细胞核钙信号研究进展

钙离子作为细胞内重要的第二信使,在许多生理过程中发挥着关键的作用。最近几年来,随着钙检测技术的不断发展和改进,细胞核钙的研究取得了许多重要的进展。本文从细胞核钙运输、核钙对核与胞质间物质运输的调节,以及核钙在基因表达、有丝分裂和细胞凋亡中的作用等几个方面综述了核钙研究的最新进展。     

细胞核存在独立的钙运输系统

细胞核具有独立的钙运输系统,可以以依赖于胞质游离钙浓度而对核内钙浓度进行调节。核膜为核钙库,其外核膜上存在钙泵和ＩＰ４受体,内核膜上存在的ＩＰ３受体和利苦丁（ｒｙｎｏｄｉｎｅ）受体,核膜经外核膜摄入钙,并在ＩＰ３或环化二磷酸腺核糖（ｃＡＤＰＲ）介导了内核膜向核内释放钙,产生核内钙信号。     

大鼠心肌细胞核钙调素入核转运与核钙调节关系的探讨

最近发现,钙调素作为细胞内钙受体,除了调节胞浆的多种功能之外,可能还参与胞浆信号向核内快速传递。本研究观察大鼠心肌细胞核对钙调素的入核转运与钙浓度的关系,并初步探讨其调节机制。大鼠心肌细胞核采用差速离心和密度梯度离心分离提纯。用荧光分光光度计测定荧光标记钙调素向细胞核转入量发现,大鼠心肌细胞核对核外的CaM向核孔转运量具有[Ca~(2+)]浓度依赖性,随核外[Ca~(2+)]浓度的增加而增加(P<0.001),在[Ca~(2+)]浓度为10~(-3)mol/L时,ryanodine受体的拮抗剂rutheniumred和cADP ribose受体拮抗剂8-Br cADP ribose显著抑制CaM的细胞核孔转运(分别降低20％和18％,P<0.05),而IP_3受体拮抗剂heparin和Ca~(2+)-ATPase抑制剂thapsigargin抑制CaM的细胞核孔转运更显著(分别降低90％和89％,P<0.001)。上述结果表明心肌细胞核对CaM的向核转运,受核外[Ca~(2+)]和核钙摄取、释放所调节。     

Caspase-3细胞定位信号的检测与分析

Caspase-3是凋亡过程中的重要作用蛋白。凋亡过程中,胞质定位的Caspase3被激活并进入细胞核中执行功能,但该定位变化的分子机制至今仍不清楚。分析caspase3中的细胞定位信号可以为深入了解该过程提供重要的线索。我们通过构建一系列含Caspase-3不同区段的截短突变体,与GFP融合表达,观察这些突变体在细胞中的定位,以鉴定Caspase-3中的核外运信号NES（Nuclear Export Signal）和核定位信号NLS（Nuclear Localization Signal）。Caspase-3中不存在明显的核定位信号NLS,但存在一个明显的核外运信号NES,该NES信号定位在caspase-3小亚基的C端（氨基酸220-245）。     

用于细胞核钙成像的新型钙指示剂的构建与鉴定

细胞核钙离子是基因转录等细胞核反应过程重要的调控因子.然而,细胞核内钙离子信号的调控机制尚不清楚.缺乏稳定的、敏感的细胞核钙指示剂,是导致其调控机制难以研究的重要原因之一.针对这一问题,设计了能够在细胞核内特异性表达的、具有核定位功能的钙指示剂.以基因编码钙指示剂（GECIs）家族成员GCaMP6为模板,首先融合了对钙离子不敏感的红色荧光蛋白tdTomato来对局部的钙信号进行量化,其次融合了核定位信号（NLS）,使GCaMP6能够特异定位于细胞核中.结果表明,NLS-GCaMP6-tdTomato能够在细胞核中有效发挥作用,并且在钙敏感性与动力学上,也与GCaMP6相当. 这一新型细胞核钙指示剂将为研究细胞核钙离子的功能及其调控机制提供新的方法与途径.     

蛋白质的入核运送和它在基因表达中调节作用

蛋白质进入细胞核是蛋白质分子内部的核定位信号引导的,ＮＬＳ蛋白首先与ＮＬＳ受体结合,然后在多种胞浆因子及核孔复合物蛋白的作用下穿过核孔,转位入核、蛋白质上存在ＮＬＳ并不一定总能够引导蛋白质入核。当ＮＬＳ被修饰或遮掩时,它们便不能被核转运装置所识别,因而,ＮＬＳ的遮掩被解除之前,蛋白质一直被扣留在胞浆中,以调节转录因子的入核运送来控制转录因子的活性是基因表达调节的一个新概念,了是细胞生长和分化拐一水     

钙闪烁——引导细胞定向运动的微区钙信号

细胞迁移在机体发育、器官重塑以及损伤修复等过程中都起着重要作用。在极化的迁移细胞中,存在"前沿低、后沿高"的胞内钙离子浓度梯度,但是,长期以来一直困扰人们的一个问题是:在迁移细胞的引导前沿,钙信号如何控制细胞的定向运动呢?我们发现,前沿低钙信号背景中,存在大量随机发生的微区、动态高钙信号——钙闪烁(Calcium flickers)。钙闪烁由细胞膜上牵张通道TRPM7所介导的钙内流,经内质网膜钙释放通道——Ⅱ型IP3受体——的放大而产生。在趋化运动中,钙闪烁集中于趋化因子浓度较高的一侧,引导细胞转向该侧。阻断钙闪烁,细胞仍能直线迁移,但丧失了在趋化因子作用下转向的能力。因此,钙闪烁的发现及相关研究揭示了微区钙信号是如何通过时空整合,并最终精细地调节细胞转向等复杂的生物学过程。     

蛋白质的入核运送和它在基因表达中的调节作用

蛋白质进入细胞核是由蛋白质分子内部的核定位信号（nuclear localization signal, NLS）引导的.NLS蛋白首先与NLS受体结合,然后在多种胞浆因子及核孔复合物蛋白的作用下穿过核孔、转位入核.蛋白质上存在NLS并不一定总能够引导蛋白质入核.当NLS被修饰或遮掩时,它们便不能被核转运装置所识别.因而,NLS的遮掩被解除之前,蛋白质一直被扣留在胞浆中.以调节转录因子的入核运送来控制转录因子的活性是基因表达调节的一个新概念,也是细胞生长和分化的另一水平的调节.     

核定位信号研究进展

核定位信号是一种存在于细胞内众多蛋白质中,介导其由细胞质向细胞核转运的氨基酸序列。核定位信号具有形式多样性,其作用机制也不尽相同,对于入核蛋白发挥正常的生理功能有着重要的作用。因其功能异常引发的多种疾病越来越受关注,核定位信号研究成了众多疾病病理机制研究的方向和寻求治疗的手段。简要概述近年来关于核定位信号的类型、相关疾病和应用研究的情况,并列举了几个常用预测软件与网站,供读者参考。     

钙在被子植物受精过程中的作用

近年来,花粉管中的钙信号和生理功能的研究又取得了明显的进展,同时在雌蕊系统中有关钙分布的研究也初步显示了其时、空特征与被子植物的受精作用密切相关。该文总结了花粉萌发和花粉管生长过程中外源钙的作用机制,结合雌蕊组织中钙分布的特征,进一步探讨了钙在被子植物受精过程中的功能。     

Potentiation of a sodium-calcium exchanger in the nuclear envelope by nuclear GM1 ganglioside

Calcium is recognized as an important intracellular messenger with a pivotal role in the regulation of many cytosolic and nuclear processes. Gangliosides of various types, especially GM1, are known to have a role in some aspects of Ca2+ regulation, operating through a variety of mechanisms that are gradually coming to light. The present study provides evidence for a sodium-calcium exchanger in the nuclear envelope of NG108-15 neuroblastoma cells that is potently and specifically activated by GM1. Immunoblot analysis revealed an unusually tight association of GM1 with the exchanger in the nuclear envelope but not with that in the plasma membrane. Exchanger and associated GM1 were located in the inner membrane of the nuclear envelope, suggesting this system could function to transfer Ca2+ between nucleoplasm and the envelope lumen. The GM1-enhanced exchange was blocked by cholera toxin B subunit while C2-ceramide, a recently discovered inhibitor of the exchanger, blocked all transfer. Exchanger activity was significantly elevated in nuclei isolated from cells that were induced to differentiate by KCl + dibutyryl-cAMP, a treatment previously shown to promote up-regulation of nuclear GM1 in conjunction with axonogenesis. Similar enhancement was achieved by addition of exogenous GM1 to nuclei from undifferentiated cells. These results suggest a prominent role for nuclear GM1 in regulation of nuclear Ca2+ homeostasis.     

Calcium regulation of nucleocytoplasmic transport

Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calcium-regulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule super-resolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.     

Gangliosides of the nuclear membrane: a crucial locus of cytoprotective modulation

The original concept of gangliosides as localized components of the plasma membrane has broadened in recent years with recognition of their presence in various intracellular pools as well. The nuclear envelope (NE), consisting of two unique membranes, is one such structure shown to contain members of the gangliotetraose family and possibly other sialoglycolipids. GM1 situated in the inner membrane of the NE is tightly associated with a Na+/Ca2+ exchanger whose activity it potentiates in the transfer of Ca2+ from nucleoplasm to the NE lumen. This is in contrast to Na+/Ca2+ exchangers of the plasma membrane which bind GM1 less avidly or not at all. This is believed due to different isoforms of exchanger, and a difference in topology of the exchanger relative to GM1. Cultured neurons from mice genetically engineered to lack gangliotetraose gangliosides such as GM1 were highly vulnerable to Ca2+-induced apoptosis. They were rescued to some extent by GM1 but more effectively by LIGA-20, a membrane-permeant derivative of GM1 that traverses the plasma membrane more effectively than GM1 and inserts into the NE. As further indication of Ca2+ dysregulation, the mutant mice were highly susceptible to kainite-induced seizures which were attenuated by LIGA-20. This correlated with the ability of LIGA-20 to cross the blood-brain barrier, enter brain cells, insert into the NE, and potentiate the nuclear exchanger. GM1 in the NE, in association with nuclear Na+/Ca2+ exchanger, is thus seen as contributing to Ca2+ regulation within the nucleus and in the process exerting a cytoprotective role.     

钙信号在卵母细胞激活中的作用

钙信号是胞内主要的第二信使之一,发挥广泛的作用如细胞分裂、细胞凋亡等,对细胞的生命活动起着非常重要的作用。在精子和卵母细胞中,钙信号对精子获能、顶体反应、卵母细胞成熟、受精及卵裂等一系列复杂的过程有非常重要的影响。现就Ca2 在卵母细胞中的释放机制、信号转导途径、调控功能作一综述。     

Nuclear sphingolipids: metabolism and signaling

Sphingolipids are most prominently expressed in the plasma membrane, but recent studies have pointed to important signaling and regulatory roles in the nucleus. The most abundant nuclear sphingolipid is sphingomyelin (SM), which occurs in the nuclear envelope (NE) as well as intranuclear sites. The major metabolic product of SM is ceramide, which is generated by nuclear sphingomyelinase and triggers apoptosis and other metabolic changes. Ceramide is further hydrolyzed to free fatty acid and sphingosine, the latter undergoing conversion to sphingosine phosphate by action of a specific nuclear kinase. Gangliosides are another type of sphingolipid found in the nucleus, members of the a-series of gangliotetraose gangliosides (GM1, GD1a) occurring in the NE and endonuclear compartments. GM1 in the inner membrane of the NE is tightly associated with a Na(+)/Ca(2+) exchanger whose activity it potentiates, thereby contributing to regulation of Ca(2+) homeostasis in the nucleus. This was shown to exert a cytoprotective role as absence or inactivation of this nuclear complex rendered cells vulnerable to apoptosis. This was demonstrated in the greatly enhanced kainite-induced seizure activity in knockout mice lacking gangliotetraose gangliosides. The pathology included apoptotic destruction of neurons in the CA3 region of the hippocampus. Ca(2+) homeostasis was restored in these animals with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger. Some evidence suggests the presence of uncharged glycosphingolipids in the nucleus.     

植物钙感受器及其介导的逆境信号途径

钙信号是植物生长发育和逆境响应的重要调控因子, 是植物生理与逆境生物学研究领域中的热点之一。当植物细胞受到外界逆境刺激时, 其胞内会产生具有时空特异性的Ca2+信号变化, 这种变化首先被胞内钙感受器所感知并解码, 再由钙感受器互作蛋白将信号传递到下游, 从而激活下游早期响应基因的表达或相关离子通道的活性, 最终产生特异性逆境响应。植物细胞通过感知胞内钙信号的变化如何识别来自外界不同性质或不同强度的刺激, 是近几年植物生物学家所关注的科学问题。文章主要总结了近几年在植物钙感受器研究领域中的最新进展, 包括钙依赖蛋白激酶(CDPKs)、钙调素(CaMs)、类钙调素蛋白(CMLs)、类钙调磷酸酶B蛋白(CBLs)及其互作蛋白激酶(CIPKs)等的结构、功能及其介导的逆境信号途径, 并提供新的见解和展望。     

Regulation of nuclear Ca2+ signaling by translocation of the Ca2+ messenger synthesizing enzyme ADP-ribosyl cyclase during neuronal depolarization

In neurons, voltage-gated Ca(2+) channels and nuclear Ca(2+) signaling play important roles, such as in the regulation of gene expression. However, the link between electrical activity and biochemical cascade activation involved in the generation of the nuclear Ca(2+) signaling is poorly understood. Here we show that depolarization of Aplysia neurons induces the translocation of ADP-ribosyl cyclase, a Ca(2+) messenger synthesizing enzyme, from the cytosol into the nucleus. The translocation is dependent on Ca(2+) influx mainly through the voltage-dependent L-type Ca(2+) channels. We report also that specific nucleoplasmic Ca(2+) signals can be induced by three different calcium messengers, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate (NAADP), both produced by the ADP-ribosyl cyclase, and inositol 1,4,5-trisphosphate (IP(3)). Moreover, our pharmacological data show that NAADP acts on its own receptor, which cooperates with the IP(3) and the ryanodine receptors to generate nucleoplasmic Ca(2+) oscillations. We propose a new model where voltage-dependent L-type Ca(2+) channel-induced nuclear translocation of the cytosolic cyclase is a crucial step in the fine tuning of nuclear Ca(2+) signals in neurons.     

Large-conductance cationic channels in the nuclear envelope of Purkinje neurons from the rat cerebellum

Using a patch-clamp technique, we studied the biophysical properties of large-conductance channels in the nuclear envelope of rat cerebellar Purkinje neurons. Our experiments showed that channels with identical conductance, selectivity, and kinetics are expressed in the external and internal nuclear membranes of these cells. These channels connect the perinuclear space with the cyto-and nucleoplasm; they are not channels of the complex of the nuclear pores for passive diffusion of ions and small molecules, as was believed earlier [17]. We hypothesize that large-conductance cationic channels in the membranes of the nuclear envelope are identical to ion channels of the endoplasmic reticulum and are necessary for functioning of the intermembrane space of the envelope as a calcium store. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 113–118, March–April, 2007.