前纤维蛋白点突变对粗糙脉孢菌生长的影响及其生化机制研究

【目的】为进一步了解前纤维蛋白(profilin,PFN)在丝状真菌中的功能,本文以粗糙脉孢菌(Neurospora crassa)为研究对象,进行了前纤维蛋白对其菌落生长和肌动蛋白(actin)聚合特性影响的探究。【方法】通过采用定点突变、同源重组、分生孢子过膜和PCR等技术,获得粗糙脉孢菌前纤维蛋白F78 (F78A和F78D)和V113 (V113E、V113R和V113W)的点突变体。利用平板生长法、竞争性生长管和显微镜观察检测表型变化,并结合多聚脯氨酸亲和层析纯化、荧光分光光度技术和高速共沉淀等技术分析点突变的前纤维蛋白对肌动蛋白聚合特性的影响。【结果】获得的粗糙脉孢菌前纤维蛋白点突变株F78A、F78D、V113E、V113R和V113W,与对照菌株ku70RIP相比,突变株生长均明显减慢(P<0.05),其中PFN (F78D)和PFN (V113W)的突变体在生长的12–48 h,菌落直径分别仅为对照的20.0%–75.7%和12.7%–39.2%。竞争性生长管分析表明,PFN (F78D)和PFN(V113W)突变株菌丝生长速度受到显著抑制,分生孢子形成的节律并...     

形成素结合蛋白调节粗糙脉孢菌菌丝极性生长发育

微丝骨架在真菌菌丝极性生长中具有重要的功能,而其动态解聚 聚合特性是其实现功能的前提.形成素作为肌动蛋白结合蛋白,是微丝骨架动态调控因子之一,而形成素结合蛋白对于形成素发挥功能非常关键,但是对其在真菌极性生长发育中的功能还未见报道.本文以丝状真菌粗糙脉孢菌为材料,利用同源重组基因敲除技术,通过电击转化、分生孢子过膜以及PCR鉴定的方法,获得了形成素结合蛋白基因缺失突变菌株（FBPKO）.进一步利用平板生长方法并结合细胞壁染色对突变菌株的表型进行分析. 结果显示, 与野生型相比,在分生孢子接种后24 h内突变菌株FBPKO的菌丝生长明显减慢且分支异常.这些结果表明, 形成素结合蛋白调节着粗糙脉孢菌菌丝早期的极性生长发育.     

植物细胞中的前纤维蛋白

肌动蛋白组成的微丝骨架是真核细胞中的重要结构,在体内处于高度动态变化之中,受多种肌动蛋白结合蛋白（actin-binding proteins）的调节。前纤维蛋白（profilin）是一种单体肌动蛋白结合蛋白,存在于所有的真核细胞中,在植物细胞中也得到较多的研究。前纤维蛋白除可以结合单体肌动蛋白之外,还可以与磷脂酰肌醇及富含多聚脯氨酸的蛋白质等多种分子结合,在细胞信号转导中行使着重要的功能。本文结合本实验室的研究结果,概述了前纤维蛋白的最新研究进展。     

微丝骨架的构成及其对花粉管极性生长的调控作用

微丝骨架是细胞骨架的重要组成部分,它由肌动蛋白和肌动蛋白结合蛋白组成,广泛存在于真核细胞中。近年来,大量研究表明植物花粉及花粉管中存在丰富的微丝骨架。目前,在微丝骨架作为信号转导途径的靶标参与对花粉管极性生长的调控、微丝骨架在花粉和花粉管中的分布及其在花粉管生长过程中与其他信号分子之间的相互作用等方面取得了一系列突破性进展。     

应用水稻花粉离体萌发体系观察花粉管内微丝分布

采用非固定、DMSO渗透和异硫氰酸标记的鬼笔环肽（FITC—Ph）染色方法,观察水稻花粉离体萌发过程中花粉管内肌动蛋白微丝的形态和分布。结果表明：（1）水稻花粉水合2min后即可萌发,花粉管生长速度在600～1500μm／h之间。（2）水合而未萌发的花粉粒中,大量较短的梭形微丝束构成微丝网络结构,萌发过程中花粉粒内的梭形微丝束松解,部分微丝转移至萌发的花粉管内沿花粉管纵轴呈束状结构;随着花粉管的伸长,微丝束主要分布在花粉管中前端,但在花粉管顶端区域始终未见明显的微丝束。（3）水合后不能正常萌发的花粉粒内肌动蛋白微丝呈弥散不规则分布,在相同萌发时间生长迟缓的花粉管中,微丝束较少,且主要位于花粉管近萌发孔的部位。表明微丝骨架的形态和分布影响水稻花粉管的萌发和生长。     

前纤维蛋白影响花粉肌动蛋白聚合分析

利用超速离心沂淀及紫外分光光度测定等技术,研究了不同比例的玉米（Ｚｅａ ｎａｙｓ Ｌ．）花粉内泊前纤维蛋白对玉米（Ｚｅａ ｍａｙｓ Ｌ．）花烩肌动蛋白（前纤维蛋白与肌动蛋白摩尔数比分别为２：１,１．５：１,１：１,０．５：１,０．１：１）聚合与解聚的影响。初步实验结果显示,前纤维蛋白在各种比例下均可与Ｍｇ＝ＡＴＰ肌动蛋白结合并抑制肌动蛋白的聚实验条件下尚水见玻有前纤维蛋白促进植物肌动蛋白聚合的作用     

MACF1与成骨细胞骨架之间的相互关系研究

采用激光共聚焦显微术研究微管微丝交联因子（MACF1）与成骨样细胞（MD63及MC3T3）微丝／微管骨架、黏着斑之间的相互关系．结果表明,MACF1不连续地分布于微管纤维上,与微丝骨架部分共定位于胞质中,在很多的成骨细胞中可见MACF1分布于骨架相关的粘着斑处：细胞松弛素B影响了MACF1在成骨细胞中的分布,并有使其向细胞核周围及核内转位的趋势．秋水仙素对MACF1的分布无明显的影响．转染了siRNA—MACFl的MG．63细胞微丝骨架纤维分布不连续、微管骨架纤维分布紊乱．这些结果提示MACF1不仅起交联微丝及微管细胞骨架的作用．而且还可稳定细胞骨架：成骨细胞MACF1的分布更依赖于微丝骨架的完整性．     

肌动蛋白解聚因子在动物生殖和胚胎发育中的作用

肌动蛋白解聚因子家族Cofilin/ADF(AC蛋白家族)属于肌动蛋白结合蛋白,是微丝骨架的一个重要调节者。AC蛋白家族能够截断微丝,促进肌动蛋白单体的解离和循环以及微丝解聚,调控微丝骨架的重建,进而影响与微丝骨架相关的一些生理功能如细胞增殖、迁移、凋亡及胚胎发育等。本文将着重介绍AC蛋白家族在动物生殖诸如精子发生、卵巢发育、卵子发生、卵裂,以及胚胎发育等过程中的调节与功能。     

植物微丝骨架动态变化的调节

由球形肌动蛋白聚合而成的微丝骨架,又称肌动蛋白纤维,它在细胞运动、细胞形态建成以及物质运输等诸多生命活动中发挥重要作用。细胞内微丝的解聚和聚合动态特性是微丝骨架行使功能的重要基础,并受到如微丝结合蛋白、金属离子、小G蛋白等各种因素的严格控制。植物细胞微丝骨架的研究虽然晚于动物细胞,但也取得了飞速发展。本文对植物细胞内微丝骨架动态变化的作用机制及一些主要调节因子的最新研究进展做一介绍。     

Profilin is essential for tip growth in the moss Physcomitrella patens

The actin cytoskeleton is critical for tip growth in plants. Profilin is the main monomer actin binding protein in plant cells. The moss Physcomitrella patens has three profilin genes, which are monophyletic, suggesting a single ancestor for plant profilins. Here, we used RNA interference (RNAi) to determine the loss-of-function phenotype of profilin. Reduction of profilin leads to a complete loss of tip growth and a partial inhibition of cell division, resulting in plants with small rounded cells and fewer cells. We silenced all profilins by targeting their 3' untranslated region sequences, enabling complementation analyses by expression of profilin coding sequences. We show that any moss or a lily (Lilium longiflorum) profilin support tip growth. Profilin with a mutation in its actin binding site is unable to rescue profilin RNAi, while a mutation in the poly-l-proline binding site weakly rescues. We show that moss tip growing cells contain a prominent subapical cortical F-actin structure composed of parallel actin cables. Cells lacking profilin lose this structure; instead, their F-actin is disorganized and forms polarized cortical patches. Plants expressing the actin and poly-l-proline binding mutants exhibited similar F-actin disorganization. These results demonstrate that profilin and its binding to actin are essential for tip growth. Additionally, profilin is not needed for formation of F-actin, but profilin and its interactions with actin and poly-l-proline ligands are required to properly organize F-actin.     

Lack of the GTPase RHO-4 in Neurospora crassa causes a reduction in numbers and aberrant stabilization of microtubules at hyphal tips

The multinucleate hyphae of the filamentous ascomycete fungus Neurospora crassa grow by polarized hyphal tip extension. Both the actin and microtubule cytoskeleton are required for maximum hyphal extension, in addition to other vital processes. Previously, we have shown that the monomeric GTPase encoded by the N. crassa rho-4 locus is required for actin ring formation during the process of septation; rho-4 mutants lack septa. However, other phenotypic aspects of the rho-4 mutant, such as slow growth and cytoplasmic bleeding, led us to examine the hypothesis that the microtubule (MT) cytoskeleton of the rho-4 mutant was affected in morphology and dynamics. Unlike a wild-type strain, the rho-4 mutant had few MTs and these few MTs originated from nuclear spindle pole bodies. rho-4 mutants and rho-4 strains containing a GTP-locked (activated) rho-4 allele showed a reduction in numbers of cytoplasmic MTs and microtubule stabilization at hyphal tips. Strains containing a GDP-biased (negative) allele of rho-4 showed normal numbers of MTs and minor effects on microtubule stabilization. An examination of nuclear dynamics revealed that rho-4 mutants have large, and often, stretched or broken nuclei. These observations indicate that RHO-4 plays important roles in regulating both the actin and MT cytoskeleton, which are essential for optimal hyphal tip growth and in nuclear distribution and morphology.     

Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants

Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.     

Coronin is a component of the endocytic collar of hyphae of Neurospora crassa and is necessary for normal growth and morphogenesis

Coronin plays a major role in the organization and dynamics of actin in yeast. To investigate the role of coronin in a filamentous fungus (Neurospora crassa), we examined its subcellular localization using fluorescent proteins and the phenotypic consequences of coronin gene (crn-1) deletion in hyphal morphogenesis, Spitzenk?rper behavior and endocytosis. Coronin-GFP was localized in patches, forming a subapical collar near the hyphal apex; significantly, it was absent from the apex. The subapical patches of coronin colocalized with fimbrin, Arp2/3 complex, and actin, altogether comprising the endocytic collar. Deletion of crn-1 resulted in reduced hyphal growth rates, distorted hyphal morphology, uneven wall thickness, and delayed establishment of polarity during germination; it also affected growth directionality and increased branching. The Spitzenk?rper of Δcrn-1 mutant was unstable; it appeared and disappeared intermittently giving rise to periods of hyphoid-like and isotropic growth respectively. Uptake of FM4-64 in Δcrn-1 mutant indicated a partial disruption in endocytosis. These observations underscore coronin as an important component of F-actin remodeling in N. crassa. Although coronin is not essential in this fungus, its deletion influenced negatively the operation of the actin cytoskeleton involved in the orderly deployment of the apical growth apparatus, thus preventing normal hyphal growth and morphogenesis.     

Purification of a 47-kDa calmodulin-binding polypeptide as an actin-binding protein from Neurospora crassa

We have enriched a 47-kDa polypeptide (p47) from Neurospora crassa on the basis of its affinity to calmodulin. The p47 was purified to homogeneity by chromatography on a Mono S cation exchange column and evidence is presented that the polypeptide co-sediments specifically with F-actin. The intracellular distribution of p47 and actin was also examined using indirect double immunofluorescence staining of cells at different stages of development. Our results suggest that by altering the conformation binding site of actin to p47, calmodulin could play a regulatory role in the polarized hyphal growth of N. crassa.     

Tumor suppressor activity of profilin requires a functional actin binding site

Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1.     

Involvement of Elk-1 in L6E9 skeletal muscle differentiation

The binding of phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P(2)) to profilin at a region distinct from the actin interaction surface is demonstrated by experiments with covalently cross-linked profilin:beta-actin. The result is in agreement with observations made with several mutant profilins and provides strong evidence for two regions on mammalian profilin mediating electrostatic interaction with phosphatidylinositol lipids; one close to the binding site for poly(L-proline), and one partially overlapping with the actin-binding surface. Congruent with this, two plant profilins, which have a reduced number of positive amino acids in one of these regions, displayed a dramatically lower binding to PI(4,5)P(2) compared to human profilin I.     

Profilin binding to poly-L-proline and actin monomers along with ability to catalyze actin nucleotide exchange is required for viability of fission yeast

We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-L-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2-5% wild-type affinity for actin or poly-L-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-L-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast.     

A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization

Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the filamentous fungus Neurospora crassa, we have identified genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding.