Karen M. Page

Gene Regulatory Logic for Reading the Sonic Hedgehog Signaling Gradient in the Vertebrate Neural Tube

Summary Secreted signals, known as morphogens, provide the positional information that organizes gene expression and cellular differentiation in many developing tissues. In the vertebrate neural tube, Sonic Hedgehog (Shh) acts as a morphogen to control the pattern of neuronal subtype specification. Using an in vivo reporter of Shh signaling, mouse genetics, and systems modeling, we show that a spatially and temporally changing gradient of Shh signaling is interpreted by the regulatory logic of a downstream transcriptional network. The design of the network, which links three transcription factors to Shh signaling, is responsible for differential spatial and temporal gene expression. In addition, the network renders cells insensitive to fluctuations in signaling and confers hysteresis—memory of the signal. Our findings reveal that morphogen interpretation is an emergent property of the architecture of a transcriptional network that provides robustness and reliability to tissue patterning.

Complement Fragment C3a Controls Mutual Cell Attraction during Collective Cell Migration

Summary Collective cell migration is a mode of movement crucial for morphogenesis and cancer metastasis. However, little is known about how migratory cells coordinate collectively. Here we show that mutual cell-cell attraction (named here coattraction) is required to maintain cohesive clusters of migrating mesenchymal cells. Coattraction can counterbalance the natural tendency of cells to disperse via mechanisms such as contact inhibition and epithelial-to-mesenchymal transition. Neural crest cells are coattracted via the complement fragment C3a and its receptor C3aR, revealing an unexpected role of complement proteins in early vertebrate development. Loss of coattraction disrupts collective and coordinated movements of these cells. We propose that coattraction and contact inhibition act in concert to allow cell collectives to self-organize and respond efficiently to external signals, such as chemoattractants and repellents.

The spatial ultimatum game.

In the ultimatum game, two players are asked to split a certain sum of money. The proposer has to make an offer. If the responder accepts the offer, the money will be shared accordingly. If the responder rejects the offer, both players receive nothing. The rational solution is for the proposer to offer the smallest possible share, and for the responder to accept it. Human players, in contrast, usually prefer fair splits. In this paper, we use evolutionary game theory to analyse the ultimatum game. We first show that in a non–spatial setting, natural selection chooses the unfair, rational solution. In a spatial setting, however, much fairer outcomes evolve.

Evidence for Two Concentration-Dependent Processes for β-Subunit Effects on α1B Calcium Channels

beta-Subunits of voltage-dependent Ca(2+) channels regulate both their expression and biophysical properties. We have injected a range of concentrations of beta3-cDNA into Xenopus oocytes, with a fixed concentration of alpha1B (Ca(V)2.2) cDNA, and have quantified the corresponding linear increase of beta3 protein. The concentration dependence of a number of beta3-dependent processes has been studied. First, the dependence of the a1B maximum conductance on beta3-protein occurs with a midpoint around the endogenous concentration of beta3 (approximately 17 nM). This may represent the interaction of the beta-subunit, responsible for trafficking, with the I-II linker of the nascent channel. Second, the effect of beta3-subunits on the voltage dependence of steady-state inactivation provides evidence for two channel populations, interpreted as representing alpha1B without or with a beta3-subunit, bound with a lower affinity of 120 nM. Third, the effect of beta3 on the facilitation rate of G-protein-modulated alpha1B currents during a depolarizing prepulse to +100 mV provides evidence for the same two populations, with the rapid facilitation rate being attributed to Gbetagamma dissociation from the beta-subunit-bound alpha1B channels. The data are discussed in terms of two hypotheses, either binding of two beta-subunits to the alpha1B channel or a state-dependent alteration in affinity of the channel for the beta-subunit.

Dominant-Negative Calcium Channel Suppression by Truncated Constructs Involves a Kinase Implicated in the Unfolded Protein Response

Expression of the calcium channel CaV2.2 is markedly suppressed by coexpression with truncated constructs of CaV2.2. Furthermore, a two-domain construct of CaV2.1 mimicking an episodic ataxia-2 mutation strongly inhibited CaV2.1 currents. We have now determined the specificity of this effect, identified a potential mechanism, and have shown that such constructs also inhibit endogenous calcium currents when transfected into neuronal cell lines. Suppression of calcium channel expression requires interaction between truncated and full-length channels, because there is inter-subfamily specificity. Although there is marked cross-suppression within the CaV2 calcium channel family, there is no cross-suppression between CaV2 and CaV3 channels. The mechanism involves activation of a component of the unfolded protein response, the endoplasmic reticulum resident RNA-dependent kinase (PERK), because it is inhibited by expression of dominant-negative constructs of this kinase. Activation of PERK has been shown previously to cause translational arrest, which has the potential to result in a generalized effect on protein synthesis. In agreement with this, coexpression of the truncated domain I of CaV2.2, together with full-length CaV2.2, reduced the level not only of CaV2.2 protein but also the coexpressed α2δ-2. Thapsigargin, which globally activates the unfolded protein response, very markedly suppressed CaV2.2 currents and also reduced the expression level of both CaV2.2 and α2δ-2 protein. We propose that voltage-gated calcium channels represent a class of difficult-to-fold transmembrane proteins, in this case misfolding is induced by interaction with a truncated cognate CaV channel. This may represent a mechanism of pathology in episodic ataxia-2.

Calcium Channel β Subunit Promotes Voltage-Dependent Modulation of α1B by Gβγ

Abstract Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming α 1 subunit and auxiliary subunits, including the intracellular β subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G βγ dimer concentration. Here we have examined the interaction between the regulation of N-type ( α 1B) channels by their β subunits and by G βγ dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC β subunit, we found a significantly larger G βγ -dependent inhibition of α 1B channel activation when the VDCC α 1B and β subunits were coexpressed. In the absence of coexpressed VDCC β subunit, the G βγ dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC β subunit coexpression restored all of the hallmarks of G βγ modulation. These results suggest that the VDCC β subunit must be present for G βγ to induce voltage-dependent modulation of N-type calcium channels.

β-Subunits Promote the Expression of CaV2.2 Channels by Reducing Their Proteasomal Degradation

The β-subunits of voltage-gated calcium channels regulate their functional expression and properties. Two mechanisms have been proposed for this, an effect on gating and an enhancement of expression. With respect to the effect on expression, β-subunits have been suggested to enhance trafficking by masking an unidentified endoplasmic reticulum (ER) retention signal. Here we have investigated whether, and how, β-subunits affect the level of CaV2.2 channels within somata and neurites of cultured sympathetic neurons. We have used YFP-CaV2.2 containing a mutation (W391A), that prevents binding of β-subunits to its I-II linker and found that expression of this channel was much reduced compared with WT CFP-CaV2.2 when both were expressed in the same neuron. This effect was particularly evident in neurites and growth cones. The difference between the levels of YFP-CaV2.2(W391A) and CFP-CaV2.2(WT) was lost in the absence of co-expressed β-subunits. Furthermore, the relative reduction of expression of CaV2.2(W391A) compared with the WT channel was reversed by exposure to two proteasome inhibitors, MG132 and lactacystin, particularly in the somata. In further experiments in tsA-201 cells, we found that proteasome inhibition did not augment the cell surface CaV2.2(W391A) level but resulted in the observation of increased ubiquitination, particularly of mutant channels. In contrast, we found no evidence for selective retention of CaV2.2(W391A) in the ER, in either the soma or growth cones. In conclusion, there is a marked effect of β-subunits on CaV2.2 expression, particularly in neurites, but our results point to protection from proteasomal degradation rather than masking of an ER retention signal.

The novel product of a five-exon stargazin-related gene abolishes CaV2.2 calcium channel expression

We have cloned and characterized a new member of the voltage‐dependent Ca2+ channel γ subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential γ subunits identified by their homology to the stargazin gene, CACNG7 is a five‐, and not four‐exon gene whose mRNA encodes a protein we have designated γ7. Expression of human γ7 has been localized specifically to brain. N‐type current through CaV2.2 channels was almost abolished when co‐expressed transiently with γ7 in either Xenopus oocytes or COS‐7 cells. Furthermore, immunocytochemistry and western blots show that γ7 has this effect by causing a large reduction in expression of CaV2.2 rather than by interfering with trafficking or biophysical properties of the channel. No effect of transiently expressed γ7 was observed on pre‐existing endogenous N‐type calcium channels in sympathetic neurones. Low homology to the stargazin‐like γ subunits, different gene structure and the unique functional properties of γ7 imply that it represents a distinct subdivision of the family of proteins identified by their structural and sequence homology to stargazin.

The α1B Ca2+ channel amino terminus contributes determinants for β subunit‐mediated voltage‐dependent inactivation properties

1 Co‐expression of auxiliary β subunits with the α1B Ca2+ channel subunit in COS‐7 cells resulted in an increase in current density and a hyperpolarising shift in the mid‐point of activation. Amongst the β subunits, β2a in particular, but also β4 and β1b caused a significant retardation of the voltage‐dependent inactivation compared to currents with α1B alone, whilst no significant changes in inactivation properties were seen for the β3 subunit in this system. 2 Prevention of β2a palmitoylation, by introducing cysteine to serine mutations (β2a(C3,4S)), greatly reduced the ability of β2a to retard voltage‐dependent inactivation. 3 Deletion of the proximal half of the α1B cytoplasmic amino terminus (α1BΔ1‐55) differentially affected β subunit‐mediated voltage‐dependent inactivation properties. These effects were prominent with the β2a subunit and, to a lesser extent, with β1b. For β2a, the major effects of this deletion were a partial reversal of β2a‐mediated retardation of inactivation and the introduction of a fast component of inactivation, not seen with full‐length α1B. Deletion of the amino terminus had no other major effects on the measured biophysical properties of α1B when co‐expressed with β subunits. 4 Transfer of the whole α1B amino terminus into α1C (α1bCCCC) conferred a similar retardation of inactivation on α1C when co‐expressed with β2a to that seen in parental α1B. 5 Individual (α1B(Q47A) and α1B(R52A)) and double (α1B(R52,54A)) point mutations within the amino terminus of α1B also opposed the β2a‐mediated retardation of α1B inactivation kinetics. 6 These results indicate that the α1B amino terminus contains determinants for β subunit‐mediated voltage‐dependent inactivation properties. Furthermore, effects were β subunit selective. As deletion of the α1B amino terminus only partially opposed β subunit‐mediated changes in inactivation properties, the amino terminus is likely to contribute to a complex site necessary for complete β subunit function.

Diversity, Dilemmas, and Monopolies of Niche Construction

The behavior of organisms can contribute to the transformation of their environments. When organismal impacts on the environment feed back to influence organismal density, viability, fertility, or persistence, the environment can be construed as an extension of the organism. This process of fitness‐enhancing environmental transformation has been called niche construction. We focus on the relationship of niche construction with species or strain diversity and on the variability of investment in niche construction versus reproduction. We demonstrate a fundamental dilemma of niche construction, whereby the construction of a shared resource leads to a tragedy of the commons, with competition tending to eliminate niche construction strategies. The ability to monopolize a niche, either through spatial proximity or through preferential exploitation, can stabilize niche construction and promote ecological coexistence among polymorphic constructors. We consider both sympatric and allopatric origins of niche construction. Under a variety of different construction mechanisms, variability in the investment in niche construction versus reproduction suggests reproductive altruism but is fully consistent with selfish behavior. We discuss the implications of niche‐construction theory on the evolution of life cycles and development, behavioral plasticity, the division of labor, and long‐term macroevolutionary trends.