Centrifugation causes adaptation of microfilaments Studies on the transport of statoliths in gravity sensingChara rhizoids

Summary The actin cytoskeleton is involved in the positioning of statoliths in tip growingChara rhizoids. The balance between the acropetally acting gravity force and the basipetally acting net out-come of cytoskeletal force results in the dynamically stable position of the statoliths 10–30 m above the cell tip. A change of the direction and/or the amount of one of these forces in a vertically growing rhizoid results in a dislocation of statoliths. Centrifugation was used as a tool to study the characteristics of the interaction between statoliths and microfilaments (MFs). Acropetal and basipetal accelerations up to 6.5 g were applied with the newly constructed slow-rotating-centrifuge-microscope (NIZEMI). Higher accelerations were applied by means of a conventional centrifuge, namely acropetally 10–200 g and basipetally 10–70 g. During acropetal accelerations (1.4–6 g), statoliths were displaced to a new stable position nearer to the cell vertex (12–6.5 m distance to the apical cell wall, respectively), but they did not sediment on the apical cell wall. The original position of the statoliths was reestablished within 30 s after centrifugation. Sedimentation of statoliths and reduction of the growth rates of the rhizoids were observed during acropetal accelerations higher than 50 g. When not only the amount but also the direction of the acceleration were changed in comparison to the natural condition, i.e., during basipetal accelerations (1.0–6.5 g), statoliths were displaced into the subapical zone (up to 90 m distance to the apical cell wall); after 15–20 min the retransport of statoliths to the apex against the direction of acceleration started. Finally, the natural position in the tip was reestablished against the direction of continuous centrifugation. Retransport was observed during accelerations up to 70 g. Under the 1 g condition that followed the retransported statoliths showed an up to 5-fold increase in sedimentation time onto the lateral cell wall when placed horizontally. During basipetal centrifugations 70 g all statoliths entered the basal vacuolar part of the rhizoid where they were cotransported in the streaming cytoplasm. It is concluded that the MF system is able to adapt to higher mass accelerations and that the MF system of the polarly growing rhizoid is polarly organized.Abbreviations g gravitational acceleration (9.81 m/s²) - MF microfilament - NIZEMI Niedergeschwindigkeits-Zentrifugen-Mikroskop (slow-rotating-centrifuge-microscope)     

荧光素酶mRNA的构建及电穿孔介导的mRNA体内表达特性北大核心CSCD

为构建一种非复制型mRNA平台并探究电穿孔介导的mRNA对小鼠健康状况的影响及蛋白的表达情况,以荧光素酶作为靶标基因,用T7 RNA聚合酶体外转录及酶法加帽加尾的策略制备mRNA,用活体基因导入仪通过电穿孔的方式体内递送mRNA,借助小动物活体成像系统观测荧光素酶蛋白在小鼠体内的表达强度和持续时间。结果表明,使用该非复制型mRNA平台得到的mRNA成功在体内外表达,电穿孔介导的mRNA对小鼠健康体征无明显影响,所有的小鼠均成功表达了荧光素酶蛋白,蛋白表达在电穿孔后第1天达到峰值,在第4天迅速下降,但蛋白表达强度和持续时间存在较大的小鼠个体间差异。研究对非复制型mRNA的构建及其应用于疫苗或肿瘤药物研发具有重要参考价值。     

The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion (Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek (Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the -oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.Abbreviations BDM 2,3-butanedione monoxime - DAPI 4,6-diamidino-2-phenylindole - ER endoplasmic reticulum - GFP green fluorescent protein     

Studying nuclear disassembly in vitro using Xenopus egg extract

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.     

Induction of anterior neural fates in the ascidian Ciona intestinalis

The sensory vesicle of ascidians is thought to be homologous to the vertebrate forebrain and midbrain (Development 125 (1998) 1113). Here we report the isolation of two sensory vesicle markers in the ascidian Ciona intestinalis, which are homologs of vertebrate otx and gsx homeobox genes. By using these markers to analyze the induction of anterior neural tissue in Ciona, we find that the restriction of anterior neural fate to the progeny of the anterior animal blastomeres is due to a combination of two factors. The vegetal blastomeres show a differential inducing activity along the anterior-posterior axis, while the competence to respond to this inducing signal is markedly higher in the anterior animal blastomeres than in the posterior animal blastomeres. This differential competence to respond is also observed in response to bFGF, a candidate neural inducer in ascidians (J. Physiol. 511.2 (1998) 347) and can be detected by the gastrula stage. Our results, however, indicate that bFGF can only induce a subset of the responses of the endogenous inducer, suggesting that additional signals in the embryo are necessary to induce a fully patterned nervous system.     

Nuclear behavior during branch formation in a centrifugedAdiantum protonema and the nuclear polarity

A new branch was induced on the side wall of fern protonema by cell centrifugation and subsequent polarized red light irradiation after the induction of cell division under white light. Nuclear behavior during the branch formation was analyzed. Immediately after cell division, the two daughter nuclei moved away from the division site in both red and dark conditions. Under continuous irradiation with polarized red light, cell swelling occurred as an early step of branching near the cell dividing wall, even though the nucleus was localized far from the branching site at the beginning of the swelling. After a new branch started to grow, the nucleus returned to the branching site and moved into the new branch from its basipetal end. When a protonema incubated in the dark was centrifuged again acropetally or basipetally just before the irradiation of polarized red light, the rate of apical growth or branch formation was increased, respectively. Moreover, growth of a branched protonema was altered from its former apex or from the branch again by dislocating the nucleus acropetally or basipetally by centrifugation, respectively. These facts suggest that the nucleus has no polarity physiologically, i.e. head and tail, namely either end of the spindle-shaped nucleus can be the nuclear front in a tip-growing protonema.     

Nuclear pore distribution and relation to adjacent cytoplasmic organelles in cotyledon cells of developing Vicia faba

Following a zinc iodine-osmium tetroxide fixation, nuclear pore distribution was studied in 0.3-m sections from cotyledons of developing Vicia faba L. Localised absence of nuclear pores was found to be associated with proximity of organelles to the nucleus. Golgi cisternae and mitochondria are associated with areas of pore absence while cisternal endoplasmic reticulum and tubular endoplasmic reticulum are linked with areas showing reduction in pore density. Pores were seen in the nuclear membrane adjacent to vacuoles. Pattern analysis of pore distribution indicated possible clustering within an overall regularity.Abbreviations ER endoplasmic reticulum - ZIO zinc iodine-osmium tetroxide     

The relation between translation and mRNA degradation in the lacZ gene

The technique of gene fusion, in which the gene of interest, severed from its 3' end, is in-phase fused to a reporter gene--usually lacZ--is widely used to study translational regulation in Escherichia coli. Implicit in these approaches is the assumption that the activity of the ribosome binding site (RBS) fused in-phase with lacZ, does not per se modify the steady-state level of the lacZ mRNA. Herein, we have tested this hypothesis, using a model system in which the RBS of the lamB gene is fused to lacZ. Several point mutations affecting translation initiation have been formerly characterized in this RBS, and we used Northern blots to study their effect upon the lacZ mRNA pattern. Two series of constructs were assayed: in the first one, a 51-bp fragment centered around the lamB initiator codon, was inserted in front of lacZ within the natural lactose operon, whereas in the second the lacZ gene was fused to the genuine malK-lamB operon just downstream from the lamB RBS. We observed that in the first series, the concentration and average molecular weight of the lacZ mRNA dropped sharply as the efficiency of the RBS decreased. This apparently arose from a decreased stability of the message, since the mRNA patterns are equalized when the endonuclease RNase E is inactivated. We suggest that in this case the rate limiting step in the decay process is an RNase E cleavage that is outcompeted by translation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The functional analysis of Type I postplasmic/PEM mRNAs in embryos of the ascidian Halocynthia roretzi

Maternal factors, such as a muscle determinant macho-1 mRNA that is localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, are crucial for embryonic axis formation and cell fate specification. Maternal mRNAs that show an identical posterior localization pattern to that of macho-1 in eggs and embryos are called Type I postplasmic/PEM mRNAs. We investigated the functions of five of the nine Type I mRNAs so far known in Halocynthia roretzi: Hr-Wnt-5, Hr-GLUT, Hr-PEM3, Hr-PEN1, and Hr-PEN2. Suppression of their functions with specific antisense morpholino oligonucleotides (MOs) had effects on the formation of various tissues: Hr-Wnt-5 on notochord, muscle, and mesenchyme, although zygotic function of Hr-Wnt-5 is responsible for notochord formation; Hr-GLUT on notochord, mesenchyme, and endoderm; and Hr-PEN2 on muscle, mesenchyme, and endoderm. On the other hand, Hr-PEM3 and Hr-PEN1 MOs seemed to have no effect. We conclude that the functions of at least some localized maternal Type I postplasmic/PEM mRNAs are necessary for early embryonic patterning in ascidians.     

Establishment and dispersal of the biological control weevil Rhinoncomimus latipes on mile-a-minute weed, Persicaria perfoliata

Mile-a-minute weed, Persicaria perfoliata (L.) H. Gross (Polygonaceae), is an annual vine from Asia that has invaded the eastern US where it can form dense monocultures and outcompete other vegetation in a variety of habitats. The host-specific Asian weevil Rhinoncomimus latipes Korotyaev (Coleoptera: Curculionidae) was first released in the US in 2004 as part of a classical biological control program. The weevil was intensively monitored in three release arrays over 4 years, and field cages at each site were used to determine the number of generations produced. The weevil established at all three sites and produced three to four generations before entering a reproductive diapause in late summer. Weevils dispersed at an average rate of 1.5–2.9 m wk⁻¹ through the 50 m diameter arrays, which had fairly contiguous mile-a-minute cover. Weevils dispersing in the broader, more variable landscape located both large monocultures and small isolated patches of mile-a-minute 600–760 m from the release within 14 months. Weevil density ranged from fewer than 10 to nearly 200 weevils m⁻² mile-a-minute weed. Mile-a-minute cover decreased at the site with the highest weevil density. The production of P. perfoliata seed clusters decreased with increasing weevil populations at two sites, and seedling production declined over time at two sites by 75% and 87%. The ability of the weevil to establish, produce multiple generations per season, disperse to new patches, and likelihood of having an impact on plants in the field suggests that R. latipes has the potential to be a successful biological control agent.     

Trypanosoma cruzi: Identification of DNA targets of the nuclear periphery coiled-coil protein TcNUP-1

The nuclear lamina is a structure that lines the inner nuclear membrane. In metazoans, lamins are the primary structural components of the nuclear lamina and are involved in several processes. Eukaryotes that lack lamins have distinct proteins with homologous functions. Some years ago, a coiled-coil protein in Trypanosoma brucei, NUP-1, was identified as the major filamentous component of its nuclear lamina. However, its precise role has not been determined. We characterized a homologous protein in Trypanosoma cruzi, TcNUP-1, and identified its in vivo DNA binding sites using a chromatin immunoprecipitation assay. We demonstrate for the first time that TcNUP-1 associates with chromosomal regions containing large non-tandem arrays of genes encoding surface proteins. We therefore suggest that TcNUP-1 is a structural protein that plays an essential role in nuclear organization by anchoring T. cruzi chromosomes to the nuclear envelope.     

Actin cytoskeleton and budding pattern are altered in the yeast rvs161 mutant: the Rvs161 protein shares common domains with the brain protein amphiphysin

The actin cytoskeleton cells is altered in rvs161 mutant yeast, with the defect becoming more pronounced under unfavorable growth conditions, as described for the rvs167 mutant. The cytoskeletal alteration has no apparent effect on invertase secretion and polarized growth. Mutations in RTVS161, just as in RI/S167, lead to a random budding pattern in a/ diploid cells. This behavior is not observed in a/a diploid cells homozygous for the rvs161-1 or rvs167-1 mutations. In addition, sequence comparisons revealed that amphiphysin, a protein first found in synaptic vesicles of chicken and shown to be the autoantigen of Stiff Man syndrome, presents similarity with both Rvs proteins. Furthermore, limited similarities with myosin heavy chain and tropomyosin alpha chain from higher eukaryotic cells allow for the definition of a possible consensus sequence. The finding of related sequences suggests the existence of a function for these proteins that is conserved among eukaryotic organisms.     

A cytochemical and immunofluorescence study of endocrine cells in the gut of the ascidian Styela clava

Summary Strong secretin-like immunofluorescence has been demonstrated in endocrine-like cells from the gastric epithelium of Styela. These cells also stain with lead haematoxylin and exhibit a brilliant formaldehyde-induced fluorescence, but do not show any other cytochemical features characteristic of the mammalian APUD series. Tests with antisera to glucagon, gastrin and somatostatin all proved negative. In the oesophagus tests with all four antisera proved negative. The significance of these results is discussed in relation to the phylogeny of vertebrate gastro-intestinal hormones.     

The branched actin nucleator Arp2/3 promotes nuclear migrations and cell polarity in the C. elegans zygote

Regulated movements of the nucleus are essential during zygote formation, cell migrations, and differentiation of neurons. The nucleus moves along microtubules (MTs) and is repositioned on F-actin at the cellular cortex. Two families of nuclear envelope proteins, SUN and KASH, link the nucleus to the actin and MT cytoskeletons during nuclear movements. However, the role of actin nucleators in nuclear migration and positioning is poorly understood. We show that the branched actin nucleator, Arp2/3, affects nuclear movements throughout embryonic and larval development in C. elegans, including nuclear migrations in epidermal cells and neuronal precursors. In one-cell embryos the migration of the male pronucleus to meet the female pronucleus after fertilization requires Arp2/3. Loss of Arp2/3 or its activators changes the dynamics of non-muscle myosin, NMY-2, and alters the cortical accumulation of posterior PAR proteins. Reduced establishment of the posterior microtubule cytoskeleton in Arp2/3 mutants correlates with reduced male pronuclear migration. The UNC-84/SUN nuclear envelope protein that links the nucleus to the MT and actin cytoskeleton is known to regulate later nuclear migrations. We show here it also positions the male pronucleus. These studies demonstrate a global role for Arp2/3 in nuclear migrations. In the C. elegans one-cell embryo Arp2/3 promotes the establishment of anterior/posterior polarity and promotes MT growth that propels the anterior migration of the male pronucleus. In contrast with previous studies emphasizing pulling forces on the male pronucleus, we propose that robust MT nucleation pushes the male pronucleus anteriorly to join the female pronucleus.