Each week we highlight a hot new cell signaling paper. Original research published by Nature Publishing Group will be made freely available for three months.
Cell-specific signaling networks during the interaction between cells.
How signals are processed by interacting cells is largely unknown. In a new study in Science, Pawson, Linding and colleagues have dissected cell-specific signaling networks during the interaction between cells expressing transmembrane Eph receptor Tyr kinases (EphRs) and cells expressing their membrane-bound ephrin ligands. They found that the receptor- and ligand-expressing cells use different Tyr kinases and phosphorylation targets to process signals induced by cell-cell contacts.
To study bidirectional EphR-ephrin signaling in the context of direct cell-cell interactions, the authors labeled cells expressing EphB2 or ephrin B1 using different amino acid isotopes, which enable relative quantification of Tyr phosphorylation peptides (and therefore signaling events) by mass spectrometry in a cell line-specific manner. They found that global changes in Tyr phosphorylation induced by cell–cell contact differ between the two cell types. The comparison of cell-specific modulation of the 100 Tyr phosphorylation sites present in both cell types identified asymmetric regulation of proteins that have a wide range of molecular and cellular functions. For example, the phosphorylation of adaptor proteins is preferentially increased in EphB2-expressing cells, suggesting that they have cell-specific regulatory roles.
When cells expressing EphB2 and ephrin B1 contact each other in cell culture they segregate and form distinct colonies with well-defined boundaries. To study the role of selected signaling proteins during this process, a small interfering RNA (siRNA) screen was performed. Among the 200 targets that affected EphB2-ephrin B1 cell segregation, 37 were significantly modulated in EphB2-expressing cells and 26 were significantly modulated in ephrin-expressing cells. The siRNA data were combined with the quantified modulation of Tyr phosphorylation in the two cell types. The authors then used the NetPhorest and NetworKIN algorithms to derive a computational model of cell-specific dynamic signaling networks during cell segregation. The analysis revealed that the asymmetric regulation of Tyr phosphorylation events in EphB2- and ephrin B1-expressing cells is achieved through the alternative use of kinases and adaptor proteins.
Cells expressing a variant of ephrin B1 that lacks the cytoplasmic region (ephrin B1ΔIC) elicit a unidirectional signal in EphB2-expressing cells but do not induce signaling in the cell in which they are expressed. Notably, the analysis of EphB2-ephrin B1ΔIC signaling revealed that the cytoplasmic region of ephrin B1 is required for cell sorting and also affects signaling in neighboring cells that express EphB2. This shows that EphR-ephrin signaling is not entirely cell autonomous.
Using proteomic and computational approaches, this study provides important new insights into cell-specific signaling networks in two populations of cells, while they contact each other. Similar integrative network biology studies will enable more accurate studies of the effects of cell–cell interactions and signaling networks during normal and pathological processes.
References:
Jørgensen, C. et al. Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells Science326, 1502-1509 (2009) Abstract | PDF
A clever yeast-based system clears up confusion over store-operated calcium entry in mammals
The past three years have seen a revolution in our understanding of store-operated calcium entry, with the discoveries of the two key molecular players: STIM1, in the endoplasmic reticulum, and ORAI1, in the plasma membrane. But the question of whether STIM1 and ORAI1 are sufficient for gating has been difficult to answer. Now Patrick Hogan and colleagues report in Nature Structural & Molecular Biology a clever yeast-based system that resolves this issue.
Calcium entry into cells is essential for many processes, including transcription and response to stimuli. One major route by which calcium ions enter the cell is through the calcium release-activated calcium (CRAC) channel: an initial release of calcium from internal cellular stores triggers sustained calcium signaling and opening of CRAC channels in the plasma membrane.
STIM1 senses depletion of calcium from internal stores in the endoplasmic reticulum, it oligomerizes and then recruits ORAI1, a pore subunit of a plasma membrane calcium channel, which allows calcium in through its open channel. Structural and biochemical studies have uncovered the mechanism of STIM1 calcium sensing and recruitment of ORAI1, but establishing whether STIM1 and ORAI1 are both necessary and sufficient for calcium influx has been hampered by the fact that mammalian cells used to express store-operated channels have their own store-operated pathways, complicating the results.
Now Hogan and colleagues resolve this issue. They provide compelling data that STIM1 and ORAI1 interact directly, and that this is sufficient to open the ORAI1 channels. Their system uses the yeast Saccharomyces cerevisiae, because it does not have a conventional store-operated calcium entry system and does not express ORAI1 or STIM1, or proteins with significant similarity to them.
The authors expressed ORAI1 in a mutant strain of S. cerevisiae, sec6-4, that cannot traffic vesicles from the Golgi to the plasma membrane and so it was possible to isolate these vesicles and use them as a model system. Having established, using a membrane-flotation assay, that STIM1 binds directly to ORAI1, they looked at whether this interaction was sufficient to open the ORAI1 channel. Experiments to detect calcium efflux indicated that both proteins are needed; mutant ORAI1 proteins that do not support calcium influx in mammalian cells also did not support calcium release in this in vitro assay.
By exploiting the lack of STIM1–ORAI1 signaling in S. cerevisiae, this work indicates that native STIM1 in cells interacts directly with ORAI1 to bridge the 8–17 nanometer distance that separates the endoplasmic reticulum and plasma membrane at their closest approach. This in vitro assay, with its defined protein reagents, will be a useful system to further characterize store-operated calcium gating.
Maria Hodges Nature Structural & Molecular Biology
Reference:
Zhou, Y. et al. STIM1 gates the store-operated calcium channel ORAI1 in vitro Nature Structural & Molecular Biology17, 112-116 (2009) Full text | PDF | Subscribe to Nature Structural & Molecular Biology
Germ cells go it alone: E-cadherin, Rac 'n' Rho
Rac1- and RhoA-mediated actin dynamics and E-cadherin-mediated cell-cell adhesion are all required for single-cell motility in vivo
Molecular mechanisms regulating single-cell motility in vitro are well characterized, but less is known about the mechanisms involved in vivo. Erez Raz and colleagues now report in Nature Cell Biology that germ-cell motility and migration in vivo are maintained by the orchestrated effects of the Rho GTPases Rac1 and RhoA on actin dynamics and E-cadherin-mediated cell–cell adhesion.
The authors first asked where active RhoGTPases are located in migrating zebrafish germ cells. Using in vivo fluorescence resonance energy transfer (FRET) to monitor Rac1 and RhoA activity, they showed that Rac1 is activated predominantly at the leading edge and its distribution coincides with actin-rich 'brushes', whereas RhoA activation was detected at the cell front and to a lesser extent at the rear. To investigate the roles of Rac1 and RhoA in cell migration, the authors modified the activity levels of the RhoGTPases. Lowering Rac1 activity resulted in loss of actin brushes from the leading edge and loss of cell polarity. Conversely, expression of a constitutively active form of Rac1 promoted actin brush formation. For RhoA, reducing its activity resulted in stationary actin brush formation at the cell's periphery, whereas expression of a constitutively active form of RhoA increased retrograde flow of actin, which accumulates at the cell rear and resulted in abnormal motility. Importantly, Rac1 or RhoA activity was not affected by chemokine signaling. These findings suggest that Rac1 mediates formation of actin-rich brushes and RhoA regulates retrograde actin flow. Together, they contribute to the maintenance of general cell motility, rather than directional migration.
Next, the authors investigated how the actin cytoskeleton within the cells interacted with the extracellular environment during migration. In vivo time-lapse imaging of isolated germ cells migrating on different extracellular matrix substrates demonstrated that the cells generate protrusions, but are not motile and do not adhere to the surfaces. Importantly, knocking down integrins with morpholino (MO) antisense nucleotides or blocking their function with RGD peptides did not affect normal migration of germ cells toward their targets. This suggested that cell–matrix interactions are not essential for germ-cell motility and migration.
In vivo confocal imaging revealed that the migrating cells interact with surrounding cells, indicating a possible involvement of the cell adhesion molecule E-cadherin in germ-cell migration. Indeed, when Raz and colleagues knocked-down E-cadherin, they found that the adhesion between the germ cells and the somatic cells was reduced. Interestingly, imaging of germ cells that have increased Rho activity and express GFP-tagged E-cadherin showed an accumulation of E-cadherin at the cell rear where actin brushes were found, suggesting that the two molecules interact. Inhibition of E-cadherin with a dominant-negative mutant that lacks key extracellular domains or with an MO knock-down strongly inhibited cell motility, with no effect on retrograde actin flow or cell polarity. This indicates that these processes are not sufficient to maintain proper migration.
Overall, this study provides a model for how actin dynamics and cell adhesion are coordinated to drive single-cell migration in vivo, through Rac-dependent actin brush formation, Rho-dependent retrograde actin flow and E-cadherin-mediated cell–cell adhesion. It also provides an interesting example of integrin-independent migration, where cell–cell adhesion may instead provide traction for the migrating cell.
Kardash, E. et al. A role for Rho GTPases and cell–cell adhesion in single-cell motility in vivo Nature Cell Biology12, 47-53 (2009) Full text | PDF | Subscribe to Nature Cell Biology
Further reading:
Baumann, K. GTPase activation at the leading edge: Advancing technologies Cell Migration Gateway (September 2009) Full text
Heasman, S. J. & Ridley, A. J. Mammalian Rho GTPases: new insights into their functions from in vivo studies Nature Reviews Molecular Cell Biology9, 690-701 (2009) Full text | PDF | Subscribe to Nature Reviews Molecular Cell Biology
Fukata, M. & Kaibuchi, K. Rho-family GTPases in cadherin-mediated cell–cell adhesion Nature Reviews Molecular Cell Biology2, 887-897 (2001) Full text | PDF | Subscribe to Nature Reviews Molecular Cell Biology
Senescence: A key role for Cdk2
The loss of Cdk2 promotes cellular senescence in response to oxidative stress or Myc signaling
Oncogenes promote growth, but their expression can also induce apoptosis or cellular senescence. However, the signaling pathways through which oncogenes effect proliferation, apoptosis or senescence have not yet been defined. In Nature Cell Biology, Bruno Amati and colleagues now identify an important role for the cyclin-dependent kinase Cdk2 in regulating the induction of Myc-mediated senescence.
Expression of the oncogene Myc activates Cdk2 and promotes cell-cycle progression. As previous reports have shown that proliferation is independent of Cdk2, Amati and colleagues sought to understand the role of Cdk2 in Myc-induced proliferation. As expected, transient Myc expression induced hyperproliferation of Cdk2-null and wild-type cells. However, prolonged Myc expression in Cdk2-/- cells induced senescence. Re-expression of wild-type, but not kinase-dead, Cdk2 averted Myc-induced senescence in the Cdk2-deficient cells. Furthermore, expression of Wnt3a — which is thought to signal through Myc — elicited senescence in Cdk2-/- cells, whereas activated Ras induced senescence equally in Cdk2-/- and wild-type cells. These data suggest that Cdk2 attenuates senescence in response to oncogenic Wnt-Myc signaling.
Myc expression in Cdk2-/- cells upregulated the expression of components of the ARF–p53–p21Cip1 and p16INK4a–Rb pathways, and each of these proteins was required to induce Myc-mediated senescence in the absence of Cdk2. But what physiological signals promote Myc-induced senescence in Cdk2-null cells? Oxidative stress, but not other genotoxic stressors, induced p53-, p21Cip1- and p16INK4a-dependent senescence selectively in Cdk2-/- cells. In addition, anti-oxidant treatment prevented Myc-induced senescence in Cdk2-null cells.
Because Cdk2 inhibits senescence, the authors next determined whether the loss of Cdk2 affects lymphoma progression in Eμ-Myc transgenic mice, which overexpress Myc in the B-cell lineage. Indeed, Cdk2-null Eμ-Myc mice experienced a significant delay in the onset of lymphoma compared with heterozygous or wild-type mice. In murine fibroblast and human cancer cell lines, pharmacologic inhibition of Cdk2 with concomitant expression of Myc induced senescence, confirming the importance of Cdk2 in circumventing Myc-mediated senescence and suggesting that Cdk2 inhibitors might be useful anti-cancer therapeutics.
Thus, Cdk2 suppresses senescence following oxidative stress and oncogenic Myc and Wnt signaling. In mice, the ARF–p53–p21Cip1 and p16INK4a–Rb pathways both seem to be crucial for mediating Myc-mediated senescence following the loss of Cdk2. It is intriguing that, although the function of Cdk2 in many cellular processes is redundant with other cyclin-dependent kinases, the loss of Cdk2 but not other Cdks affected Myc-induced senescence. It will be important to identify the molecular target of Cdk2 in this process.
Emily J. Chenette Signaling Gateway
Reference:
Campaner, S. et al. Cdk2 suppresses cellular senescence induced by the c-myc oncogene Nature Cell Biology12, 54-59 (2009) Full text | PDF | Subscribe to Nature Cell Biology
Further reading:
Malumbres, M. & Barbacid, M. Cell cycle, CDKs and cancer: a changing paradigm Nature Reviews Cancer9, 153-166 (2009) Full text | PDF | Subscribe to Nature Reviews Cancer
