David J. Wiley

Oscillatory Dynamics of Cdc42 GTPase in the Control of Polarized Growth

Pole to Pole How do fission yeast cells decide when to grow at a single end (or pole) of the cell or whether to grow in a multipolar manner? Das et al. (p. 239, published online 17 May) found that accumulation of the active form of the small guanine nucleotide–binding protein Cdc42 at the growing tip of the cell oscillated with a period of a few minutes. In cells growing at one pole, the oscillations were primarily present at that pole and during bipolar growth symmetrical anticorrelated oscillations were observed. Dynamic competition for Cdc42 between multiple growth zones could represent a flexible mechanism to modulate cell growth asymmetry. The regulation of a yeast cell-growth enzyme is dynamic rather than on-off. Cells promote polarized growth by activation of Rho-family protein Cdc42 at the cell membrane. We combined experiments and modeling to study bipolar growth initiation in fission yeast. Concentrations of a fluorescent marker for active Cdc42, Cdc42 protein, Cdc42-activator Scd1, and scaffold protein Scd2 exhibited anticorrelated fluctuations and oscillations with a 5-minute average period at polarized cell tips. These dynamics indicate competition for active Cdc42 or its regulators and the presence of positive and delayed negative feedbacks. Cdc42 oscillations and spatial distribution were sensitive to the amounts of Cdc42-activator Gef1 and to the activity of Cdc42-dependent kinase Pak1, a negative regulator. Feedbacks regulating Cdc42 oscillations and spatial self-organization appear to provide a flexible mechanism for fission yeast cells to explore polarization states and to control their morphology.

Mob2p interacts with the protein kinase Orb6p to promote coordination of cell polarity with cell cycle progression.

The molecular mechanisms that temporally and spatially coordinate cell morphogenesis with the cell cycle remain poorly understood. Here we describe the characterization of fission yeast Mob2p, a novel protein required for regulating cell polarity and cell cycle control. Deletion of mob2 is lethal and causes cells to become spherical, with depolarized actin and microtubule cytoskeletons. A decrease in Mob2p protein level results in a defect in the activation of bipolar growth. This phenotype is identical to that of mutants defective in the orb6 protein kinase gene, and we find that Mob2p physically interacts with Orb6p. In addition, overexpression of Mob2p, like that of Orb6p, results in a delay in the onset of mitosis. Mob2p localizes to the cell periphery and cytoplasm throughout the cell cycle and to the division site during late anaphase and telophase. Mob2p is unable to localize to the cell middle in mutants defective in actomyosin ring and septum formation. Our results suggest that Mob2p, along with Orb6p, is required for coordinating polarized cell growth during interphase with the onset of mitosis.

Fission yeast MO25 protein is localized at SPB and septum and is essential for cell morphogenesis

Cell morphogenesis is of fundamental significance in all eukaryotes for development, differentiation, and cell proliferation. In fission yeast, Drosophila Furry‐like Mor2 plays an essential role in cell morphogenesis in concert with the NDR/Tricornered kinase Orb6. Mutations of these genes result in the loss of cell polarity. Here we show that the conserved proteins, MO25‐like Pmo25, GC kinase Nak1, Mor2, and Orb6, constitute a morphogenesis network that is important for polarity control and cell separation. Intriguingly, Pmo25 was localized at the mitotic spindle pole bodies (SPBs) and then underwent translocation to the dividing medial region upon cytokinesis. Pmo25 formed a complex with Nak1 and was required for both the localization and kinase activity of Nak1. Pmo25 and Nak1 in turn were essential for Orb6 kinase activity. Further, the Pmo25 localization at the SPBs and the Nak1‐Orb6 kinase activities during interphase were under the control of the Cdc7 and Sid1 kinases in the septation initiation network (SIN), suggesting a functional linkage between SIN and the network for cell morphogenesis/separation following cytokinesis.

The conserved NDR kinase Orb6 controls polarized cell growth by spatial regulation of the small GTPase Cdc42.

The conserved NDR kinase regulates cell morphogenesis and polarized cell growth in different eukaryotic cells ranging from yeast to neurons. Although studies have unraveled the mechanism of regulation of NDR kinase activity, the mechanism of morphology control by NDR and the effectors that mediate NDR function are unknown. Via a chemical genetic approach, we show that the fission yeast NDR homolog, Orb6 kinase, maintains polarized cell growth at the cell tips by spatially regulating the localization of Cdc42 GTPase, a key morphology regulator. Loss of Orb6 kinase activity leads to the recruitment of Cdc42 GTPase and the Cdc42-dependent formin For3, normally found only at the cell tips, to the cell sides. Furthermore, we show that loss of Orb6 kinase activity leads to ectopic lateral localization of the Cdc42 guanine nucleotide exchange factor (GEF) Gef1, but not of the other Cdc42 GEF, Scd1. Consistent with these observations, gef1 deletion suppresses the increased cell diameter phenotype of orb6 mutants. In contrast, the microtubule cytoskeleton and the localization of the microtubule-dependent polarity markers Tea1 and Tea4 are not altered by loss of Orb6 kinase activity. Our findings indicate that the conserved NDR kinase Orb6 regulates cell polarity by spatially restricting the localization and activity of Cdc42 GTPase.

Eukaryotic Initiation Factor 2 (eIF2) Signaling Regulates Proinflammatory Cytokine Expression and Bacterial Invasion

Background: eIF2 is a critical point of stress-induced regulation of translation in eukaryotic cells. Results: eIF2 signaling is activated by bacterial pathogens and regulates two key infection-associated processes. Conclusion: Regulation of translation in eukaryotic cells is involved in innate immune responses. Significance: These findings enlarge the possible targets for therapeutic interventions against bacterial pathogens. In eukaryotic cells, there are two well characterized pathways that regulate translation initiation in response to stress, and each have been shown to be targeted by various viruses. We recently showed in a yeast-based model that the bacterial virulence factor YopJ disrupts one of these pathways, which is centered on the α-subunit of the translation factor eIF2. Here, we show in mammalian cells that induction of the eIF2 signaling pathway occurs following infection with bacterial pathogens and that, consistent with our yeast-based findings, YopJ reduces eIF2 signaling in response to endoplasmic reticulum stress, heavy metal toxicity, dsRNA, and bacterial infection. We demonstrate that the well documented activities of YopJ, inhibition of NF-κB activation and proinflammatory cytokine expression, are both dependent on an intact eIF2 signaling pathway. Unexpectedly, we found that cells with defective eIF2 signaling were more susceptible to bacterial invasion. This was true for pathogenic Yersinia, a facultative intracellular pathogen, as well as for the intracellular pathogens Listeria monocytogenes and Chlamydia trachomatis. Collectively, our data indicate that the highly conserved eIF2 signaling pathway, which is vitally important for antiviral responses, plays a variety of heretofore unrecognized roles in antibacterial responses.

The Activities of the Yersinia Protein Kinase A (YpkA) and Outer Protein J (YopJ) Virulence Factors Converge on an eIF2α Kinase

The Yersinia protein kinase A (YpkA) and outer protein J (YopJ) are co-expressed from a single transcript and are injected directly into eukaryotic cells by the plague bacterium Yersinia pestis. When overexpressed in vertebrate or yeast cells, YpkA disrupts the actin-based cytoskeletal system by an unknown mechanism, whereas YopJ obstructs inductive chemokine expression by inhibiting MAPK and NF-κB signaling. Previously, we showed that the fission yeast Schizosaccharomyces pombe was sensitive to the kinase activity of YpkA. Here, we screened yeast for cellular processes important for YpkA activity and found that the eIF2α kinases mollify the toxicity imparted by the kinase activity of YpkA. Specifically, strains lacking the eIF2α kinase Hri2 were particularly sensitive to YpkA. Unexpectedly, the activity of YopJ, which conferred a phenotype consistent with its inhibitory effect on MAPK signaling, was also found to be dependent on Hri2. When expressed in S. pombe, YopJ sensitized cells to osmotic and oxidative stresses through a Hri2-dependent mechanism. However, when co-expressed with YpkA, YopJ protected cells from YpkA-mediated toxicity, and this protection was entirely dependent on Hri2. In contrast, YopJ did not confer protection against the toxic effects of the Yersinia virulence factor YopE. These findings are the first to functionally link YpkA and YopJ and suggest that eIF2α kinases, which are critically important in antiviral defenses and protection against environmental stresses, also play a role in bacterial virulence.

Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

The 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, spatially regulating Cdc42 activity during cell morphogenesis. Gef1 is sequestered in the cytoplasm upon 14-3-3 interaction, mediated by Orb6 kinase. The resulting competition for Gef1 promotes anticorrelated Cdc42 oscillations at cell tips.

Yeast Augmented Network Analysis (YANA): a new systems approach to identify therapeutic targets for human genetic diseases

Genetic interaction networks that underlie most human diseases are highly complex and poorly defined. Better-defined networks will allow identification of a greater number of therapeutic targets. Here we introduce our Yeast Augmented Network Analysis (YANA) approach and test it with the X-linked spinal muscular atrophy (SMA) disease gene UBA1. First, we express UBA1 and a mutant variant in fission yeast and use high-throughput methods to identify fission yeast genetic modifiers of UBA1. Second, we analyze available protein-protein interaction network databases in both fission yeast and human to construct UBA1 genetic networks. Third, from these networks we identified potential therapeutic targets for SMA. Finally, we validate one of these targets in a vertebrate (zebrafish) SMA model. This study demonstrates the power of combining synthetic and chemical genetics with a simple model system to identify human disease gene networks that can be exploited for treating human diseases.

Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy

Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 ( UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1’s enzymatic activity with Ubiquitin, to other roles such as altering UBA1 mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners. These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations’ biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.

Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis. DOI: http://dx.doi.org/10.7554/eLife.14216.001