A tip-high, Ca2+-interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes

We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca2+](cyt) gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca2+](cyt) gradient. Such modulation of intracellular [Ca2+](cyt) signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.

The terminal tail region of a yeast myosin-V mediates its attachment to vacuole membranes and sites of polarized growth

The Saccharomyces cerevisiae myosin-V, Myo2p, has been implicated in the polarized movement of several organelles and is essential for yeast viability. We have shown previously that Myo2p is required for the movement of a portion of the lysosome (vacuole) into the bud and consequently for proper inheritance of this organelle during cell division. Class V myosins have a globular carboxyl terminal tail domain that is proposed to mediate localization of the myosin, possibly through interaction with organelle-specific receptors. Here we describe a myo2 allele whose phenotypes support this hypothesis. vac15–1/myo2–2 has a single mutation in this globular tail domain, causing defects in vacuole movement and inheritance. Although a portion of wild-type Myo2p fractionates with the vacuole, the myo2–2 gene product does not. In addition, the mutant protein does not concentrate at sites of active growth, the predominant location of wild-type Myo2p. Although deletion of the tail domain is lethal, the myo2–2 gene product retains the essential functions of Myo2p. Moreover, myo2–2 does not cause the growth defects and lethal genetic interactions seen in myo2–66, a mutant defective in the actin-binding domain. These observations suggest that the myo2–2 mutation specifically disrupts interactions with selected myosin receptors, namely those on the vacuole membrane and those at sites of polarized growth.

The Yck2 Yeast Casein Kinase 1 Isoform Shows Cell Cycle-specific Localization to Sites of Polarized Growth and Is Required for Proper Septin Organization

Casein kinase 1 protein kinases are ubiquitous and abundant Ser/Thr-specific protein kinases with activity on acidic substrates. In yeast, the products of the redundant YCK1 and YCK2 genes are together essential for cell viability. Mutants deficient for these proteins display defects in cellular morphogenesis, cytokinesis, and endocytosis. Yck1p and Yck2p are peripheral plasma membrane proteins, and we report here that the localization of Yck2p within the membrane is dynamic through the cell cycle. Using a functional green fluorescent protein (GFP) fusion, we have observed that Yck2p is concentrated at sites of polarized growth during bud morphogenesis. At cytokinesis, GFP–Yck2p becomes associated with a ring at the bud neck and then appears as a patch of fluorescence, apparently coincident with the dividing membranes. The bud neck association of Yck2p at cytokinesis does not require an intact septin ring, and septin assembly is altered in a Yck-deficient mutant. The sites of GFP–Yck2p concentration and the defects observed for Yck-deficient cells together suggest that Yck plays distinct roles in morphogenesis and cytokinesis that are effected by differential localization.

The Syntaxin Tlg1p Mediates Trafficking of Chitin Synthase III to Polarized Growth Sites in Yeast

Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers. We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p. Remarkably, growth is possible in the absence of all four proteins. In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64. However, although Pep12p is required for the ligand-induced internalization of the α factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not. In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p. In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme. Pep12p is neither necessary nor sufficient for this process. We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.

Identification of Two Type V Myosins in Fission Yeast, One of Which Functions in Polarized Cell Growth and Moves Rapidly in the Cell

We characterized the novel Schizosaccharomyces pombe genes myo4+ and myo5+, both of which encode myosin-V heavy chains. Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm. The mutant cells were rounder than normal, although the sites for cell polarization were still established. Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation. Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures. In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions. The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules. Moreover, the motor activity of Myo4 was essential for its function. These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization. In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells. This and other data suggest that Myo5 has a role distinct from that of Myo4.

Characterization of small GTPase Cdc42 from the ectomycorrhizal fungus Suillus bovinus and Agrobacterium tumefaciens-mediated transformation of fungi

Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic fungus, Helminthosporium turcicum, to hygromycin B resistance. Small GTPases Cdc42 and Rac1, the regulators of actin cytoskeleton in eukaryotes were isolated from S. bovinus. Sbcdc42 and Sbrac1, are both expressed in vegetative and in the symbiotic hyphae of S. bovinus . Using IIF microscopy, Cdc42 and actin were co-localized at the tips of vegetative hyphae and were visualized in association with the plasma membrane in swollen cells typical to the symbiotic hyphae. These results suggest that the small GTPases Cdc42 may play a significant role in the polarized growth of S. bovinus hyphae and regulate fungal morphogenesis during ectomycorrhiza formation through reorganization of the actin cytoskeleton. The functional equality of Cdc42 was tested in yeast complementation experiments using a Saccharomyces cerevisiae temperature sensitive mutant, cdc42-1ts. The genomic clone of CDC42 was isolated from S. bovinus genomic DNA via specific primers for Cdc42. The analogous S. cerevisiae cdc42 mutations, dominant active G12V and dominant negative D118A, were generated in the Sbcdc42 gene by in-vitro mutagenesis. The ectomycorrhizal fungi, S. bovinus, P. involutus and H. cylindroporum were transformed using ATMT and phleomycin as a selectable marker. PCR screeing suggested that the T-DNA was inserted in all the three fungal genomes but the fate of integration could not be proved by Southern blot analysis. An alternative Agrobacterium strain, AGL-1 and selection marker, hygromycin was used to transform our model fungus S. bovinus. PCR and Southern analysis suggested an improved efficiency of transformation. All the transformed fungal colonies selected for hygromycin gave positives in PCR and the Southerns showed multiple or single copy T-DNA integrations into the S. bovinus genome. Using the same Agrobacterium strain and the selectable marker, a maize pathogen, H. turcicum was also subjected to ATMT. The H. turcicum transformation data suggested the single copy T-DNA integrations into the genome of the screened transformants that further confirms wider applicability of the ATMT. The plasmids carrying the wild-type (pHGCDC42) and the mutated Sbcdc42 alleles (pHGGV; pHGDA) under Agaricus bisporus gpd promoter were constructed in an A. tumefaciens vector. ATMT was used to transform S. bovinus with the plasmids carrying the wild-type and mutated Sbcdc42 alleles. The isolation of Sbcdc42 and Sbrac1 genes and some other functionally related genes from ectomycorrhizal fungus, S. bovinus will form the basis of future work to resolve the signalling pathway leading to ectomycorrhizal symbiosis. The development of ATMT system will be a valuable tool in analysing the exact function of signalling pathway components in ectomycorrhizal symbiosis or in plant pathogenic interactions. The transformation frequency and broad applicability along with the simplicity of T-DNA integration make Agrobacterium a valuable, new and a powerfull tool for targeted and insertional mutagenesis in these plant associated fungi. The developed ATMT systems should therefore make it possible to generate large number of transformants with tagged genes which could then be screened for their specific roles in symbiosis and pathogenecity, respectively.

三氟拉嗪抑制钙-钙调素对白杄花粉管生长的调节

花粉管是种子植物受精过程中雄性生殖单位的载体，具有典型的极性顶端生长模式，因此成为研究细胞极性生长机理的理想模式体系。本研究以裸子植物白杄（Picea meyeri Rehd.et Wils）花粉为材料，并以对花粉萌发和花粉管生长起关键作用的Ca2+作为切入点，分析钙-钙调素在花粉萌发及花粉管极性生长中的作用，同时也为进一步探讨它们在其他植物细胞中的作用机理研究提供重要参考。 通过细胞化学定位证明，白杄花粉中含有丰富的游离钙离子和钙调素，在花粉管顶端呈现明显的梯度分布;钙调素特异拮抗剂三氟拉嗪（trifluoperazine，简称TFP）可以在钙离子存在的情况下与钙调素特异性结合，从而抑制钙-钙调素复合物对下游效应蛋白的激活。微摩尔浓度的TFP明显抑制白杄花粉萌发以及花粉管的生长，并导致大部分花粉管畸形生长。TFP处理后的花粉管（约80%以上）中游离钙离子梯度消失或梯度不明显，由此说明钙调素参与花粉管顶端游离钙离子梯度的维持。抑制剂处理显著影响钙调素在花粉管顶端的梯度分布模式，梯度落差明显减小。 应用鬼笔环肽标记花粉管微丝骨架表明，正常生长的花粉管中微丝骨架沿花粉管长轴平行的方向呈网络状分布，但是在花粉管顶端仅有杂乱的微丝片断分布;低浓度TFP处理之后，微丝骨架分布的方向性丧失并开始卷曲，花粉管顶端的微丝片断消失，高浓度TFP处理之后微丝骨架完全断裂，聚集成短粗的束状。FM4-64标记花粉管后发现，经TFP处理的花粉管顶端胞吞速度明显加快，最终染料集中分布在紧贴质膜下很小的区域内，同时胞吞过程加快主要表现在染料进入花粉管细胞的速度加快，而随后染料在细胞内的扩散速度并无明显变化。以酸性磷酸酶为标志的胞吐活性也显著下降。通过MitoTracker染色发现，TFP处理之后花粉管中线粒体的形态和分布都发生了显著变化;在电子显微镜下观察显示，抑制剂处理的花粉管中液泡化现象严重，线粒体膨大变形，其内嵴的结构遭到严重破坏，同时高尔基体和内质网的形态也都发生了不同程度的异常变化，另外线粒体和液泡还出现了类似于自体吞噬的现象。 在荧光显微镜下观察发现，在标准培养基中培养的花粉管经苯胺兰染色后，胼胝质分布于整根花粉管侧壁上，而顶端区域胼胝质分布却很少或不存在。但经TFP处理之后，在花粉管细胞壁的个别区域有胼胝质大量沉积，同时在花粉管中还出现能被苯胺兰特异染色的许多颗粒状物质。此时花粉管顶端细胞壁中的纤维素含量明显减少。以单克隆抗体JIM5、JIM7标记果胶质，在激光扫描共聚焦显微镜下观察发现，标准培养基中培养的花粉管，酸性果胶质分布于整根花粉管的侧壁中，但在其顶端的含量很低或不存在，与此相反，酯化果胶质只分布在花粉管的顶端;而经TFP处理的花粉管中，酸性果胶质均匀分布于花粉管细胞壁上，酯化果胶质仅出现在花粉管基部的细胞壁中。单克隆抗体LM2和LM6标记结果显示，正常生长的花粉管细胞壁中AGPs呈周期性的环状分布，TFP处理后AGPs仅仅分布在花粉管基部的细胞壁中。SDS-PAGE电泳分析显示，抑制剂处理之后花粉管细胞壁蛋白的表达也发生明显变化。由FT-IR分析进一步证实了上述两种果胶质及纤维素在花粉管顶端细胞壁中相对含量的变化趋势。 利用双向电泳技术分离花粉管全蛋白，结果发现正常生长和TFP处理后的花粉管的大部分蛋白斑点都处于pI 4-8以及分子量在14-97 KD的范围内，主要是一些中等分子量大小、微酸性和中性的蛋白类群。由软件分析显示，除其中76个蛋白外，大部分蛋白质的表达并未发生变化。将上述76个表达量发生变化的差异蛋白进行胶内酶解，并经ESI-MS/MS分析鉴定，以及质谱数据库搜索，最终鉴定出57个蛋白，其中23个表达量上调，其余34个表达量下调。根据其生物学功能可以分为碳水化合物及能量代谢、胁迫及防御反应、细胞扩展、信号转导等功能蛋白类群。经TFP处理后，花粉管中碳水化合物及能量代谢过程整体水平下降，氧化磷酸化水平减低，但是丙酮酸脱羧酶旁路代谢水平却略有上升。由此暗示，花粉管在生长停滞的环境条件下，该途径可作为能量供应的替代机制;参与转一碳单位反应的蛋白表达量普遍上调，参与细胞延展（如细胞骨架重构、细胞壁多糖合成）的蛋白表达量下调，此项研究结果与上述的细胞生物学分析结论基本一致。 综上所述，当钙调素蛋白功能受到抑制后，顶端游离钙离子浓度梯度消失同时胞质钙离子浓度显著升高;细胞代谢水平（糖酵解和三羧酸酸循环）整体下降，而可能通过丙酮酸脱羧酶旁路来维持最低限度的能量供应;同时花粉管微丝骨架发生解聚，花粉管细胞壁组成成分合成水平下降，细胞延展相关的能力减弱，最终导致花粉管生长的停滞。钙-钙调素信号存在于白杄花粉萌发和花粉管生长这一特定的细胞生物学事件中，并参与花粉管顶端游离钙离子梯度的维持和定向生长。

Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34cdc2 mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

Dominant Negative Alleles of SEC10 Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeast Saccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.

Ras Regulates the Polarity of the Yeast Actin Cytoskeleton through the Stress Response Pathway

Polarized growth in yeast requires cooperation between the polarized actin cytoskeleton and delivery of post-Golgi secretory vesicles. We have previously reported that loss of the major tropomyosin isoform, Tpm1p, results in cells sensitive to perturbations in cell polarity. To identify components that bridge these processes, we sought mutations with both a conditional defect in secretion and a partial defect in polarity. Thus, we set up a genetic screen for mutations that conferred a conditional growth defect, showed synthetic lethality with tpm1Δ, and simultaneously became denser at the restrictive temperature, a hallmark of secretion-defective cells. Of the 10 complementation groups recovered, the group with the largest number of independent isolates was functionally null alleles of RAS2. Consistent with this, ras2Δ and tpm1Δ are synthetically lethal at 35°C. We show that ras2Δ confers temperature-sensitive growth and temperature-dependent depolarization of the actin cytoskeleton. Furthermore, we show that at elevated temperatures ras2Δ cells are partially defective in endocytosis and show a delocalization of two key polarity markers, Myo2p and Cdc42p. However, the conditional enhanced density phenotype of ras2Δ cells is not a defect in secretion. All the phenotypes of ras2Δ cells can be fully suppressed by expression of yeast RAS1 or RAS2 genes, human Ha-ras, or the double disruption of the stress response genes msn2Δmsn4Δ. Although the best characterized pathway of Ras function in yeast involves activation of the cAMP-dependent protein kinase A pathway, activation of the protein kinase A pathway does not fully suppress the actin polarity defects, suggesting that there is an additional pathway from Ras2p to Msn2/4p. Thus, Ras2p regulates cytoskeletal polarity in yeast under conditions of mild temperature stress through the stress response pathway.

The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions in Saccharomyces cerevisiae

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C. rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ and kar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.

Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG

Modulation of the immune response is an important step in the induction of protective humoral and cellular immunity against pathogens. In this study, we investigated the possibility of using a nanomaterial conjugated with the toll-like receptor (TLR) ligand CpG to modulate the immune response towards the preferred polarity. MgAl-layered double hydroxide (LDH) nanomaterial has a very similar chemical composition to Alum, an FDA approved adjuvant for human vaccination. We used a model antigen, ovalbumin (OVA) to demonstrate that MgAl-LDH had comparable adjuvant activity to Alum, but much weaker inflammation. Conjugation of TLR9 ligand CpG to LDH nanoparticles significantly enhanced the antibody response and promoted a switch from Th2 toward Th1 response, demonstrated by a change in the IgG2a:IgG1 ratio. Moreover, immunization of mice with CpG-OVA-conjugated LDH before challenge with OVA-expressing B16/F10 tumor cells retarded tumor growth. Together, these data indicate that LDH nanomaterial can be used as an immune adjuvant to promote Th1 or Th2 dominant immune responses suitable for vaccination purposes.

Electrical control of dynamic spin splitting induced by exchange interaction as revealed by time-resolved Kerr rotation in a degenerate spin-polarized electron gas

Manipulation of the spin degree of freedom has been demonstrated in a spin-polarized electron plasma in a heterostructure by using exchange-interaction-induced dynamic spin splitting rather than the Rashba and Dresselhaus types, as revealed by time-resolved Kerr rotation. The measured spin splitting increases from 0.256 meV to 0.559 meV as the bias varies from -0.3 V to -0.6 V. Both the sign switch of the Kerr signal and the phase reversal of Larmor precessions have been observed with biases, which all fit into the framework of exchange-interaction-induced spin splitting. The electrical control of it may provide a new effective scheme for manipulating spin-selected transport in spin FET-like devices. Copyright (C) EPLA, 2008.

InAs nanostructure grown with different growth rate in InAlAs matrix on InP (001) substrate

InAs self-organized nanostructures in In0.52Al0.48As matrix have been grown on InP (001) substrates by molecular beam epitaxy. The morphologies of the nanostructures are found to be strongly dependent on the growth rate of the InAs layer. By increasing the growth rate from 0.005 to 0.35 ML/s, the morphology of the nanostructure changes from wire to elongated dot and then changes back to wire again. Polarized photoluminescence of the InAs quantum wires and quantum dots are performed at 77 K, which are characterized by strong optical anisotropies. (C) 2003 Elsevier B.V. All rights reserved.