Members of the Arabidopsis dynamin-like gene family,ADL1, are essential for plant cytokinesis and polarized cell growth

Polarized membrane trafficking during plant cytokinesis and cell expansion are critical for plant morphogenesis, yet very little is known about the molecular mechanisms that guide this process. Dynamin and dynamin-related proteins are large GTP binding proteins that are involved in membrane trafficking. Here, we show that two functionally redundant members of the Arabidopsis dynamin-related protein family, ADL1A and ADL1E, are essential for polar cell expansion and cell plate biogenesis. adl1A-2 adl1E-1 double mutants show defects in cell plate assembly, cell wall formation, and plasma membrane recycling. Using a functional green fluorescent protein fusion protein, we show that the distribution of ADL1A is dynamic and that the protein is localized asymmetrically to the plasma membrane of newly formed and mature root cells. We propose that ADL1-mediated membrane recycling is essential for plasma membrane formation and maintenance in plants.     

Arabidopsis dynamin-like protein 2a (ADL2a), like ADL2b, is involved in plant mitochondrial division

The Arabidopsis genome has two similar dynamin-like proteins, ADL2a and ADL2b (76.7% identity). ADL2a is reported to be localized in chloroplasts [Kang et al. (1998) Plant Mol. Biol. 38: 437], while ADL2b functions in mitochondrial division [Arimura and Tsutsumi (2002) PROC: Natl. Acad. Sci. USA 99: 5727]. Using GFP fusion proteins, we observed both ADL2a and ADL2b in portions of mitochondria but not in chloroplasts. Furthermore, cells transformed with ADL2a and ADL2b with a defective GTPase domain had normal chloroplasts but elongated mitochondria. These results imply that both ADL2b and ADL2a are involved in the division of plant mitochondria.     

A dynamin-like protein, ADL1, is present in membranes as a high-molecular-mass complex in Arabidopsis thaliana.

Dynamin, a GTP-binding protein, is involved in endocytosis in animal cells. We found that a dynamin-like protein, ADL1, is present in multiple forms in Arabidopsis leaf tissue. Subcellular fractionation experiments, together with gel-filtration and nondenaturing-gel electrophoresis revealed that most of ADL1 is present as a high-molecular-mass complex of 400 to 600 kD in the membrane or pellet fraction, whereas ADL1 is present in the soluble fraction as a monomer. The subcellular distribution of ADL1 is affected by various agents such as Ca2+, cyclosporin A, GTP, and ATP. Ca2+ increases the amount of ADL1 present in the membrane fraction, whereas cyclosporin A inhibits the membrane association. Furthermore, Ca2+ and GTP change the migration pattern of ADL1 in nondenaturing polyacrylamide gels, indicating that these chemicals influence either the complex formation and/or the conformation of the ADL1 complex. Our results demonstrate that ADL1 has characteristics that are similar to Dynamin I, which is found in animal cells. Therefore, it is possible that ADL1 is also involved in biological processes that require vesicle formation.     

The arabidopsis serrate gene encodes a zinc-finger protein required for normal shoot development

Organogenesis in plants depends upon the proper regulation of many genes, but how such necessary changes in gene expression are coordinated is largely unknown. The serrate (se) mutant of Arabidopsis displays defects in the initiation and elaboration of cotyledons and post-embryonic lateral organs. Cloning the SE gene revealed that it encodes a protein with a single, C(2)H(2)-type, zinc finger related to genes in other eukaryotes. Consistent with a role in organogenesis, the SE gene is transcribed in shoot meristems and in emerging organ primordia throughout development. Expression of the SE cDNA under the control of a heterologous promoter caused both accelerated and arrested plant growth, and these phenotypes were due to overexpression and co-suppression of the SE gene, respectively. Our analysis of the se mutant and the SE gene suggests a role for the SE gene product in regulating changes in gene expression via chromatin modification. Consistent with this proposed function, a synergistic double mutant phenotype was seen for plants mutant at both the SE locus and the locus encoding the largest subunit of chromatin assembly factor I.     

The Arabidopsis dynamin-like proteins ADL1C and ADL1E play a critical role in mitochondrial morphogenesis

Dynamin-related proteins are high molecular weight GTP binding proteins and have been implicated in various biological processes. Here, we report the functional characterization of two dynamin homologs in Arabidopsis, Arabidopsis dynamin-like 1C (ADL1C) and Arabidopsis dynamin-like 1E (ADL1E). ADL1C and ADL1E show a high degree of amino acid sequence similarity with members of the dynamin family. However, both proteins lack the C-terminal Pro-rich domain and the pleckstrin homology domain. Expression of the dominant-negative mutant ADL1C[K48E] in protoplasts obtained from leaf cells caused abnormal mitochondrial elongation. Also, a T-DNA insertion mutation at the ADL1E gene caused abnormal mitochondrial elongation that was rescued by the transient expression of ADL1C and ADL1E in protoplasts. In immunohistochemistry and in vivo targeting experiments in Arabidopsis protoplasts, ADL1C and ADL1E appeared as numerous speckles and the two proteins colocalized. These speckles were partially colocalized with F1-ATPase-gamma:RFP, a mitochondrial marker, and ADL2b localized at the tip of mitochondria. These results suggest that ADL1C and ADL1E may play a critical role in mitochondrial fission in plant cells.     

The single dynamin-like protein of Trypanosoma brucei regulates mitochondrial division and is not required for endocytosis

Members of the evolutionarily conserved dynamin-related GTPase family mediate numerous cellular membrane remodeling events. Dynamin family functions include the scission of clathrin-coated pits from the plasma membrane, mitochondrial fission, and chloroplast division. Here we report that the divergent eukaryote Trypanosoma brucei possesses a single dynamin family gene, which we have designated TbDLP. Furthermore, a single dynamin family gene is also found in the Leishmania major and Trypanosoma vivax genomes, indicating that this is a conserved feature among the kinetoplastida. TbDLP is most homologous to the DMN/DRP family of dynamin-like proteins. Indirect immunofluorescence microscopy reveals that TbDLP is distributed in punctate structures within the cell that partially co-localize with the mitochondrion when labeled with MitoTracker. To define TbDLP function, we have used RNA interference to silence the TbDLP gene. Reduction of TbDLP protein levels causes a profound alteration in mitochondrial morphology without affecting the structure of other membrane-bound compartments, including the endocytic and exocytic apparatus. The mitochondrial profiles present in wild type trypanosomes fuse and collapse in the mutant cells, and by electron microscopy the mitochondria are found to contain an accumulation of constriction sites. These findings demonstrate TbDLP functions in division of the mitochondrial membrane. Most significantly, as TbDLP is the sole member of the dynamin family in this organism, scission of clathrin-coated pits involved in protein trafficking through the highly active endocytic system in trypanosomes must function in the absence of dynamin. The evolutionary implications of these findings are discussed.     

The RNA-binding protein Vg1 RBP is required for cell migration during early neural development

After mid-blastula transition, populations of cells within the Xenopus embryo become motile. Using antisense morpholino oligonucleotides, we find that Vg1 RBP, an RNA-binding protein implicated in RNA localization in oocytes, is required for the migration of cells forming the roof plate of the neural tube and, subsequently, for neural crest migration. These cells are properly determined but remain at their site of origin. Consistent with a possible role in cell movement, Vg1 RBP asymmetrically localizes to extended processes in migrating neural crest cells. Given that Vg1 RBP is a member of the conserved VICKZ family of proteins, expressed in embryonic and neoplastic cells, these data shed light on the likely role of these RNA-binding proteins in regulating cell movements during both development and metastasis.     

The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.     

The Arabidopsis ARCP protein, CSI1, which is required for microtubule stability, is necessary for root and anther development

Armadillo repeat-containing proteins (ARCPs) are conserved across eukaryotic kingdoms and function in various processes. Regulation of microtubule stability by ARCPs exists widely in mammals and algae, but little is known in plants. Here, we present the functional characterization of an Arabidopsis thaliana ARCP, which was previously identified as Cellulose synthase-interactive protein1 (CSI1), and prove its crucial role in anther and root development. CSI1 is highly expressed in floral tissues, and knockout mutants of CSI1 (three allelic lines) accordingly exhibit defective anther dehiscence, which can be partially rescued by mammalian microtubule-stabilizer MAP4, suggesting that CSI1 functions by stabilizing the microtubular cytoskeleton. CSI1 binds microtubules in vitro, and immunofluorescence and coimmunoprecipitation studies confirmed the physical interactions between CSI1 and microtubules in vivo. Analysis using oryzalin, a microtubule-disrupting drug, further revealed the destabilized microtubules under CSI1 deficiency and confirmed the crucial role of CSI1 in microtubule stability. The dynamic change of CSI1 in response to dehydration strongly suggests the important function of CSI1 in dehydration-induced microtubule depolymerization and reorganization, which is crucial for anther development. These results indicate the pivotal role of CSI1 in anther development by regulating microtubule stability and hence cell morphogenesis.     

The actin-interacting protein AIP1 is essential for actin organization and plant development

Cell division, growth, and cytoplasmic organization require a dynamic actin cytoskeleton. The filamentous actin (F-actin) network is regulated by actin binding proteins that modulate actin dynamics. These actin binding proteins often have cooperative interactions. In particular, actin interacting protein 1 (AIP1) is capable of capping F-actin and enhancing the activity of the small actin modulating protein, actin depolymerising factor (ADF) in vitro. Here, we analyze the effect of the inducible expression of AIP1 RNAi in Arabidopsis plants to assess AIP1s role in vivo. In intercalary growing cells, the normal actin organization is disrupted, and thick bundles of actin appear in the cytoplasm. Moreover, in root hairs, there is the unusual appearance of actin cables ramifying the root hair tip. We suggest that the reduction in AIP1 results in a decrease in F-actin turnover and the promotion of actin bundling. This distortion of the actin cytoskeleton causes severe plant developmental abnormalities. After induction of the Arabidopis RNAi lines, the cells in the leaves, roots, and shoots fail to expand normally, and in the severest phenotypes, the plants die. Our data suggest that AIP1 is essential for the normal functioning of the actin cytoskeleton in plant development.     

Vacuole membrane protein 1 is an endoplasmic reticulum protein required for organelle biogenesis, protein secretion, and development

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1(-) Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1(-) cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.     

The Arabidopsis DDB1 interacting protein WDR55 is required for vegetative development

The CULLIN family of E3 ubiquitin ligases are important regulators of plant development and function. A newly identified class of CULLIN4-RING-E3 ligases (CRL4s) interacts with substrate receptors referred to as DDB1-CUL4 ASSOCIATED FACTORS (DCAFs) via a DDB1 linker protein. We have previously reported that the WD40 protein WDR55 interacts with DDB1A and is thus a putative DCAF. Mutants of WDR55 are embryo lethal, suggesting that a DDB1^WDR55 complex could regulate embryo and endosperm development. Here we report that a weak allele homozygous for wdr55 display pleiotropic phenotypes in the seedling and adult stages, suggesting a novel regulatory role for WDR55 in vegetative development.     

LAP3, a novel plant protein required for pollen development, is essential for proper exine formation

We isolated lap3-1 and lap3-2 mutants in a screen for pollen that displays abnormal stigma binding. Unlike wild-type pollen, lap3-1 and lap3-2 pollen exine is thinner, weaker, and is missing some connections between their roof-like tectum structures. We describe the mapping and identification of LAP3 as a novel gene that contains a repetitive motif found in β-propeller enzymes. Insertion mutations in LAP3 lead to male sterility. To investigate possible roles for LAP3 in pollen development, we assayed the metabolite profile of anther tissues containing developing pollen grains and found that the lap3-2 defect leads to a broad range of metabolic changes. The largest changes were seen in levels of a straight-chain hydrocarbon nonacosane and in naringenin chalcone, an obligate compound in the flavonoid biosynthesis pathway. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DEMETER,a DNA glycosylase domain protein,is required for endosperm gene imprinting and seed viability in arabidopsis

We isolated mutations in Arabidopsis to understand how the female gametophyte controls embryo and endosperm development. For the DEMETER (DME) gene, seed viability depends only on the maternal allele. DME encodes a large protein with DNA glycosylase and nuclear localization domains. DME is expressed primarily in the central cell of the female gametophyte, the progenitor of the endosperm. DME is required for maternal allele expression of the imprinted MEDEA (MEA) Polycomb gene in the central cell and endosperm. Ectopic DME expression in endosperm activates expression of the normally silenced paternal MEA allele. In leaf, ectopic DME expression induces MEA and nicks the MEA promoter. Thus, a DNA glycosylase activates maternal expression of an imprinted gene in the central cell.