Adaptor Protein Complex 2–Mediated Endocytosis Is Crucial for Male Reproductive Organ Development in Arabidopsis

Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2–dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.     

Class I α-Mannosidases Are Required for N-Glycan Processing and Root Development in Arabidopsis thaliana

In eukaryotes, class I α-mannosidases are involved in early N-glycan processing reactions and in N-glycan–dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ER-type α-mannosidase I (MNS3) and the two Golgi-α-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected α1,2-mannose residue from Man₉GlcNAc₂, MNS1/2 readily removed three α1,2-mannose residues from Man₈GlcNAc₂. Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man₈GlcNAc₂ accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man₉GlcNAc₂, demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I α-mannosidases in wild-type seedlings resulted in a similar root phenotype. These findings show that class I α-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis.N-glycosylation is a major co- and posttranslational modification of proteins in eukaryotic cells. The biosynthesis of protein N-linked glycans starts in the endoplasmic reticulum (ER) when the oligosaccharyltransferase complex catalyzes the transfer of the Glc₃Man₉GlcNAc₂ oligosaccharide from the lipid-linked precursor to Asn residues (N-X-S/T) of nascent polypeptide chains. Subsequent N-glycan processing involves a series of highly coordinated step-by-step enzymatic conversions occurring in the ER and Golgi apparatus (Kornfeld and Kornfeld, 1985). In the first trimming reactions, α-glucosidases I (GCSI) and GCSII cleave off three glucose residues from Glc₃Man₉GlcNAc₂ to generate Man₉GlcNAc₂ (Figure 1A). The next steps of the pathway are the removal of four α1,2-linked mannose residues to provide the Man₅GlcNAc₂ substrate for the formation of complex N-glycans in the Golgi apparatus. In mammals, these mannose trimming reactions are catalyzed by class I α-mannosidases (glycosyl hydrolase family 47 of the Carbohydrate Active Enzymes database; http://www.cazy.org/). These enzymes are inverting glycosyl hydrolases that are highly specific for α1,2-mannose residues, require Ca²⁺ for catalytic activity, and are sensitive to inhibition by pyranose analogs such as 1-deoxymannojirimycin and kifunensine (Lipari et al., 1995; Gonzalez et al., 1999). Class I α-mannosidases are conserved through eukaryotic evolution and do not share sequence homology with class II α-mannosidases, such as Golgi α-mannosidase II and the catabolic lysosomal and cytoplasmic α-mannosidases (Gonzalez et al., 1999; Herscovics, 2001).Open in a separate windowFigure 1.Cartoon of Important Oligosaccharide Structures.(A) Man₉GlcNAc₂ oligosaccharide (Man9): the substrate for ER-MNSI.(B) Man₈GlcNAc₂ isomer Man8.1 according to Tomiya et al. (1991): the product of ER-MNSI and substrate for Golgi-MNSI.(C) Man₅GlcNAc₂ (Man5.1): the product of the mannose trimming reactions.The linkage of the sugar residues is indicated.[See online article for color version of this figure.]The mammalian class I α-mannosidase family consists of three protein subgroups, which have been distinguished based on their sequence similarity and proposed function: ER-α1,2-mannosidases I (ER-MNSIs), Golgi-α-mannosidases I (Golgi-MNSIs), and ER degradation-enhancing α-mannosidase (EDEM)-like proteins (Mast and Moremen, 2006). In humans, there is a single ER-MNSI, which cleaves the terminal mannose residue from the b-branch of the Man₉GlcNAc₂ oligosaccharide to create the Man₈GlcNAc₂ isomer Man8.1 (Figure 1B). Subsequently, Golgi-MNSI (three isoforms, Golgi-MNSIA, Golgi-MNSIB, and Golgi-MNSIC, are present in humans) catalyze the removal of the remaining three α1,2-linked mannose residues to generate Man₅GlcNAc₂ (Figure 1C). The three human EDEM proteins are not directly involved in N-glycan processing but play a role in ER-associated degradation of glycoproteins (Mast et al., 2005; Hirao et al., 2006; Olivari et al., 2006).The formation of the Man₈GlcNAc₂ isomer (Man8.1), which is catalyzed by ER-MNSI, is the last N-glycan processing step that is conserved in yeast and mammals. Apart from its N-glycan processing function, ER-MNSI plays a key role in ER-mediated quality control of glycoproteins in yeasts and mammals (Mast and Moremen, 2006; Lederkremer, 2009). It has been proposed that ER-MNSI cooperates with mammalian EDEM1 to 3 or the yeast α1,2-mannosidase HTM1 to generate the signal that marks misfolded glycoproteins for degradation through the ER-associated protein degradation (ERAD) pathway. This quality control process, which finally leads to retrotranslocation to the cytoplasm and hydrolysis by the 26S proteasome, serves to prevent the secretion of aberrantly folded cargo proteins and is required to maintain protein homeostasis in the ER. Initially it was proposed that the Man₈GlcNAc₂ isomer Man8.1 (Figure 1B) flags aberrantly folded glycoproteins for degradation; however, recent evidence suggests that further mannose trimming to Man₇GlcNAc₂ in yeast and Man_5-6GlcNAc₂ in mammals is required to trigger ERAD (Avezov et al., 2008; Clerc et al., 2009). In addition, these mannose cleavage reactions serve also to release glycoproteins from the calnexin/calreticulin quality control cycle (Caramelo and Parodi, 2008).Unlike for animals and yeast, much less is known about the biological function of plant class I α-mannosidases. Processing mannosidases have been purified and characterized from mung bean (Vigna radiata) seedlings and castor bean (Ricinus communis) cotyledons (Forsee, 1985; Szumilo et al., 1986; Kimura et al., 1991). These preparations were a mixture of different α-mannosidases, and no evidence for ER-MNSI-like activity was provided. A putative Golgi-α-mannosidase I has been cloned from soybean (Glycine max) (Nebenführ et al., 1999). A green fluorescent protein (GFP)-tagged fusion protein of the soybean enzyme has been shown to reside in the cis-stacks of the Golgi apparatus (Nebenführ et al., 1999; Saint-Jore-Dupas et al., 2006), but its role in N-glycan processing and its enzymatic properties have not been reported so far. Thus, the involvement of class I α-mannosidases in N-glycan processing as well as in glycoprotein quality control in plants is still unclear, and the existence of a plant ER-MNSI has so far been inferred only from the presence of Man₈GlcNAc₂ oligosaccharides on ER-resident glycoproteins (Pagny et al., 2000).Here, we report the molecular cloning and biochemical characterization of the enzymes accounting for ER-MNSI and Golgi-MNSI activities in Arabidopsis thaliana. We also demonstrate that disruption of these genes leads to severe cell expansion defects in roots as well as to distinct cell wall alterations. Hence, the identification of the Arabidopsis ER-type and Golgi class I α-mannosidases not only establishes the molecular basis for the missing steps in the plant N-glycan processing pathway but also provides unprecedented insights into the role of N-glycans in plant development.     

Structural Analysis of Mutations in the Drosophila β2-Tubulin Isoform Reveals Regions in the β-Tubulin Molecule Required for General and for Tissue-Specific Microtubule Functions

We have determined the lesions in a number of mutant alleles of βTub85D, the gene that encodes the testis-specific β2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the β2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all β-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t(6) contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate β-tubulins. Correspondingly, B2t(6) disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that β3, a developmentally regulated Drosophila β-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type β2-tubulin. We show here by complementation analysis that β3 and the B2t(6) product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the β2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the β2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the β2 variant lacking the carboxy terminus and the B2t(6) variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate β-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type β-tubulins. We propose that the integrity of this structure in the Drosophila testis β2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.     

The plant-specific G protein γ subunit AGG3 influences organ size and shape in Arabidopsis thaliana

? Control of organ size and shape by cell proliferation and cell expansion is a fundamental developmental process, but the mechanisms that set the size and shape of determinate organs are largely unknown in plants. ? Molecular, genetic, cytological and biochemical approaches were used to characterize the roles of the Arabidopsis thaliana G protein γ subunit (AGG3) gene in organ growth. ? Here, we describe A. thaliana AGG3, which promotes petal growth by increasing the period of cell proliferation. Both the N-terminal region and the C-terminal domains of AGG3 are necessary for the function of AGG3. By contrast, analysis of a series of AGG3 derivatives with deletions in specific domains showed that the deletion of any of these domains cannot completely abolish the function of AGG3. The GFP-AGG3 fusion protein is localized to the plasma membrane. The predicted transmembrane domain plays an important role in the plasma membrane localization of AGG3. Genetic analyses revealed that AGG3 action requires a functional G protein α subunit (GPA1) and G protein β subunit (AGB1). ? Our findings demonstrate that AGG3, GPA1 and AGB1 act in the same genetic pathway to influence organ size and shape in A. thaliana.     

Mutations in TUBGCP4 Alter Microtubule Organization via the γ-Tubulin Ring Complex in Autosomal-Recessive Microcephaly with Chorioretinopathy

We have identified TUBGCP4 variants in individuals with autosomal-recessive microcephaly and chorioretinopathy. Whole-exome sequencing performed on one family with two affected siblings and independently on another family with one affected child revealed compound-heterozygous mutations in TUBGCP4. Subsequent Sanger sequencing was performed on a panel of individuals from 12 French families affected by microcephaly and ophthalmic manifestations, and one other individual was identified with compound-heterozygous mutations in TUBGCP4. One synonymous variant was common to all three families and was shown to induce exon skipping; the other mutations were frameshift mutations and a deletion. TUBGCP4 encodes γ-tubulin complex protein 4, a component belonging to the γ-tubulin ring complex (γ-TuRC) and known to regulate the nucleation and organization of microtubules. Functional analysis of individual fibroblasts disclosed reduced levels of the γ-TuRC, altered nucleation and organization of microtubules, abnormal nuclear shape, and aneuploidy. Moreover, zebrafish treated with morpholinos against tubgcp4 were found to have reduced head volume and eye developmental anomalies with chorioretinal dysplasia. In summary, the identification of TUBGCP4 mutations in individuals with microcephaly and a spectrum of anomalies in eye development, particularly photoreceptor anomalies, provides evidence of an important role for the γ-TuRC in brain and eye development.     

G Protein–Coupled Receptor-Type G Proteins Are Required for Light-Dependent Seedling Growth and Fertility in Arabidopsis

G protein–coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein–coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1_pro:GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development.     

The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.     

Oct4 Is Required ～E7.5 for Proliferation in the Primitive Streak

Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ∼E6.0–E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ∼E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ∼E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.     

Arabidopsis PTD Is Required for Type Ⅰ Crossover Formation and Affects Recombination Frequency in Two Different Chromosomal Regions

In eukaryotes,crossovers together with sister chromatid cohesion maintain physical association between homologous chromosomes,ensuring accurate chromosome segregation during meiosis I and resulting in exchange of genetic information between homologues.The Arabidopsis PTD(Parting Dancers)gene affects the level of meiotic crossover formation,but its functional relationships with other core meiotic genes,such as AtSPO11-1,AtRAD51,and AtMSH4,are unclear;whether PTD has other functions in meiosis is also unknown.To further analyze PTD function and to test for epistatic relationships,we compared the meiotic chromosome behaviors of Atspol 1-1 ptd and AtradSl ptd double mutants with the relevant single mutants.The results suggest that PTD functions downstream of AtSPOll-1 and AtRAD51 in the meiotic recombination pathway.Furthermore,we found that meiotic defects in rck ptd and Atmsh4 ptd double mutants showed similar meiotic phenotypes to those of the relevant single mutants,providing genetic evidences for roles of PTD and RCK in the type I crossovers pathway.Moreover,we employed a pollen tetrad-based fluorescence method and found that the meiotic crossover frequencies in two genetic intervals were significantly reduced from 6.63%and 22.26%in wild-type to 1.14%and 6.36%,respectively,in the ptd-2 mutant.These results revealed new aspects of PTD function in meiotic crossover formation.     

The mRNA-stabilizing Factor HuR Protein Is Targeted by β-TrCP Protein for Degradation in Response to Glycolysis Inhibition

The mRNA-stabilizing protein HuR acts a stress response protein whose function and/or protein stability are modulated by diverse stress stimuli through posttranslational modifications. Here, we report a novel mechanism by which metabolic stress facilitates proteasomal degradation of HuR in cancer cells. In response to the glucose transporter inhibitor CG-5, HuR translocates to the cytoplasm, where it is targeted by the ubiquitin E3 ligase β-TrCP1 for degradation. The cytoplasmic localization of HuR is facilitated by PKCα-mediated phosphorylation at Ser-318 as the Ser-318 → alanine substitution abolishes the ability of the resulting HuR to bind PKCα and to undergo nuclear export. The mechanistic link between β-TrCP1 and HuR degradation was supported by the ability of ectopically expressed β-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of β-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by β-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a β-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that β-TrCP1 recognizes an unconventional motif, ²⁹⁶EEAMAIAS³⁰⁴, in the RNA recognition motif 3. Furthermore, mutational analysis indicates that IKKα-dependent phosphorylation at Ser-304 is crucial to the binding of HuR to β-TrCP1. Mechanistically, this HuR degradation pathway differs from that reported for heat shock and hypoxia, which underlies the complexity in the regulation of HuR turnover under different stress stimuli. The ability of glycolysis inhibitors to target the expression of oncogenic proteins through HuR degradation might foster novel strategies for cancer therapy.     

γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

Background

There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex.

Principal Findings

We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis.

Conclusions/Significance

These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein.