A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.     

Vaccinia virus F12L protein is required for actin tail formation, normal plaque size, and virulence

Vaccinia virus gene F12L is shown to encode a 65-kDa protein that is synthesized early and late during infection and that is not modified by glycosylation. Computational sequence comparison revealed that related proteins are encoded by all sequenced chordopoxviruses. A virus deletion mutant lacking the F12L gene (vDeltaF12L) and a revertant virus with the F12L gene reinserted into the deletion mutant (vF12L-rev) were constructed and analyzed. A version of the F12L gene with a C-terminal amino acid tag derived from the influenza virus hemagglutinin and that is recognized by a monoclonal antibody was also inserted into the F12L locus of vDeltaF12L. Loss of the F12L protein reduced the formation of IMV 2-fold, but there was a dramatic (99.5%) reduction in actin tail formation, and the levels of cell-associated enveloped virus and extracellular enveloped virus were reduced 8- to 11-fold and 7-fold, respectively. Consistent with the lack of actin tail formation, vDeltaF12L produced a very small plaque. The vDeltaF12L virus was severely attenuated in vivo, such that a dose of vDeltaF12L 10,000-fold greater than the dose of wild-type virus that induced severe disease was unable to induce disease in mice infected intranasally.     

The actin gene ACT1 is required for phagocytosis, motility, and cell separation of Tetrahymena thermophila

A previously identified Tetrahymena thermophila actin gene (C. G. Cupples and R. E. Pearlman, Proc. Natl. Acad. Sci. USA 83:5160-5164, 1986), here called ACT1, was disrupted by insertion of a neo3 cassette. Cells in which all expressed copies of this gene were disrupted exhibited intermittent and extremely slow motility and severely curtailed phagocytic uptake. Transformation of these cells with inducible genetic constructs that contained a normal ACT1 gene restored motility. Use of an epitope-tagged construct permitted visualization of Act1p in the isolated axonemes of these rescued cells. In ACT1Delta mutant cells, ultrastructural abnormalities of outer doublet microtubules were present in some of the axonemes. Nonetheless, these cells were still able to assemble cilia after deciliation. The nearly paralyzed ACT1Delta cells completed cleavage furrowing normally, but the presumptive daughter cells often failed to separate from one another and later became reintegrated. Clonal analysis revealed that the cell cycle length of the ACT1Delta cells was approximately double that of wild-type controls. Clones could nonetheless be maintained for up to 15 successive fissions, suggesting that the ACT1 gene is not essential for cell viability or growth. Examination of the cell cortex with monoclonal antibodies revealed that whereas elongation of ciliary rows and formation of oral structures were normal, the ciliary rows of reintegrated daughter cells became laterally displaced and sometimes rejoined indiscriminately across the former division furrow. We conclude that Act1p is required in Tetrahymena thermophila primarily for normal ciliary motility and for phagocytosis and secondarily for the final separation of daughter cells.     

Phospholipase D activity is required for actin stress fiber formation in fibroblasts

Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined function. In order to explore its cellular actions, we inactivated the rat PLD1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). This was stably expressed in Rat-2 fibroblasts to see if it acted as a dominant-negative mutant for PLD activity. Three clones that expressed rPLD1-V5 were selected (Rat2V16, Rat2V25, and Rat2V29). Another clone (Rat2V20) that lost expression of rPLD1-V5 was also obtained. In the three clones expressing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by ~50%, while the PLD activity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in response to LPA or PMA were examined in these clones. All three clones expressing rPLD1-V5 failed to form actin stress fibers after treatment with LPA. However, Rat2V20 cells formed stress fibers in response to LPA to the same extent as wild-type Rat-2 cells. In contrast, there was no significant change in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. Since Rho is an activator both of rPLD1 and stress fiber formation, the activation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin mediated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also showed normal vinculin-containing focal adhesions. However, the translocation of alpha-actinin to the cytoplasm and to the detergent-insoluble fraction in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form stress fibers and that PLD might be involved in the cross-linking of actin filaments mediated by alpha-actinin.     

Choline kinase beta is required for normal endochondral bone formation

Background

Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb^−/− mice display neonatal forelimb bone deformations.

Methods

To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb^−/− mice.

Results

The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb^−/− mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb^−/− mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb^−/− mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb^−/− mice contained fewer osteoclasts along the cartilage/bone interface.

Conclusions

Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice.

General Significance

Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

SCARFACE encodes an ARF-GAP that is required for normal auxin efflux and vein patterning in Arabidopsis

To identify molecular mechanisms controlling vein patterns, we analyzed scarface (sfc) mutants. sfc cotyledon and leaf veins are largely fragmented, unlike the interconnected networks in wild-type plants. SFC encodes an ADP ribosylation factor GTPase activating protein (ARF-GAP), a class with well-established roles in vesicle trafficking regulation. Quadruple mutants of SCF and three homologs (ARF-GAP DOMAIN1, 2, and 4) showed a modestly enhanced vascular phenotype. Genetic interactions between sfc and pinoid and between sfc and gnom suggest a possible function for SFC in trafficking of auxin efflux regulators. Genetic analyses also revealed interaction with cotyledon vascular pattern2, suggesting that lipid-based signals may underlie some SFC ARF-GAP functions. To assess possible roles for SFC in auxin transport, we analyzed sfc roots, which showed exaggerated responses to exogenous auxin and higher auxin transport capacity. To determine whether PIN1 intracellular trafficking was affected, we analyzed PIN1:green fluorescent protein (GFP) dynamics using confocal microscopy in sfc roots. We found normal PIN1:GFP localization at the apical membrane of root cells, but treatment with brefeldin A resulted in PIN1 accumulating in smaller and more numerous compartments than in the wild type. These data suggest that SFC is required for normal intracellular transport of PIN1 from the plasma membrane to the endosome.     

ACT-5 is an essential Caenorhabditis elegans actin required for intestinal microvilli formation

Investigation of Caenorhabditis elegans act-5 gene function revealed that intestinal microvillus formation requires a specific actin isoform. ACT-5 is the most diverged of the five C. elegans actins, sharing only 93% identity with the other four. Green fluorescent protein reporter and immunofluorescence analysis indicated that act-5 gene expression is limited to microvillus-containing cells within the intestine and excretory systems and that ACT-5 is apically localized within intestinal cells. Animals heterozygous for a dominant act-5 mutation looked clear and thin and grew slowly. Animals homozygous for either the dominant act-5 mutation, or a recessive loss of function mutant, exhibited normal morphology and intestinal cell polarity, but died during the first larval stage. Ultrastructural analysis revealed a complete loss of intestinal microvilli in homozygous act-5 mutants. Forced expression of ACT-1 under the control of the act-5 promoter did not rescue the lethality of the act-5 mutant. Together with immuno-electron microscopy experiments that indicated ACT-5 is enriched within microvilli themselves, these results suggest a microvillus-specific function for act-5, and further, they raise the possibility that specific actins may be specialized for building microvilli and related structures.     

Living markers for actin block myosin-dependent motility of plant organelles and auxin

Expression-based techniques using recombinant actin-binding proteins (ABPs) have been developed as advantageous means of visualising actin filaments. As actin function is linked to the movement of cellular cargoes, and overexpression of ABPs may compete with endogenous cytoskeletal proteins, such as myosins, secondary effects on cellular motility might be observed during actin visualisation. Cytoplasmic streaming and auxin transport were chosen as examples of cargo movement and investigated in two Arabidopsis thaliana lines stably transformed with fluorescently labelled talin (GFP-mTn) or fimbrin (GFP-FABD2). In both lines, the maximal streaming velocity of organelles was reduced to 80% in hypocotyl epidermal cells, where actin was broadly equally labelled by both ABPs. In contrast, observations of streaming and actin organisation during treatments with cytochalasin D (CD) suggested GFP-mTn-labelled actin to remain more stable. Furthermore, basipetal auxin transport was undisturbed in the GFP-FABD2 line but reduced by GFP-mTn. Remarkably, treatments with CD and 2,3-butanedione monoxime, which immobilizes myosin by impairing its ATPase, produced not only failures in organelle movement but also in basipetal auxin transport in the wild-type. These observations suggest that myosin is involved in processes of auxin translocation. In parallel, reduced motility in transgenic plants may be explained by a disturbed acto-myosin interplay, if overexpressed ABPs block the processive movement of myosin along actin filaments. This report shows that the use of live markers for actin visualisation may affect motility of cellular compounds and underlines the general need for critical investigation of actin-related processes in wild-type as well as transgenic plants prior to further interpretation.     

Optimal copper supply is required for normal plant iron deficiency responses

Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. Understanding crosstalk between Fe and Cu nutrition could lead to strategies for improved growth on soils with low or excess metals, with implications for agriculture and phytoremediation. Here, we show that Cu and Fe nutrition interact to increase or decrease Fe and/or Cu accumulation in leaves and Fe uptake processes. Leaf Cu concentration increased under low Fe supply, while high Cu lowered leaf Fe concentration. Ferric reductase activity, an indicator of Fe demand, was inhibited at insufficient or high Cu supply. Surprisingly, plants grown without Fe were more susceptible to Cu toxicity.     

Bacillus subtilis alpha-phosphoglucomutase is required for normal cell morphology and biofilm formation

Mutations designated gtaC and gtaE that affect alpha-phosphoglucomutase activity required for interconversion of glucose 6-phosphate and alpha-glucose 1-phosphate were mapped to the Bacillus subtilis pgcA (yhxB) gene. Backcrossing of the two mutations into the 168 reference strain was accompanied by impaired alpha-phosphoglucomutase activity in the soluble cell extract fraction, altered colony and cell morphology, and resistance to phages phi29 and rho11. Altered cell morphology, reversible by additional magnesium ions, may be correlated with a deficiency in the membrane glycolipid. The deficiency in biofilm formation in gtaC and gtaE mutants may be attributed to an inability to synthesize UDP-glucose, an important intermediate in a number of cell envelope biosynthetic processes.     

Drosophila fasciclinII is required for the formation of odor memories and for normal sensitivity to alcohol.

Drosophila fasciclinII (fasII) mutants perform poorly after olfactory conditioning due to a defect in encoding, stabilizing, or retrieving short-term memories. Performance was rescued by inducing the expression of a normal transgene just before training and immediate testing. Induction after training but before testing failed to rescue performance, showing that Fas II does not have an exclusive role in memory retrieval processes. The stability of odor memories in fasII mutants are indistinguishable from control animals when initial performance is normalized. Like several other mutants deficient in odor learning, fasII mutants exhibit a heightened sensitivity to ethanol vapors. A combination of behavioral and genetic strategies have therefore revealed a role for Fas II in the molecular operations of encoding short-term odor memories and conferring alcohol sensitivity. The preferential expression of Fas II in the axons of mushroom body neurons furthermore suggests that short-term odor memories are formed in these neurites.     

Exopolysaccharide production is required for biofilm formation and plant colonization by the nitrogen-fixing endophyte Gluconacetobacter diazotrophicus

The genome of the endophytic diazotrophic bacterial species Gluconacetobacter diazotrophicus PAL5 (PAL5) revealed the presence of a gum gene cluster. In this study, the gumD gene homologue, which is predicted to be responsible for the first step in exopolysaccharide (EPS) production, was insertionally inactivated and the resultant mutant (MGD) was functionally studied. The mutant MGD presented normal growth and nitrogen (N(2)) fixation levels but did not produce EPS when grown on different carbon sources. MGD presented altered colony morphology on soft agar plates (0.3% agar) and was defective in biofilm formation on glass wool. Most interestingly, MGD was defective in rice root surface attachment and in root surface and endophytic colonization. Genetic complementation reverted all mutant phenotypes. Also, the addition of EPS purified from culture supernatants of the wild-type strain PAL5 to the mutant MGD was effective in partially restoring wild-type biofilm formation and plant colonization. These data provide strong evidence that the PAL5 gumD gene is involved in EPS biosynthesis and that EPS biosynthesis is required for biofilm formation and plant colonization. To our knowledge, this is the first report of a role of EPS in the endophytic colonization of graminaceous plants by a nitrogen-fixing bacterium.     

A novel peripherin isoform generated by alternative translation is required for normal filament network formation

Peripherin is a type III neuronal intermediate filament protein detected within the intraneuronal inclusions characteristic of amyotrophic lateral sclerosis. The constitutively expressed peripherin isoform is encoded by all nine exons of the human and mouse peripherin genes to generate a protein species of ∼58 kDa on sodium dodecyl sulfate–polyacrylamide gels. Expression of this isoform, termed Per-58, generates a filament network in transfected SW13 vim cells. On immunoblots of cell lysates derived from these transfected cells, we have consistently observed a second peripherin species of ∼45 kDa. In this study, we show that this species is a novel peripherin isoform generated through the use of an in-frame downstream initiation codon. This isoform, that we have designated Per-45, is co-expressed together with Per-58 and, thus, constitutive in both human and mouse. Using mutational analysis, we show that Per-45 is required for normal network formation, with the absence of Per-45 leading to irregular filamentous structures. We further show that peripherin expression in the normal nervous system is characterized by tissue-specific Per-58 : Per-45 isoform ratios. Taken together, these results identify novel processing requirements for peripherin expression and indicate a hitherto unrecognized role for neuronal intermediate filament network formation through intra-isoform associations.     

The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.     

Calpain is required for normal osteoclast function and is down-regulated by calcitonin

Osteoclast motility is thought to depend on rapid podosome assembly and disassembly. Both mu-calpain and m-calpain, which promote the formation and disassembly of focal adhesions, were observed in the podosome belt of osteoclasts. Calpain inhibitors disrupted the podosome belt, blocked the constitutive cleavage of the calpain substrates filamin A, talin, and Pyk2, which are enriched in the podosome belt, induced osteoclast retraction, and reduced osteoclast motility and bone resorption. The motility and resorbing activity of mu-calpain(-/-) osteoclast-like cells were also reduced, indicating that mu-calpain is required for normal osteoclast activity. Histomorphometric analysis of tibias from mu-calpain(-/-) mice revealed increased osteoclast numbers and decreased trabecular bone volume that was apparent at 10 weeks but not at 5 weeks of age. In vitro studies suggested that the increased osteoclast number in the mu-calpain(-/-) bones resulted from increased osteoclast survival, not increased osteoclast formation. Calcitonin disrupted the podosome ring, induced osteoclast retraction, and reduced osteoclast motility and bone resorption in a manner similar to the effects of calpain inhibitors and had no further effect on these parameters when added to osteoclasts pretreated with calpain inhibitors. Calcitonin inhibited the constitutive cleavage of a fluorogenic calpain substrate and transiently blocked the constitutive cleavage of filamin A, talin, and Pyk2 by a protein kinase C-dependent mechanism, demonstrating that calcitonin induces the inhibition of calpain in osteoclasts. These results indicate that calpain activity is required for normal osteoclast activity and suggest that calcitonin inhibits osteoclast bone resorbing activity in part by down-regulating calpain activity.     

Agrin is highly expressed by chondrocytes and is required for normal growth

Agrin is a heparan sulfate proteoglycan that is best known for its crucial involvement in the organization and maintenance of postsynaptic structures at the neuromuscular junction. Consistent with this role, mice deficient of agrin die at birth due to respiratory failure. Here we examined the early postnatal development of agrin-deficient mice in which perinatal death was prevented by transgenic expression of neural agrin in motor neurons. Such transgenic, agrin-deficient mice were born at Mendelian ratio but exhibited severe postnatal growth retardation. Growth plate morpholgy was markedly altered in these mice, with changes being most prominent in the hypertrophic zone. Compression of this zone was not caused by reduced viability of hypertrophic chondrocytes, as no differences in the apoptosis rates could be observed. Furthermore, deposition of the major cartilage matrix components collagen type II and aggrecan was slightly reduced in these mice. Consistent with a role for agrin in skeletal development, we show for the first time that agrin is highly expressed by chondrocytes and localizes to the growth plate in wild-type mice. Our data show that agrin is expressed in cartilage and that it plays a critical role in normal skeletal growth.