Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the research highlight will be accessible via the Updates page.
Akt membrane localization and activation is dependent on the presence of cholesterol- and sphingolipid-rich nanodomains.
Membrane rafts are nanodomains within cell membranes that are rich in sphingolipids and cholesterol. Although they are thought to have a role in signal transduction, their suboptical size has complicated efforts to study their function in live cells, and their importance remains a matter of debate. Reporting in Nature Chemical Biology, Lasserre et al. employ a unique fluorescence correlation spectroscopy (FSC) strategy to detect raft nanodomains in the plasma membrane of living cells and show that membrane rafts are required for both membrane recruitment and activation of the protein kinase Akt.
The authors had previously used FSC to reveal plasma membrane lateral organizations as these structures can constrain the lateral diffusion of associated lipids and proteins. In the current study, FCS was used to examine the existence of raft nanodomains and role of specific lipid components in their formation. Myriocin and zaragozic acid treatment, which block sphingolipid and cholesterol biosynthesis, respectively, de-repressed the lateral diffusion of putative raft-associated proteins, indicating the existence of rafts in vivo and importance of sphingolipids and cholesterol in the formation of these structures.
It has been proposed that cholesterol-rich membrane domains regulate Akt activation. Could the lipid rafts identified by Lasserre et al. affect phosphoinositide 3-kinase (PI(3)K)-Akt signaling? Membrane receptors such as CD28 and the insulin growth factor (IGF) receptor stimulate PI(3)K to generate a pool of membrane-localized phosphatidylinositol-3,4,5-triphosphate (PIP3). Akt binds to these phospholipids and is recruited to the plasma membrane, where it is phosphorylated and activated by membrane-localized kinases. Combined myriocin and zaragozic acid treatment blocked CD28-mediated activation of the PI(3)K-Akt pathway and inhibited Akt membrane association and phosphorylation in T cells, thus supporting the necessity for rafts in Akt signaling. These compounds also blocked IGF-stimulated Akt membrane recruitment and phosphorylation in COS-7 fibroblasts. Intriguingly, depletion of cholesterol and sphingolipids had no effect on CD28 or PI(3)K activation in T cells or IGF receptor activation in COS-7 cells, indicating a specific effect on Akt.
Further support for this observation came from studies in Jurkat cells, which contain constitutively high levels of PIP3. In these cells, inhibition of cholesterol and sphingolipid biosynthesis did not affect PIP3 levels but nevertheless blocked Akt membrane association and activation, suggesting a direct link between formation of PIP3-containing raft nanodomains and Akt signaling. Indeed, raft reconstitution in cholesterol- and sphingolipid-deficient cells restored Akt membrane recruitment and phosphorylation. Thus, membrane rafts are critical for Akt membrane association and activation in a variety of cell types. The authors propose that this Akt-specific effect reflects a potential role for Akt in promoting formation of the PIP3-containing rafts. Further studies are necessary to determine what part, if any, Akt or another PI(3)K pathway component plays in formation of these specific raft nanodomains.
Emily J. Chenette Signaling Gateway
Original References:
Lasserre, R. et al. Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation. Nature Chemical Biology4, 538-547 (2008) Full text | PDF | Subscribe to Nature Chemical Biology
Cell adhesion: Talin shifts cell spreading into high gear
The cytoskeletal protein talin functions as a molecular clutch that links the contracting actin cytoskeleton to extracellular matrix-bound integrins.
When suspended fibroblasts encounter a substratum such as fibronectin, they spread and then generate stable sites of cell-substratum contact, or focal adhesions (FAs). The FA protein talin links actin microfilaments to integrins, increasing adhesion by stimulating integrin-fibronectin binding. Talin-1-deficient cells spread and adhere normally due to the compensatory upregulation of talin-2, complicating efforts to understand its role in cell adhesion. In Nature Cell Biology, Zhang et al. now report that talin is a molecular clutch that links the actin cytoskeleton to fibronectin-bound integrins and facilitates the formation of mature FAs.
Mouse embryonic fibroblasts that lacked talin-1 and talin-2 were generated to study the role of talin in cell adhesion. These cells were capable of initial cell spreading mediated by integrin-fibronectin interactions, but the leading-edge protrusions later retracted. In talin-deficient cells, focal adhesion kinase (FAK) was not activated upon cell adhesion and the talin-binding FA proteins vinculin and paxillin were mislocalized; this defect inhibited FA and stress fiber formation and resulted in the detachment of the cell membrane from the extracellular matrix (ECM). Exogenous expression of the isolated head domain of talin-1 (talin-1H), which interacts with integrins and increases their affinity for fibronectin, stabilized adherence to the ECM. However, talin-H1 did not promote the activation of FAK or reconstitute mature FA complexes. Thus, talin is required for FA formation and sustained ECM adhesion.
Following initial spreading, a myosin-II-driven contractile force generates a rearward flow of actin that is constrained to the cell periphery by FAs and stress fiber formation. The contractile force generates tension that causes the maturation of FAs and initiates force-dependent signaling events. In talin-deficient cells, however, the lack of such cytoskeletal features accelerated and expanded the rearward actin flow. Furthermore, the loss of FAs effectively uncoupled the cellular myosin II 'motor' from the ECM 'track', and the resulting loss of traction caused retraction of the leading edge and cell rounding.
These studies suggest that talin is dispensable for initial cell spreading but is required for sustained cell-substratum contact, FA formation and traction. The authors predict that talin is a molecular clutch that links fibronectin-bound integrins to the contracting actin cytoskeleton. Previous studies have shown that calpain-mediated talin cleavage, which liberates the head domain, is necessary for the disassembly of FAs. The work of Zhang et al. suggests that cleavage would disengage the talin clutch but still stimulate integrin-fibronectin binding and weak adhesion. It will be important to determine if calpain-mediated cleavage can be successfully incorporated into this model of talin function.
Emily J. Chenette Signaling Gateway
Original References:
Zhang, X., Jiang, G., Cai, Y., Monkley, S. J., Critchley, D. R. & Sheetz, M. P. Talin depletion reveals independence of initial cell spreading from integrin activation and traction. Nature Cell Biology10, 1062-1068 (2008) Full text | PDF | Subscribe to Nature Cell Biology
Frame, M. & Norman, J. A tal(in) of cell spreading. Nature Cell Biology10, 1017-1019 (2008) Full text | PDF | Subscribe to Nature Cell Biology
Neuronal migration: A direct road from Neurog2 to Rnd2
The transcription factor Neurogenin 2 promotes the radial migration of newly born neurons by directly activating the small Rho GTPase Rnd2.
A key step in nervous system development is the migration of neurons from their birthplace to a final permanent location. Proneural transcription factors are known to control the differentiation of neural stem cells into neurons, as well as promoting neuronal radial migration in the embryonic cerebral cortex. However, the mechanisms underlying this migration-promoting activity have remained unclear. Reporting in Nature, François Guillemot and colleagues have identified the small Rho GTPase Rnd2 as a direct target of the transcription factor Neurogenin 2 (Neurog2), which is involved in neuronal migration.
The authors used microarray expression analysis to identify Rnd2 as a gene that is downregulated in Neurog2-mutant mice embryos, and upregulated when Neurog2 is overexpressed. Expression studies revealed that Rnd2 is restricted to migrating cortical neurons and their immediate precursors, and confirmed that this expression was dependent on Neurog2.
Rnd2 silencing resulted in the disruption of all stages of migration through the cortex and the alteration of neuronal morphology. Conversely, other features such as progenitor proliferation and cortical neuron specification, as well as the organization of radial glia along which neurons migrate, were unaffected. These findings indicate that Rnd2 functions cell-autonomously to regulate both the shape and migration of cortical neurons.
Expression of Rnd2 via the NeuroD1 promoter, which is transiently and moderately active in newborn cortical neurons, rescued the early radial migration defects seen in Neurog2 mutant neurons, but did not rescue the final migration phase for correct neuronal positioning in the cortical plate. Thus, Rnd2 appears to be the main downstream effector of Neurog2 that promotes radial migration, whereas other factors might regulate later stages of these processes.
But is Rnd2 a direct target of Neurog2 or is it possible that its expression is induced via a transcriptional cascade? Using a luciferase reporter assay, the authors showed that Neurog2 was able to efficiently activate transcription from an Rnd2 3' enhancer containing conserved E-boxes specific to Neurog2. Furthermore, Neurog2 bound to the Rnd2 3' enhancer in cortical cells in vivo. Together these results show that Neurog2 directly induces Rnd2 expression in the embryonic cortex.
This work reports for the first time the spatio-temporal regulation of a small GTPase at the transcriptional level. Unlike other Rho family members, Rnd proteins are not regulated by guanine nucleotide exchange factors (GEFs) or GTPase-activated proteins (GAPs), and their expression is thought to have an important regulatory role. Interestingly, whereas Neurog2 is downregulated when neuronal progenitors stop dividing, Rnd2 continues to be expressed. This suggests that other factors might be responsible for the maintenance of Rnd2 expression.
Kim Baumann Cell Migration Gateway
Original References:
Heng, J. I-T. et al. Neurogenin 2 controls cortical neuron migration through regulation of Rnd2 Nature455, 114-118 (2008) Full text | PDF | Subscribe to Nature
A single microRNA molecule can regulate the translation of hundreds of proteins by inducing degradation and inhibiting translation of mRNA targets.
MicroRNAs (miRNAs) control protein levels by inhibiting translation and/or inducing degradation of messenger RNA (mRNA). So far there has been no large-scale analysis of the relative importance of these two mechanisms. Reporting in Nature, Selbach et al. and Baek et al. measured global changes in protein and mRNA levels after ectopic expression or silencing of a handful of miRNAs. Both groups found that a given miRNA can regulate translation of hundreds of proteins, although most mRNA targets are repressed only weakly.
Cell proteomes were assessed using stable isotope labeling with amino acids in cell culture followed by mass spectrometry. mRNA levels were measured in parallel by microarray. Bioinformatic analysis confirmed that a 7–8mer matching the miRNA binding sequence in the 3' untranslated regions (UTRs) of target mRNAs strongly correlates with repression. Baek et al. showed that only one miRNA binding site can mediate inhibition. Although miRNAs can act exclusively at the translation level, the targets repressed most also showed the greatest changes in mRNA, highlighting that effective repression probably requires degradation of mRNA targets. Selbach et al. showed that let-7 miRNA expression correlated with downregulation of DICER miRNA translation, which encodes an enzyme involved in miRNA production, suggesting that let-7 also acts indirectly on many other targets. Thus, a given miRNA subtly adjusts a large fraction of the proteome.
Original References:
Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs Nature455, 58-63 (2008) Full text | PDF | Subscribe to Nature
Baek, D. et al. The impact of microRNAs on protein output Nature455, 64-71 (2008) Full text | PDF | Subscribe to Nature
