The arabidopsis cell plate-associated dynamin-like protein, ADL1Ap, is required for multiple stages of plant growth and development

Dynamin and dynamin-like proteins are GTP-binding proteins involved in vesicle trafficking. In soybean, a 68-kD dynamin-like protein called phragmoplastin has been shown to be associated with the cell plate in dividing cells (Gu and Verma, 1996). Five ADL1 genes encoding dynamin-like proteins related to phragmoplastin have been identified in the completed Arabidopsis genome. Here we report that ADL1Ap is associated with punctate subcellular structures and with the cell plate in dividing cells. To assess the function of ADL1Ap we utilized a reverse genetic approach to isolate three separate Arabidopsis mutant lines containing T-DNA insertions in ADL1A. Homozygous adl1A seeds were shriveled and mutant seedlings arrested soon after germination, producing only two leaf primordia and severely stunted roots. Immunoblotting revealed that ADL1Ap expression was not detectable in the mutants. Despite the loss of ADL1Ap, the mutants did not display any defects in cytokinesis, and growth of the mutant seedlings could be rescued in tissue culture by the addition of sucrose. Although these sucrose-rescued plants displayed normal vegetative growth and flowered, they set very few seeds. Thus, ADL1Ap is critical for several stages of plant development, including embryogenesis, seedling development, and reproduction. We discuss the putative role of ADL1Ap in vesicular trafficking, cytokinesis, and other aspects of plant growth.     

Chlorella virus Marburg topoisomerase II: high DNA cleavage activity as a characteristic of Chlorella virus type II enzymes

Although the formation of a covalent enzyme-cleaved DNA complex is a prerequisite for the essential functions of topoisomerase II, this reaction intermediate has the potential to destabilize the genome. Consequently, all known eukaryotic type II enzymes maintain this complex at a low steady-state level. Recently, however, a novel topoisomerase II was discovered in Paramecium bursaria chlorella virus-1 (PBCV-1) that has an exceptionally high DNA cleavage activity [Fortune et al. (2001) J. Biol. Chem. 276, 24401-24408]. If robust DNA cleavage is critical to the physiological functions of chlorella virus topoisomerase II, then this remarkable characteristic should be conserved throughout the viral family. Therefore, topoisomerase II from Chlorella virus Marburg-1 (CVM-1), a distant family member, was expressed in yeast, isolated, and characterized. CVM-1 topoisomerase II is 1058 amino acids in length, making it the smallest known type II enzyme. The viral topoisomerase II displayed a high DNA strand passage activity and a DNA cleavage activity that was approximately 50-fold greater than that of human topoisomerase IIalpha. High DNA cleavage appeared to result from a greater rate of scission rather than promiscuous DNA site utilization, inordinately tight DNA binding, or diminished religation rates. Despite the fact that CVM-1 and PBCV-1 topoisomerase II share approximately 67% amino acid sequence identity, the two enzymes displayed clear differences in their DNA cleavage specificity/site utilization. These findings suggest that robust DNA cleavage is intrinsic to the viral enzyme and imply that chlorella virus topoisomerase II plays a physiological role beyond the control of DNA topology.     

A Highlights from MBoC Selection: Structurally distinct endocytic pathways for B cell receptors in B lymphocytes

B lymphocytes play a critical role in adaptive immunity. On antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to these factors. Here, we studied the mechanism of soluble antigen-induced BCR clustering and internalization in a cultured human B cell line using correlative superresolution fluorescence and platinum replica electron microscopy. First, by visualizing nanoscale BCR clusters, we provide direct evidence that BCR cluster size increases with F(ab’)2 concentration. Next, we show that the physical mechanism of internalization switches in response to BCR cluster size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when cluster size increases beyond the size of a single clathrin-coated pit, B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton.     

Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis

Recent evidence suggests that low molecular weight GTP-binding proteins may play important roles in a variety of membrane transport processes. In order to address the question of whether these proteins are involved in transport processes in the nerve axon, we have assessed their presence in rapid transport membranes from rabbit optic nerve. We report the characterization of a group of low molecular weight GTP-binding proteins which are constituents of rapid transport vesicles. Although these proteins are components of rapid transport vesicles, they are apparently not major rapidly transported species. They are localized in cytosolic as well as in membrane fractions of axons, and the membrane-associated form behaves as an integral membrane protein(s). These proteins are also found in association with a variety of vesicular and organellar components of neurons including coated vesicles, synaptic vesicles, synaptic plasma membranes, and mitochondria. We discuss the possible roles of these proteins in rapid axonal transport and exocytosis.     

Light regulatory sequences are located within the 5'' portion of the Fed-1 message sequence.

We have previously shown that element(s) mediating a light-induced increase in the abundance of Fed-1 mRNA in the leaves of transgenic tobacco plants are located within the transcribed portion of the gene. As part of an effort to define the mechanism of this effect, we report here that cis-acting elements capable of mediating a 5-fold light-induced increase in the abundance of this mRNA are located within a region comprising the 5' leader and first third of the Fed-1 coding sequence. No activity was detected in the 3' untranslated region of the gene. In a gain-of-function assay, the 5' region was found to be capable of conferring light responsiveness on three different reporter sequences, although experiments with the gusA reporter were complicated by an apparent negative light effect on the stability of this mRNA. Deletion experiments show that at least one essential light regulatory element is located in the 5' untranslated region of Fed-1 between nucleotides +19 and +57. Additional Fed-1 sequences, including a portion of the protein coding region, are required to confer positive responsiveness on the gusA reporter. These additional sequences may include specific light regulatory elements or simply provide an environment in which the leader element can function normally.     

Cre-mediated recombination in mouse Clara cells

Clara cells are nonciliated secretory cells lining the respiratory epithelium and are easily identified by the expression of Clara cell secretory protein (CCSP). To investigate molecular mechanism(s) regulating Clara cell function in the lungs, Cre recombinase was inserted into exon 1 of the CCSP, generating two novel mouse models, CCSP(Cre-Neo) and CCSP(Cre). These two models differ only by the inclusion of the neomycin resistance gene. These mice were bred to the R26R reporter mouse to investigate the tissue and cell specificity of Cre-mediated recombination. The efficiency of Cre recombination in the CCSP(Cre) mouse model was higher than in the CCSP(Cre-Neo) mouse model. Recombination was detected at D 4.5 in CCSP(Cre-Neo)/R26R mice and at D 0.5 in CCSP(Cre)/R26R mice. The CCSP(Cre-Neo) and CCSP(Cre) mouse models provide valuable tools for the ablation of genes in the postnatal mouse Clara cells.     

Studies on the antifungal properties of N-thiolated beta-lactams

N-thiolated beta-lactams had previously been shown to have antibacterial activity against a narrow selection of pathogenic bacteria including Staphylococcus aureus and Bacillus anthracis, as well as apoptotic-inducing activity in a variety of human cancer cell lines. We now have found that these lactams also possess antifungal activity against Candida and other fungi by exerting powerful cytostatic effects that disrupt the structural integrity of cytoplasmic membranes. The mode of action and structure-activity trends of these lactams as antifungals parallel that previously seen in our antibacterial studies.