Drosophila phosphoinositide-dependent kinase-1 regulates apoptosis and growth via the phosphoinositide 3-kinase-dependent signaling pathway

Phosphoinositide-dependent kinase-1 (PDK-1) is a central mediator of the cell signaling between phosphoinositide 3-kinase (PI3K) and various intracellular serine/threonine kinases including Akt/protein kinase B (PKB), p70 S6 kinases, and protein kinase C. Recent studies with cell transfection experiments have implied that PDK-1 may be involved in various cell functions including cell growth and apoptosis. However, despite its pivotal role in cellular signalings, the in vivo functions of PDK-1 in a multicellular system have rarely been investigated. Here, we have isolated Drosophila PDK-1 (dPDK-1) mutants and characterized the in vivo roles of the kinase. Drosophila deficient in the dPDK-1 gene exhibited lethality and an apoptotic phenotype in the embryonic stage. Conversely, overexpression of dPDK-1 increased cell and organ size in a Drosophila PI3K-dependent manner. dPDK-1 not only could activate Drosophila Akt/PKB (Dakt1), but also substitute the in vivo functions of its mammalian ortholog to activate Akt/PKB. This functional interaction between dPDK-1 and Dakt1 was further confirmed through genetic analyses in Drosophila. On the other hand, cAMP-dependent protein kinase, which has been proposed as a possible target of dPDK-1, did not interact with dPDK-1. In conclusion, our findings provide direct evidence that dPDK-1 regulates cell growth and apoptosis during Drosophila development via the PI3K-dependent signaling pathway and demonstrate our Drosophila system to be a powerful tool for elucidating the in vivo functions and targets of PDK-1.

T cell-specific FADD-deficient mice: FADD is required for early T cell development

FADD/Mort1, initially identified as a Fas-associated death-domain containing protein, functions as an adapter molecule in apoptosis initiated by Fas, tumor necrosis factor receptor-I, DR3, and TRAIL-receptors. However, FADD likely participates in additional signaling cascades. FADD-null mutations in mice are embryonic-lethal, and analysis of FADD−/− T cells from RAG-1−/− reconstituted chimeras has suggested a role for FADD in proliferation of mature T cells. Here, we report the generation of T cell-specific FADD-deficient mice via a conditional genomic rescue approach. We find that FADD-deficiency leads to inhibition of T cell development at the CD4−CD8− stage and a reduction in the number of mature T cells. The FADD mutation does not affect apoptosis or the proximal signaling events of the pre-T cell receptor; introduction of a T cell receptor transgene fails to rescue the mutant phenotype. These data suggest that FADD, through either a death-domain containing receptor or a novel receptor-independent mechanism, is required for the proliferative phase of early T cell development.

Altered stability of pulmonary surfactant in SP-C-deficient mice

The surfactant protein C (SP-C) gene encodes an extremely hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the lung. To discern the role of SP-C in lung function, SP-C-deficient (−/−) mice were produced. The SP-C (−/−) mice were viable at birth and grew normally to adulthood without apparent pulmonary abnormalities. SP-C mRNA was not detected in the lungs of SP-C (−/−) mice, nor was mature SP-C protein detected by Western blot of alveolar lavage from SP-C (−/−) mice. The levels of the other surfactant proteins (A, B, D) in alveolar lavage were comparable to those in wild-type mice. Surfactant pool sizes, surfactant synthesis, and lung morphology were similar in SP-C (−/−) and SP-C (+/+) mice. Lamellar bodies were present in SP-C (−/−) type II cells, and tubular myelin was present in the alveolar lumen. Lung mechanics studies demonstrated abnormalities in lung hysteresivity (a term used to reflect the mechanical coupling between energy dissipative forces and tissue-elastic properties) at low, positive-end, expiratory pressures. The stability of captive bubbles with surfactant from the SP-C (−/−) mice was decreased significantly, indicating that SP-C plays a role in the stabilization of surfactant at low lung volumes, a condition that may accompany respiratory distress syndrome in infants and adults.

In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair

Biochemical studies with model DNA heteroduplexes have implicated RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X in Escherichia coli methyl-directed mismatch correction. However, strains deficient in the four exonucleases display only a modest increase in mutation rate, raising questions concerning involvement of these activities in mismatch repair in vivo. The quadruple mutant deficient in the four exonucleases, as well as the triple mutant deficient in RecJ exonuclease, exonuclease VII, and exonuclease I, grow poorly in the presence of the base analogue 2-aminopurine, and exposure to the base analogue results in filament formation, indicative of induction of SOS DNA damage response. The growth defect and filamentation phenotypes associated with 2-aminopurine exposure are effectively suppressed by null mutations in mutH, mutL, mutS, or uvrD/mutU, which encode activities that act upstream of the four exonucleases in the mechanism for the methyl-directed reaction that has been proposed based on in vitro studies. The quadruple exonuclease mutant is also cold-sensitive, having a severe growth defect at 30°C. This phenotype is suppressed by a uvrD/mutU defect, and partially suppressed by mutH, mutL, or mutS mutations. These observations confirm involvement of the four exonucleases in methyl-directed mismatch repair in vivo and suggest that the low mutability of exonuclease-deficient strains is a consequence of under recovery of mutants due to a reduction in viability and/or chromosome loss associated with activation of the mismatch repair system in the absence of RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X.

Enhanced resistance in STAT6-deficient mice to infection with ectromelia virus

We inoculated BALB/c mice deficient in STAT6 (STAT6−/−) and their wild-type (wt) littermates (STAT6+/+) with the natural mouse pathogen, ectromelia virus (EV). STAT6−/− mice exhibited increased resistance to generalized infection with EV when compared with STAT6+/+ mice. In the spleens and lymph nodes of STAT6−/− mice, T helper 1 (Th1) cytokines were induced at earlier time points and at higher levels postinfection when compared with those in STAT6+/+ mice. Elevated levels of NO were evident in plasma and splenocyte cultures of EV-infected STAT6−/− mice in comparison with STAT6+/+ mice. The induction of high levels of Th1 cytokines in the mutant mice correlated with a strong natural killer cell response. We demonstrate in genetically susceptible BALB/c mice that the STAT6 locus is critical for progression of EV infection. Furthermore, in the absence of this transcription factor, the immune system defaults toward a protective Th1-like response, conferring pronounced resistance to EV infection and disease progression.

Isolation and Characterization of Effector-Loop Mutants of CDC42 in Yeast

The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of “effector-loop” mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.

Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomyces cerevisiae

Accumulation of misfolded proteins in the cell at high temperature may cause entry into a nonproliferating, heat-shocked state. The imino acid analog azetidine 2-carboxylic acid (AZC) is incorporated into cellular protein competitively with proline and can misfold proteins into which it is incorporated. AZC addition to budding yeast cells at concentrations sufficient to inhibit proliferation selectively activates heat shock factor (HSF). We find that AZC treatment fails to cause accumulation of glycogen and trehalose (Msn2/4-dependent processes) or to induce thermotolerance (a protein kinase C-dependent process). However, AZC-arrested cells can accumulate glycogen and trehalose and can acquire thermotolerance in response to a subsequent heat shock. We find that AZC treatment arrests cells in a viable state and that this arrest is reversible. We find that cells at high temperature or cells deficient in the ubiquitin-conjugating enzymes Ubc4 and Ubc5 are hypersensitive to AZC-induced proliferation arrest. We find that AZC treatment mimics temperature up-shift in arresting cells in G1 and represses expression of CLN1 and CLN2. Mutants with reduced G1 cyclin-Cdc28 activity are hypersensitive to AZC-induced proliferation arrest. Expression of the hyperstable Cln3–2 protein prevents G1 arrest upon AZC treatment and temperature up-shift. Finally, we find that the EXA3–1 mutation, encoding a defective HSF, prevents efficient G1 arrest in response to both temperature up-shift and AZC treatment. We conclude that nontoxic levels of misfolded proteins (induced by AZC treatment or by high temperature) selectively activate HSF, which is required for subsequent G1 arrest.

Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP

We have introduced a targeted mutation in SH2D1A/DSHP/SAP, the gene responsible for the human genetic disorder X-linked lymphoproliferative disease (XLP). SLAM-associated protein (SAP)-deficient mice had normal lymphocyte development, but on challenge with infectious agents, recapitulated features of XLP. Infection of SAP− mice with lymphocyte choriomeningitis virus (LCMV) or Toxoplasma gondii was associated with increased T cell activation and IFN-γ production, as well as a reduction of Ig-secreting cells. Anti-CD3-stimulated splenocytes from uninfected SAP− mice produced increased IFN-γ and decreased IL-4, findings supported by decreased serum IgE levels in vivo. The Th1 skewing of these animals suggests that cytokine misregulation may contribute to phenotypes associated with mutation of SH2D1A/SAP.

Carbonic Anhydrase Activity and CO2-Transfer Resistance in Zn-Deficient Rice Leaves1

It has been reported that carbonic anhydrase (CA) activity in plant leaves is decreased by Zn deficiency. We examined the effects of Zn deficiency on the activity of CA and on photosynthesis by leaves in rice plants (Oryza sativa L.). Zn deficiency increased the transfer resistance from the stomatal cavity to the site of CO2 fixation 2.3-fold and, consequently, the value of the transfer resistance relative to the total resistance in the CO2-assimilation process increased from 10% to 21%. This change led to a reduced CO2 concentration at the site of CO2 fixation, resulting in an increased gradient of CO2 between the stomatal cavity and this site. The present findings support the hypothesis that CA functions to facilitate the supply of CO2 from the stomatal cavity to the site of CO2 fixation. We also showed that the level of mRNA for CA decreased to 13% of the control level during Zn deficiency. This decrease resembled the decrease in CA activity, suggesting the possible involvement of the CA mRNA level in the regulation of CA activity.

Interaction of Cryptochrome 1, Phytochrome, and Ion Fluxes in Blue-Light-Induced Shrinking of Arabidopsis Hypocotyl Protoplasts1

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl− and K+. The postshrinking volume recovery is achieved by K+ and Cl− influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.

A New Class of Arabidopsis Mutants with Reduced Hexadecatrienoic Acid Fatty Acid Levels1

Chloroplast glycerolipids in a number of higher-plant species, including Arabidopsis thaliana, are synthesized by two distinct pathways termed the prokaryotic and eukaryotic pathways. The molecules of galactolipids produced by the prokaryotic pathway contain substantial amounts of hexadecatrienoic acid fatty acid. Here we describe a new class of mutants, designated gly1, with reduced levels of hexadecatrienoic acid. Lipid fatty acid profiles indicated that gly1 mutants exhibited a reduced carbon flux through the prokaryotic pathway that was compensated for by an increased carbon flux through the eukaryotic pathway. Genetic and biochemical approaches revealed that the gly1 phenotype could not be explained by a deficiency in the enzymes of the prokaryotic pathway. The flux of fatty acids into the prokaryotic pathway is sensitive to changes in glycerol-3-phosphate (G3P) availability, and the chloroplast G3P pool can be increased by exogenous application of glycerol to leaves. Exogenous glycerol treatment of gly1 plants allowed chemical complementation of the mutant phenotype. These results are consistent with a mutant lesion affecting the G3P supply within the chloroplast. The gly1 mutants may therefore help in determining the pathway for synthesis of chloroplast G3P.

Stepwise Photoinhibition of Photosystem II1 : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the QB-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of QA reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified QB pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.

Suppression of O-Methyltransferase Gene by Homologous Sense Transgene in Quaking Aspen Causes Red-Brown Wood Phenotypes1

Homologous sense suppression of a gene encoding lignin pathway caffeic acid O-methyltransferase (CAOMT) in the xylem of quaking aspen (Populus tremuloides Michx.) resulted in transgenic plants exhibiting novel phenotypes with either mottled or complete red-brown coloration in their woody stems. These phenotypes appeared in all independent transgenic lines regenerated with a sense CAOMT construct but were absent from all plants produced with antisense CAOMT. The CAOMT sense transgene expression was undetectable, and the endogenous CAOMT transcript levels and enzyme activity were reduced in the xylem of some transgenic lines. In contrast, the sense transgene conferred overexpression of CAOMT and significant CAOMT activity in all of the transgenic plants' leaves and sclerenchyma, where normally the expression of the endogenous CAOMT gene is negligible. Thus, our results support the notion that the occurrence of sense cosuppression depends on the degree of sequence homology and endogene expression. Furthermore, the suppression of CAOMT in the xylem resulted in the incorporation of a higher amount of coniferyl aldehyde residues into the lignin in the wood of the sense plants. Characterization of the lignins isolated from these transgenic plants revealed that a high amount of coniferyl aldehyde is the origin of the red-brown coloration—a phenotype correlated with CAOMT-deficient maize (Zea mays L.) brown-midrib mutants.

Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered Patterns of Aluminum Accumulation and Organic Acid Release from Roots1

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27).

Light Promotion of Hypocotyl Gravitropism of a Starch-Deficient Tobacco Mutant Correlates with Plastid Enlargement and Sedimentation1

Dark-grown hypocotyls of a starch-deficient mutant (NS458) of tobacco (Nicotiana sylvestris) lack amyloplasts and plastid sedimentation, and have severely reduced gravitropism. However, gravitropism improved dramatically when NS458 seedlings were grown in the light. To determine the extent of this improvement and whether mutant hypocotyls contain sedimented amyloplasts, gravitropic sensitivity (induction time and intermittent stimulation) and plastid size and position in the endodermis were measured in seedlings grown for 8 d in the light. Light-grown NS458 hypocotyls were gravitropic but were less sensitive than the wild type (WT). Starch occupied 10% of the volume of NS458 plastids grown in both the light and the dark, whereas WT plastids were essentially filled with starch in both treatments. Light increased plastid size twice as much in the mutant as in the WT. Plastids in light-grown NS458 were sedimented, presumably because of their larger size and greater total starch content. The induction by light of plastid sedimentation in NS458 provides new evidence for the role of plastid mass and sedimentation in stem gravitropic sensing. Because the mutant is not as sensitive as the WT, NS458 plastids may not have sufficient mass to provide full gravitropic sensitivity.