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