Hidden value.

The fact that it is difficult to evaluate and compare the outputs of individual researchers might actually be good for science.

The rat race.

Labmates are colleagues, friends, comforters…and competitors.

Gß Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration.

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gß and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gß. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gß regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gß and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.

How to understand and outwit adaptation.

Adaptation is the ability of a system to respond and reset itself even in the continuing presence of a stimulus. On one hand, adaptation is a physiological necessity that enables proper neuronal signaling and cell movement. On the other hand, adaptation can be a source of annoyance, as it can make biological systems resistant to experimental perturbations. Here we speculate where adaptation might live in eukaryotic chemotaxis and how it can be encoded in the signaling network. We then discuss tools and strategies that can be used to both understand and outwit adaptation in a wide range of cellular contexts.

Two distinct functions for PI3-kinases in macropinocytosis.

Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.

The antiepileptic drug valproic acid and other medium-chain fatty acids acutely reduce phosphoinositide levels independently of inositol in Dictyostelium.

Valproic acid (VPA) is the most widely prescribed epilepsy treatment worldwide, but its mechanism of action remains unclear. Our previous work identified a previously unknown effect of VPA in reducing phosphoinositide production in the simple model Dictyostelium followed by the transfer of data to a mammalian synaptic release model. In our current study, we show that the reduction in phosphoinositide [PtdInsP (also known as PIP) and PtdInsP(2) (also known as PIP(2))] production caused by VPA is acute and dose dependent, and that this effect occurs independently of phosphatidylinositol 3-kinase (PI3K) activity, inositol recycling and inositol synthesis. In characterising the structural requirements for this effect, we also identify a family of medium-chain fatty acids that show increased efficacy compared with VPA. Within the group of active compounds is a little-studied group previously associated with seizure control, and analysis of two of these compounds (nonanoic acid and 4-methyloctanoic acid) shows around a threefold enhanced potency compared with VPA for protection in an in vitro acute rat seizure model. Together, our data show that VPA and a newly identified group of medium-chain fatty acids reduce phosphoinositide levels independently of inositol regulation, and suggest the reinvestigation of these compounds as treatments for epilepsy.

The mood stabiliser lithium suppresses PIP3 signalling in Dictyostelium and human cells.

Bipolar mood disorder (manic depression) is a major psychiatric disorder whose molecular origins are unknown. Mood stabilisers offer patients both acute and prophylactic treatment, and experimentally, they provide a means to probe the underlying biology of the disorder. Lithium and other mood stabilisers deplete intracellular inositol and it has been proposed that bipolar mood disorder arises from aberrant inositol (1,4,5)-trisphosphate [IP(3), also known as Ins(1,4,5)P(3)] signalling. However, there is no definitive evidence to support this or any other proposed target; a problem exacerbated by a lack of good cellular models. Phosphatidylinositol (3,4,5)-trisphosphate [PIP(3), also known as PtdIns(3,4,5)P(3)] is a prominent intracellular signal molecule within the central nervous system (CNS) that regulates neuronal survival, connectivity and synaptic function. By using the genetically tractable organism Dictyostelium, we show that lithium suppresses PIP(3)-mediated signalling. These effects extend to the human neutrophil cell line HL60. Mechanistically, we show that lithium attenuates phosphoinositide synthesis and that its effects can be reversed by overexpression of inositol monophosphatase (IMPase), consistent with the inositol-depletion hypothesis. These results demonstrate a lithium target that is compatible with our current knowledge of the genetic predisposition for bipolar disorder. They also suggest that lithium therapy might be beneficial for other diseases caused by elevated PIP(3) signalling.

PIP3-independent activation of TorC2 and PKB at the cell's leading edge mediates chemotaxis.

BACKGROUND: Studies show that high phosphotidylinositol 3,4,5-trisphosphate (PIP(3)) promotes cytoskeletal rearrangements and alters cell motility and chemotaxis, possibly through activation of protein kinase Bs (PKBs). However, chemotaxis can still occur in the absence of PIP(3), and the identities of the PIP(3)-independent pathways remain unknown. RESULTS: Here, we outline a PIP(3)-independent pathway linking temporal and spatial activation of PKBs by Tor complex 2 (TorC2) to the chemotactic response. Within seconds of stimulating Dictyostelium cells with chemoattractant, two PKB homologs, PKBA and PKBR1, mediate transient phosphorylation of at least eight proteins, including Talin, PI4P 5-kinase, two Ras GEFs, and a RhoGap. Surprisingly, all of the substrates are phosphorylated with normal kinetics in cells lacking PI 3-kinase activity. Cells deficient in TorC2 or PKB activity show reduced phosphorylation of the endogenous substrates and are impaired in chemotaxis. The PKBs are activated through phosphorylation of their hydrophobic motifs via TorC2 and subsequent phosphorylation of their activation loops. These chemoattractant-inducible events are restricted to the cell's leading edge even in the absence of PIP(3). Activation of TorC2 depends on heterotrimeric G protein function and intermediate G proteins, including Ras GTPases. CONCLUSIONS: The data lead to a model where cytosolic TorC2, encountering locally activated small G protein(s) at the leading edge of the cell, becomes activated and phosphorylates PKBs. These in turn phosphorylate a series of signaling and cytoskeletal proteins, thereby regulating directed migration.

Changing directions in the study of chemotaxis.

Chemotaxis--the guided movement of cells in chemical gradients--probably first emerged in our single-celled ancestors and even today is recognizably similar in neutrophils and amoebae. Chemotaxis enables immune cells to reach sites of infection, allows wounds to heal and is crucial for forming embryonic patterns. Furthermore, the manipulation of chemotaxis may help to alleviate disease states, including the metastasis of cancer cells. This review discusses recent results concerning how cells orientate in chemotactic gradients and the role of phosphatidylinositol-3,4,5-trisphosphate, what produces the force for projecting pseudopodia and a new role for the endocytic cycle in movement.

Chemotaxis in the absence of PIP3 gradients.

Chemotaxing neutrophils and Dictyostelium amoebae produce in their plasma membranes the signaling lipid PI(3,4,5)P3 (PIP3) in gradients, which are orientated with the external chemotactic gradient and have been proposed to act as an internal compass, guiding movement of the cell. Evidence for and against this idea exists, but in all cases it depends on the use of inhibitors or gene knockouts, which may only incompletely abolish the PIP3 gradient. We have created a multiple gene-knockout strain in Dictyostelium lacking all five type-1 phosphoinositide 3-kinases encoded in the genome and the PTEN phosphatase and have thus removed all known ways for chemoattractant to produce PIP3 gradients in the plasma membrane. The resulting sextuple mutant is able to chemotax to cyclic-AMP with near wild-type efficiency and to trigger actin polymerization without apparent defect. There is, however, a consistent defect in movement speed in chemotaxis and especially in random movement. This work shows that polarization of membrane PIP3 is not necessary for accurate chemotaxis, but it can affect cell speed. A signaling pathway from receptor to cytoskeleton able to guide cells independently of polarized PIP3 and type-1 phosphoinositide 3-kinases must exist.

Mammalian inscuteable regulates spindle orientation and cell fate in the developing retina.

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome.

Drosophila Ric-8 is essential for plasma-membrane localization of heterotrimeric G proteins.

Heterotrimeric G proteins act during signal transduction in response to extracellular ligands. They are also required for spindle orientation and cell polarity during asymmetric cell division. We show here that, in Drosophila, both functions require the Galpha interaction partner Ric-8. Drosophila Ric-8 is a cytoplasmic protein that binds both the GDP- and GTP-bound form of the G-protein alpha-subunit Galphai. In ric-8 mutants, neither Galphai nor its associated beta-subunit Gbeta13F are localized at the plasma membrane, which leads to their degradation in the cytosol. During asymmetric cell division, this leads to various defects: apico-basal polarity is not maintained, mitotic spindles are misoriented and the size of the two daughter cells becomes nearly equal. ric-8 mutants also have defects in gastrulation that resemble mutants in the Galpha protein concertina or the extracellular ligand foldedgastrulation. Our results indicate a model in which both receptor-dependent and receptor-independent G-protein functions are executed at the plasma membrane and require the Ric-8 protein.