were plated on high concentrations of fibronectin. Not only did the cells form more organized adhesions and actin stress fibres, there was also marked increase in the directional correlation for both talin and paxillin movement relative to actin movement.

Taken together, these results indicate that the efficiency of linkage between the ECM and actin is regulated by adhesion strength. The authors propose that ECM-actin linkage is regulated at two levels: first through interactions between integrins and adhesion-related proteins, and second between adhesion-related proteins and the actin cytoskeleton. The general applicability of STICS holds promise as a powerful new tool for dissecting protein movements in other contexts such as during membrane trafficking or protein transport in polarized cell systems.

Author: Monica Hoyos-Flight

Lung cancer is still the most common cause of death from cancer worldwide with over 900,000 deaths per year. In most cases, by the time it is detected, it is incurable. In Nature Cell Biology, Yoshiru Maru and colleagues identify two chemokines in the lungs of tumour-bearing mice that directly regulate the migration of cancer cells to this organ. These findings not only provide further evidence that soluble factors released from primary tumours prepare distant tissues for malignant cell engraftment, but also indicate that the chemokines S100A8 and S100A9 might serve as prognostic indicators of metastasis or even, as targets for anticancer drug design.

Examination of the gene expression profile in lungs derived from normal, benign and malignant tumour-bearing mice revealed that the inflammatory chemoattractants S100A8 and S100A9 are strongly expressed in lung cells before tumour cells metastasize. Moreover, exposure to serum from tumour-bearing mice was sufficient to induce the upregulation of S100A8 and S100A9 in normal lung tissue. Using neutralizing antibodies and recombinant proteins, the authors showed that a combination of factors, including tumour necrosis factor (TNF), transforming growth factor- (TGF-) and vascular endothelial growth factor-A (VEGF-A), is responsible for the upregulation of these chemokines in premetastatic lungs.

S100A8 and S100A9-stimulated lung conditioned medium (S100-stimulated LCM) induced the migration of both macrophages and tumour cells in Boyden chamber assays by activating the mitogen-activated protein kinase p38, and promoting the formation of invading pseudopods. Further experiments showed that S100A8 and S100A9 induce the secretion of factors that function as migration-stimulatory factors (MSFs) such as TNF, MIP2, TGF- or VEGF-A, from lung endothelial cells and Macrophage antigen-1 (Mac1⁺)-myeloid cells. Depletion of these molecules from S100-stimulated LCM or inhibition of p38 partially blocked tumour-cell migration indicating that S100A8 and S100A9 induce the secretion of several MSFs, and that p38 signalling is required for the migration of both myeloid and cancer cells.

Finally, Yoshiru Maru and colleagues generated anti-S100A8 and anti-S100A9 antibodies, which suppressed cancer-cell migration in vitro and the formation of metastasis in vivo by decreasing the number Mac1⁺-myeloid cells, and thereby the secretion of MSFs, in the lungs of tumour-bearing mice.

Taken together, these findings indicate that the secretion of TNF, TGF- and VEGF-A from primary tumours upregulate S100A8 and S100A9 in the lungs which in turn, leads to the recruitment of Mac1⁺-myeloid cells and the secretion of MSFs. These factors activate p38 and promote tumour-cell migration. The ability of anti-S100A8 and anti-S100A9 antibodies to protect lungs from circulating tumour cells holds promise for future cancer therapies.

Author: Monica Hoyos-Flight

In zebrafish, primordial germ cells (PGCs) are guided towards the developing gonad by the chemokine SDF-1. The G-protein-coupled receptor CXCR4b is known to transduce the SDF-1 signal, but exactly how directional migration is achieved is poorly understood. A new study in Developmental Cell shows that, unlike several other types of migrating cells in which actin polymerization is enriched underneath the expanding membrane, PGC advance involves the generation of bleb-like protrusions.

Membrane blebbing occurs when a patch of membrane detaches from the submembranous cytoskeleton or cortex and is often observed during apoptosis and cytokinesis. Erez Raz's group now report that actin-free protrusions are also observed at the leading edge of migrating PGCs.

It has previously been suggested that local myosin contraction generates internal hydrostatic pressure, which could push the cytosol into cell blebs or lead to breaks in the cell cortex, leading to a decrease in adhesion with the membrane thereby initiating bleb formation. Indeed, fluorescence resonance energy transfer (FRET) experiments indicate that myosin activity is highest at the leading edge of migrating PGCs, and a cytoplasmic flow was observed to enter the cortex-free PGC protrusions. When myosin activity was increased by expressing constitutively active Rho kinase 2 (ROK2), the plasma membrane separated further from the cortex and gave rise to broad protrusions around the entire cell perimeter. Conversely, expression of dominant-negative ROK2 or treatment with the myosin inhibitor blebbistatin caused PGCs to become rounded and inhibit their migration towards their target in vivo. These results suggest that PGC protrusion is largely dependent on myosin activation.

SDF-1 signalling through CXCR4b causes an increase in intracellular calcium at the cell front, and the authors show that this localized calcium elevation is required for PGC migration. Calcium buffering inhibits PGC polarization, whereas local elevations of calcium, induced by expressing a mutant form of the calcium sensor STIM1, led to the formation of blebs at these sites. Furthermore, it appears that calcium regulates myosin activity as inhibition of myosin suppressed the STIM1-induced protrusive activity.

The authors propose that CXCR4 activation by SDF-1 stimulates calcium-dependent myosin activity locally and loosens membrane-cortex attachments at the leading edge, thereby promoting bleb formation. A flow of cytoplasm, rather than actin polymerization, then drives the protrusion forward. Zebrafish are an ideal model to examine cell migration in vivo at high resolution, and future studies may find other instances of this alternative mode of generating protrusions.

Author: Monica Hoyos-Flight