STICCS uncovers slips and grips

Intensity fluctuations in microscopy images of fluorescently-labelled molecules can be used to determine the dynamics and associations of macromolecules in both spatial and temporal domains. Recent technical advances based on this approach have allowed, for example, the identification of an intracellular molecular clutch (the ligand–integrin–actin link) that controls the extent to which cellular adhesions are coupled to retrograde movement of actin filaments. However, the approach used, spatio-temporal image correlation spectroscopy (STICS), analyzes the movement of a single molecular species and does not directly infer interactions between different components of the clutch. This prompted Toplak et al. to extend STICS to dual chromophore analysis — spatio-temporal image cross-correlation spectroscopy (STICCS) — and use this to determine whether adhesion components are moving retrograde in complexes. The authors used computer-simulated images to establish the potential and limitations of STICCS and tested it using conditions that model the dynamics thought to occur in adhesions. They then used the two-color STICCS to investigate the cotransport of paxillin and α5β1, α6β1 or αLβ2 in migrating cells imaged using total internal reflection fluorescence microscopy. Both α6β1 and αLβ2 co-fluxed with paxillin in protrusions of cells migrating on laminin and ICAM-1, respectively, showing that they moved as a complex; however, as the authors discuss, STICCS does not reveal whether they are in direct contact within the complex. By contrast, α5β1 remained static while paxillin fluxed in retracting regions of cells migrating on fibronectin. This indicates that the strength of the linkage between the extracellular matrix and actin can depend on the specific integrin–ligand pair and, more importantly, the point of slippage can be at the level of the integrin–ligand interaction as well as within the cytoplasmic molecules that comprise the linkage.

• Toplak T, Pandzic E, Chen L, Vicente-Manzanares M, Horwitz AR, Wiseman PW. STICCS Reveals Matrix-Dependent Adhesion Slipping and Gripping in Migrating Cells. Biophys J. 2012 Oct 17;103(8):1672-82. Epub 2012 Oct 16. PubMed

Lengthy and complex insights into kindlin structure and interactions

As co-activators of integrins, kindlin proteins are crucial for several physiological phenomena but, although biochemical studies indicate that kindlins bind to the membrane distal NPXY motif in integrin β tails (whereas talin, another integrin co-activator, binds to the membrane proximal NPXY motif), structural data about kindlin–integrin interactions are lacking. Yates et al. have therefore expressed and purified recombinant mouse kindlin-3 using a baculoviral system to gain further insight into its conformation and interactions. The authors used small-angle X-ray scattering to ascertain that recombinant kindlin-3 showed an extended formation, which was similar, but not identical, to that of the head domain of talin, owing to the additional presence in kindlin-3 of a long loop within the F1 subdomain and a pleckstrin homology domain within F2. Although size-exclusion chromatography failed to detect any kindlin interactions, analytical ultracentrifugation experiments uncovered a ternary complex, thought to be formed by the integrin cytoplasmic tails, of a kindlin-3 molecule, a talin head domain and an integrin β_1A cytoplasmic tail. Further analysis of the kindlin-3–integrin tail interaction carried out by nuclear magnetic resonance spectroscopy indicated that, consistent with biochemical data, kindlin-3 bound to the membrane distal NPXY motif in the tail of both β_1A and β_1D isoforms, although the data implied a stronger interaction with β_1A. Also in agreement with previous findings, a Ser/Thr-rich region upstream of the NPXY motif influences the interaction. The authors envision that these structural data regarding the conformation of full-length kindlin and its interactions will ultimately provide further insight into integrin activation.

• Yates LA, Fuzery AK, Bonet R, Campbell ID, Gilbert RJ. Biophysical analysis of kindlin-3 reveals an elongated conformation and maps integrin binding to the membrane-distal β-subunit NPxY motif. J Biol Chem. 2012 Sep 18. [Epub ahead of print] PubMed

The worm gene that turned the distal tip cell

During Caenorhabditis elegans gonadogenesis, two somatic cells — the distal tip cells (DTCs) — migrate in opposite directions along the ventral basement membrane before turning away toward dorsal and then turning again to move centripetally on the dorsal basement membrane towards the midbody region, ultimately generating U-shaped gonad arms. Martynovsky et al. previously identified F40F11.2/mig-38 as a novel gene required for DTC turning. By depleting mig-38 expression specifically in the DTCs, the authors have now shown that this gene functions cell-autonomously to regulate turning towards the midbody. DTC migration along the basement membrane and turning in response to extracellular cues requires the expression of integrin receptors, and Martynovsky et al. found that MIG-38 acted in conjunction with INA-1 integrins to regulate DTC turning, as knockdown of expression of ina-1 and mig-38 exacerbated turning defects. So, too, did a double knockdown of MIG-38 and MIG-15, the ortholog of Nck-interacting kinase (NIK), which binds to the PAT-3 integrin β subunit, indicating that MIG-38 and MIG-15 together regulate integrin-dependent DTC turning. A deficiency in talin, a protein involved in integrin activation, causes turning defects, too, but also results in premature cessation of DTC migration. Knockdown of mig-38 rescued this premature stop phenotype, but restoration of DTC migration was reversed when MIG-15 or its binding partner NCK-1 was additionally depleted. These results imply that talin and MIG-15–NCK-1 can each promote DTC migration, but by different pathways, by interacting with PAT-3/β-integrin, and that MIG-38 might negatively regulate the MIG-15–NCK-1 complex. The authors propose that such MIG-38-mediated inhibition might promote dissociation of the MIG-15–NCK-1 complex, thereby enabling MIG-15 to interact with other factors to mediate integrin-mediated turning.

• Martynovsky M, Wong MC, Byrd DT, Kimble J, Schwarzbauer JE. mig-38, a novel gene that regulates distal tip cell turning during gonadogenesis in C. elegans hermaphrodites. Dev Biol. 2012 Aug 15;368(2):404-14. PubMed

Screening strategy targets aspects of scattering

The scattering of Madin–Darby canine kidney (MDCK) cells induced by hepatocyte growth factor (HGF) provides a model system for investigating aspects of tumor invasion. Loerke et al. used a nonfluorescent, low-magnification phase–contrast microscopy approach coupled with automated cell tracking to analyse the trajectories of individual live cells in response to HGF, deriving three parameters from these data that reflected the three physical steps of cell scattering: increased cell motility, loss of intercellular adhesion, and spatial dispersion (cell redistribution during scattering). Having established that the increased average cell speed induced by HGF results from a combination of cell–cell adhesion loss and increased cell motility, the authors screened a commercially available library of small-molecule inhibitors to identify signaling pathways that contributed to either, or both, of these processes, in an effort to target epithelial cell scattering. Out of 29 efficient inhibitors of scattering that were identified, nine selectively inhibited the loss of intercellular adhesion, while eight specifically affected cell motility. Although the screen confirmed the involvement of several signaling molecules, such as Src, phosphatidylinositol 3-kinase and focal adhesion kinase, the authors validated many other targets that were less well characterized, such as ribosomal S6 kinase and cyclin-dependent kinase 1, by RNA interference, as signaling intermediates. The data also identified nonsteroidal anti-inflammatory drugs, which target cyclooxygenases, as cell–cell dissociation inhibitors, and indirubins as inhibitors of cell motility. The inhibitory effect of the majority of these drugs on HGF-induced scattering was also reproduced in an unrelated cell line from a different species and organ, confirming the suitability of this approach for widespread use in screening for inhibitors of epithelial cell scattering.

• Loerke D, le Duc Q, Blonk I, Kerstens A, Spanjaard E, Machacek M, Danuser G, de Rooij J. Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation. Sci Signal. 2012 Jul 3;5(231):rs5 PubMed

Toxoplasma gondii targets the host actin cytoskeleton during invasion

It is estimated that approximately one third of the world’s population is infected with Toxoplasma gondii. This protozoan parasite belongs to the phylum apicomplexa, which also includes Plasmodium, the agent of malaria, and Cryptosporidium, which causes cryptosporidiosis. T. gondii quickly establishes infection as a fast-replicating form, the tachyzoite, which invades numerous cell types throughout the body. The parasite can cause encephalitis and neurologic diseases and can affect the heart, liver, and eyes (chorioretinitis).

Toxoplasma, like Plasmodium, enjoys an intracellular lifestyle free from many of the host’s immune insults that are encountered by extracellular microbes, inside a sub-cellular compartment called the parasitophorous vacuole (PV). Unlike viruses and intracellular bacteria, Toxoplasma actively penetrates the host cell without using typical host-uptake pathways that involve coated pits or actin-based engulfment. During invasion, the extracellular parasite triggers the assembly of a unique zoite–cell junction, and in less than 20 sec progressively propels its ~ 2 μm long body through the junction into the growing vacuole. How the growing PV and its parasite passes through the barrier of the host cell cortical actin remains unknown.

Delorme-Walker et al. provide evidence that toxofilin, an actin-binding protein secreted by the invading parasites into the host cell cytoplasm at the onset of invasion, targets the host cortical actin cytoskeleton to facilitate vacuole folding and tachytozoite invasion. Indeed, Toxoplasma tachyzoites lacking toxofilin exhibit delayed invasion kinetics. Correlative light and electron tomography allowed tracking toxofilin in action within the host cell and showed that integrity of the host cortical actin cytoskeleton was disrupted at the site of parasite entry, suggesting that toxofilin induces actin depolymerization in close proximity to the zoite apex at the onset of invasion. Quantitative fluorescent speckle microscopy indicated that toxofilin facilitates tachyzoite invasion by upregulating host cortical actin filament turnover, consistent with toxofilin monomer sequestration and barbed-end filament capping capabilities in vitro. The limited amount of protein restricted to the site of entry is fully compatible with an actin-disassembling or actin-severing activity working at substoichiometric ratios with actin.The ability of toxofilin to expedite invasion by regulating the turnover of cortical actin filaments in the host cell is likely to confer a selective advantage to the parasite.

• Delorme-Walker V, Abrivard M, Lagal V, Anderson K, Perazzi A, Gonzalez V, Page C, Chauvet J, Ochoa W, Volkmann N, Hanein D, Tardieux I. Toxofilin upregulates the host cortical actin cytoskeleton dynamics facilitating Toxoplasma invasion. J Cell Sci. 2012 Jul 5. [Epub ahead of print] PubMed

The devil is in the details; Unmasking the function of vinculin’s isoform

Vinculin and metavinculin, key elements of multiple protein assemblies linking the cell membrane in cell–cell and cell–matrix adhesions, differ only in the presence of a 68-residue acidic insert in the carboxy (C)-terminal tail. Although the functional role of this insert remains elusive, its importance is exemplified by point mutations that are associated with dilated and hypertrophic cardiomyopathy. Having previously shown that the C-terminal tail of vinculin (Vt) binds and bundles actin filaments, Janssen et al. now show that the tail domains of both isoforms bind actin filaments in the same orientation. However, instead of inducing bundling, metavinculin (MVt) severs actin filaments. The three-dimensional reconstructions of actin filaments with bound metavinculin tail suggest that the insert spatially obscures the vinculin dimerization site, thus preventing higher-order organization of the metavinculin-bound actin filaments into bundles. The authors propose that the flexible insert (MV879–946) enables a gelsolin-like (DDY) motif present in the metavinculin insert to bind in the actin groove and induce severing. Bundling activity was, however, restored to metavinculin and severing activity was abolished in the presence of residues amino-terminal to residue 879, which, together with isoelectric point analysis, suggests that masking/unmasking of charged residues in the MV 879–946 region might regulate bundling versus severing in the full length protein. Janssen et al. hypothesize that the properties of metavinculin tail revealed in this study may be essential for modulating compliance of vinculin-induced actin bundles when exposed to rapidly increasing external forces. In this scenario, the isoform would carry a regulatory role in which its severing activity is activated upon demand when the tension passes some threshold. Application of force would unmask its severing activity, which, in turn, could be relieved once force is diminished. The triggering/ unmasking of metavinculin’s severing capabilities would provide a very efficient way to locally remodel F-actin in regions where fast reactions to sudden increases in force are needed.

• Janssen ME, Liu H, Volkmann N, Hanein D. The C-terminal tail domain of metavinculin, vinculin's splice variant, severs actin filaments. J Cell Biol. 2012 May 28;197(5):585-93. Epub 2012 May 21. PubMed

Dendritic cell pathogen receptor (DC-SIGN) immobilization in microdomains

In its role in antigen presentation, the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) interacts through its carbohydrate recognition domain (CRD) with mannose-type carbohydrates on the surface of many pathogens. Clustering of DC-SIGN on the dendritic cell (DC) plasma membrane is thought to increase both ligand avidity and specificity, but how such microdomains (in which DC-SIGN is immobilized) form and remain stable  is unclear, prompting Liu et al. to take a mutational approach to investigate which domain/motif of DC-SIGN might be responsible. Four mutants, expressed in unlabeled forms and as green fluorescent protein fusions, were generated for use in confocal imaging and fluorescence recovery after photobleaching studies to assess the existence, size and stability of resultant microdomains. Consistent with previous results, deletion of the cytoplasmic portion had little effect on microdomain formation or stability, implying that DC-SIGN clustering is not mediated by a direct interaction with cytoskeletal structures. A point mutation preventing potential N-linked glycosylation at Asn80 also failed to reduce microdomain stability, thereby ruling out any significant contribution from galectin–glycoprotein crosslinking in microdomain formation. By contrast, deletion of the seven and a half tandem repeats, which are thought to mediate tetramerization by forming coiled-coil α-helices, resulted in free membrane diffusion and ‘dynamic microdomains’. A more profound effect — the complete loss of microdomains from the cell surface — was observed following removal of the CRD domain; the deletion mutants instead showed a diffuse and homogeneous distribution within the membrane and full lateral mobility. The authors propose that the CRD might interact directly with components of the extracellular matrix, or with transmembrane adaptor proteins to indirectly link to the cytoskeleton, to thereby facilitate the extraordinary microdomain stability. Pathogens likely compete with these stabilizing interactions to facilitate their attachment to DCs.

• Liu P, Wang X, Itano MS, Neumann AK, Jacobson K, Thompson NL. The formation and stability of DC-SIGN microdomains require its extracellular moiety. Traffic. 2012 May;13(5):715-26. PubMed

Cells change tack through branch-and-pivot mechanism

During random migration, fibroblasts and other mesenchymal cells move slowly and show relatively long-lived directionality, with changes in orientation being induced by stochastic turning behavior. In The Journal of Cell Biology, Welf et al. outline a mechanism for reorientation that is mediated by phosphoinositide 3-kinase (PI3K)-dependent branching and pivoting of lamellipodial protrusions. The authors used a fluorescent biosensor to generate spatiotemporal maps of PI3K signaling ‘hotspots’, protrusion/retraction velocity and morphological extension by total internal fluorescence microscopy in randomly migrating fibroblasts. Analysis of these maps revealed that cells made significant turns by the branching of a protrusion into two followed by the pivoting of these branched protrusions, and that marked changes in cell orientation correlated with changes in PI3K activity. Inhibiting PI3K activity abrogated fibroblast reorientation by such branching and pivoting — not by inhibiting the initiation of branched protrusions but, rather, by preventing their spreading and propagation. Indeed, PI3K signaling increased after the initiation of protrusion, during the transition from nascent to mature integrin-mediated adhesions. Accordingly, inhibiting PI3K activity also failed to prevent protrusion induced by a photoactivatable form of Rac. Fibroblasts undergo chemotaxis in response to platelet-derived growth factor (PDGF), and Welf et al. showed that PDGF gradient stimulation biased the branch-and-pivot mechanism so that PI3K signaling and protrusion were greater in the branch nearest the highest concentration, resulting in better alignment of cell movement towards the attractant. Following reorientation, cells then tracked the gradient with only minor changes in morphology; or, in the absence of a gradient, again underwent random migration with stochastic turning.

• Welf ES, Ahmed S, Johnson HE, Melvin AT, Haugh JM. Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism. J Cell Biol. 2012 Apr 2;197(1):105-14 PubMed

Inter-domain associations determine talin’s cellular locations

At the plasma membrane, talin can modulate the affinity of integrin receptors for their ligands and link them to the actin cytoskeleton; however, talin is abundant in the cytosol. By combining nuclear magnetic resonance (NMR) spectroscopy with subcellular fractionation experiments, Banno et al. have structurally defined specific interactions that occur between different talin domains to regulate its subcellular localization and, consequently, its function. Talin comprises a globular head domain (THD), which contains a FERM domain made up of F1, F2 and F3 subdomains, within which are integrin-binding sites and lipid-binding sites; a short, flexible linker; and a rod domain containing a series of α-helical bundles, one of which is designated domain E. Autoinhibitory interactions occur between THD and the rod domain, so the authors mapped the interactions that regulated talin’s localization. The presence of domain E maintained talin within the cytosol by mediating an electrostatic interaction with the F3 domain of THD. However, disrupting the domain E–THD interaction did not confer plasma membrane localization; instead, it enabled talin to associate with cytoskeletal actin. Plasma membrane localization was, however, promoted by the loss of a region in the rod domain that contains two helical bundles (VBS1 and VBS2a), to which vinculin binds. NMR analysis showed that VBS1–VBS2a interacted with the F2F3 domain of THD; disrupting this interaction targeted talin to the plasma membrane. Consistent with reports that vinculin activates integrins in a talin-dependent manner, expression of the vinculin head domain, which binds to VBS1–VBS2a, resulted in talin recruitment to the plasma membrane. So, the subcellular localization of talin is regulated by specific inter-domain interactions between the THD and the rod domain and can be regulated by vinculin binding.

• Banno A, Goult BT, Lee H, Bate N, Critchley DR, Ginsberg MH. Subcellular localization of talin is regulated by inter-domain interactions. J Biol Chem. 2012 Feb 18. [Epub ahead of print] PubMed

Catching microtubules on the run

Labeling plus-end-binding proteins (+TIPs) offers a means of tracking microtubules, but provides mainly qualitative data that are limited to phases of microtubule growth; quantitative characterization of pause and shrinkage, in addition to growth, is required to gain insight into how microtubule dynamics affect processes such as cell polarization and migration. In the Journal of Structural Biology, Applegate et al. present a Matlab-based, user-friendly, open source software package that combines previously validated algorithms and published state-of-the-art technology to link collinear, sequential growth tracks to infer parameters such as shrinkage velocity or pause duration. Using only +TIP markers of microtubule growth, plusTipTracker enables automated tracking, visualization and analysis of microtubule dynamics in live cells. The researchers used a movie of a human endothelial cell expressing the fluorescently tagged +TIP EB3 to highlight a number of pivotal features of the software functionality, such as its robustness, speed and self-adaptiveness in detecting +TIP comets. Its efficiency enables thousands of reconstructed microtubule tracks to be obtained from one cell, providing an abundance of quantitative information — via several visualization modalities and bioinformatics tools — about the architecture of microtubule networks, such as the spatial regulation of microtubule growth speed and the relative subcellular distribution of growing, shrinking and pausing microtubules. These powerful properties of plusTipTracker offer not only a vast improvement in microtubule tracking, which will undoubtedly reveal insights into microtubule regulation that extend beyond cell migration, but also the possibility of developing high-content screens that assess microtubule cytoskeleton dynamics in live cells.

• Applegate KT, Besson S, Matov A, Bagonis MH, Jaqaman K, Danuser G. plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics. J Struct Biol. 2011 Nov;176(2):168-84 PubMed

Probing phosphorylation-mediated protein partnerships

The cerebral cavernous malformation (CCM) multiprotein complex cell-autonomously regulates the stability of endothelial and epithelial cell–cell junctions, and is therefore important in vascular development. KRIT1 (also known as CCM1) and CCM2 are thought to form the core of this protein complex, but to gain further insight into the mechanism(s) and regulation of complex assembly, Kim et al. attached a tandem affinity purification (TAP) tag to KRIT1/CCM1 to isolate associated proteins. They confirmed previously reported interactions of KRIT1/CCM1 with CCM2, ICAP1α (an integrin-binding protein), and heart of glass (HEG, a transmembrane receptor), before analysing phosphorylation sites within the complex. The only high-stoichiometry phosphorylation site in KRIT1/CCM1 was Ser22, which can be targeted by protein kinase A or casein kinase I. Two lower-stoichiometry phosphorylation sites — at Thr151 and Tyr252 — were also found. Tyr252, which has been implicated in CCM2 binding and nuclear retention of KRIT1/CCM1, is a putative target of Janus kinase 2. The authors postulate that the phosphorylation of multiple Ser/Thr sites, which they identified throughout CCM2, might regulate the interaction of this core complex component with other proteins. Phosphorylation of Ser164, Ser166 or Ser168 within the phosphotyrosine-binding domain domain might, for example, influence the binding of CCM2 to KRIT1/CCM1. Several phosphorylation sites were identified in the amino-terminal Ser/Thr-rich domain of ICAP1. Phosphorylation of Thr38, which is known to increase the affinity of ICAP1 for integrin β1 and thereby suppress integrin activation by talin, was not detected in this study; however, this could reflect an increased association of ICAP1 with KRIT1/CCM1 in the absence of Thr38 phosphorylation potentially favored by the TAP protocol. No phosphorylation sites were detected in HEG1.

The data associated with this publication can be accessed and manipulated through the Cell Migration Gateway, here http://www.cellmigration.org/resource/proteomics/data/phospho_index.shtml

• Kim J, Sherman NE, Fox JW, Ginsberg MH. Phosphorylation sites in the cerebral cavernous malformations complex. J Cell Sci. 2011 Dec 1;124(Pt 23):3929-32 PubMed

Retinal angiogenesis: fibronectin performs a two-fold function

During the development of the retinal vascular system, astrocytes guide the leading tips of endothelial cells by depositing a provisional extracellular matrix (ECM). The precise involvement of the major ECM component fibronectin in retinal angiogenesis has so far not been addressed. Stenzel et al. have now shown that fibronectin is expressed and deposited by astrocytes ahead of the growing retinal vasculature in mice, and have taken a genetic approach to elucidate its function. Using Cre-lox-mediated recombination, the authors specifically ablated fibronectin expression in astrocytes ahead of the growing vasculature, causing a decrease in the radial expansion of the vascular plexus through reduced tip-cell migration. Surprisingly, although endothelial cells express both integrin α5 and integrin β1, which mediate binding to fibronectin, endothelium-specific ablation of integrin α5 caused only a marginal reduction in radial migration, suggesting that endothelial α5β1 is not necessary for outgrowth; instead, it supports the alignment and adhesion of filopodia to the astrocytic scaffold. Binding of vascular endothelial growth factor (VEGF) to fibronectin was recently reported to be required for endothelial cell migration, and Stenzel et al. observed that the absence of fibronectin reduced VEGF receptor (VEGFR)2-mediated signaling through phosphoinositide 3-kinase and Akt. They also showed that the blocking peptide FnIII_13–14, which inhibits VEGF–fibronectin binding, inhibited endothelial cell migration in cell culture and significantly reduced the radial expansion of retinal vessels when injected intraocularly, coincident with a decrease in phosphorylation of VEGFR2 and Akt. VEGF also interacts with heparan-sulfate proteoglycans, the expression pattern of which resembles that of fibronectin on astrocytes, and the authors showed, using genetic deletion, that both of these ECM components synergize to promote the directional migration of endothelial tip cells. So fibronectin carries out both integrin-binding and growth-factor-binding functions during retinal angiogenesis.

• Stenzel D, Lundkvist A, Sauvaget D, Busse M, Graupera M, van der Flier A, Wijelath ES, Murray J, Sobel M, Costell M, Takahashi S, Fässler R, Yamaguchi Y, Gutmann DH, Hynes RO, Gerhardt H. Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis. Development. 2011 Oct;138(20):4451-63. PubMed