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.     

斑马鱼胚胎第一次卵裂过程中胞内钙信号的研究

钙离子作为广泛存在的细胞内信使物质,在动物胚胎早期发育过程中扮演重要角色.为了研究钙离子在斑马鱼胚胎发育过程中的空间分布和浓度变化,采用Fluo-4和Indo-1作为钙离子指示剂,利用激光共聚焦和双波长荧光比例成像技术,对斑马鱼胚胎第一次卵裂过程中的钙信号进行了详细的跟踪观察.在第一次卵裂过程中,斑马鱼胚胎的动物极顶端首先出现高钙斑,然后在分裂沟部位出现高浓度的钙信号,这一信号在卵裂过程中持续存在.利用Indo-1双波长荧光比例成像对上述过程中钙离子的时空分布进行了定量测定,表明,胞内钙离子在卵裂开始之前是均匀分布的,随着分裂沟的出现,其附近区域的钙浓度显著升高,而胞内其他区域的钙浓度则保持不变.双波长荧光比例成像排除了荧光染料分布不均匀造成的干扰,为钙信号与胚胎分裂的密切关系提供了确凿的定量依据.     

用于细胞核钙成像的新型钙指示剂的构建与鉴定（英文）

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GCa MP6为模板,首先融合了对钙离子不敏感的红色荧光蛋白td Tomato来对局部的钙信号进行量化,其次融合了核定位信号(NLS),使GCa MP6能够特异定位于细胞核中.结果表明,NLS-GCa MP6-td Tomato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GCa MP6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.     

电压门控钙通道钙依赖性易化和失活的分子机制

电压门控钙通道受钙依赖性易化和失活两种相互对立的反馈机制调节.不同浓度的钙离子,通过作为钙感受器的钙调蛋白的介导,主要与钙通道α1亚基羧基端的多个不连续片段发生复杂的相互作用,分别引发钙依赖性易化和失活.钙/钙调蛋白依赖性蛋白激酶Ⅱ及其它钙结合蛋白等也参与此调节过程.新近研究表明,钙通道的钙依赖性调节机制失衡与心律失常等的发病机制密切相关.     

圆背角无齿蚌离体培养的外套膜组织钙代谢

本次实验采用离体组织培养技术研究外套膜组织的钙代谢,它排除了蚌体内的其他因素,如神经、激素等对外套膜生理、生化等方面的影响,并用组化方法对外套膜中钙及有关粘多糖的分布进行了观察研究,期望能进一步了解外套膜组织钙代谢调控的机理,同时,为淡水珍珠养殖业提供一些参考资料。       

植物细胞中钙信号的时空多样性与信号转导

近年来,对钙信号的研究,包括对钙信号的产生,传导及最终靶蛋白的研究,越来越受到人们的重视,植物生长发育过程的信息传递,包括对各种内外刺激的反应都涉及到钙信号,钙信号的产生及传导是通过胞质自由钙离子的浓度变化来实现的,本文综述了胞质自由钙离子的测定,钙信号的时空多样性及钙信号的靶蛋白如CaM,Ca^2 依赖的蛋白激酶,钙调磷酸酶,磷脂酰肌醇－磷脂酶C等方面的一些最新进展,展望了今后钙信号研究的方向所用到的一些技术方法等。     

应用AR—CM—MIC阳离子测定系统检测单个神经元内游离钙

运用Ca~(2 )指示剂Fura-2作为细胞内钙离子的荧光探针,采用精密的AR-CM-MIC阳离子测定系统,检测了分离的单个神经细胞内游离钙离子浓度的动态变化,同时观察了钙离子载体、钙螯合剂等多种药物对细胞内钙浓度的影响,并追踪刺激前后的瞬间变化,探讨此项技术应用于检测细胞内游离钙的灵敏度及适用范围,取得了良好的效果。     

线粒体和细胞内钙自稳平衡

线粒体对胞浆钙信号调节作用的研究已经历较长时间.近年,随着研究方法和技术的不断改进,发现在绝大多数生理条件下,线粒体都能参与胞内钙通信过程.线粒体可感受其周围钙微区的存在从而摄取钙,又可以通过钠-钙交换和大分子孔道将钙释放出来,因此可以调节胞浆钙信号的时空特性,影响相关的细胞功能.但是,由于技术上的局限性,目前的研究仍然存在模糊不清和自相矛盾之处,有待于进一步研究.     

钙火花研究进展与瞻望

钙离子是最广泛而又最重要的细胞内第二信使。自1993年以来,钙火花等一系列钙信号基本单元相继发现,揭示了细胞钙信号转导的数字-模拟二元特征：纳米-微米尺度上短暂的钙信号事件（数字系统）随机叠加于连续的全细胞钙信号（模拟系统）背景中。数字模式的微区域钙信号赋予细胞钙信号在时间、空间、幅度上多尺度多层次的精细结构。对钙火花激活机制、协同机制、终止机制等方面的研究,为钙释放通道阵列的门控及调节提出了新的见解和问题。钙火花等对于高域值钙依赖性过程（如肌细胞兴奋-收缩耦联、细胞兴奋性和神经细胞分泌）的激活和时空调控具有特别重要的生理和病理意义。钙信号“激-模二元性”的研究可望进一步揭示细胞钙信号的简单性与复杂性的统一。     

植物钙吸收、转运及代谢的生理和分子机制

钙是植物必需的营养元素。酸性砂质土壤中含钙较少,导致在其土壤上生长的作物容易缺钙。另外由于果树果实、果菜类和包心叶菜类的蒸腾作用弱,导致果树和蔬菜普遍生理缺钙。根系维管束组织可能通过共质体和质外体两种途径进行钙素吸收,而果实则可通过非维管束组织直接吸收钙素。Ca2 通过Ca2 通道内流进入胞质,并通过Ca2 -ATPase和Ca2 /H 反向转运蛋白外流以保持胞质内低Ca2 浓度。为了应对植物发育和环境胁迫信号,Ca2 由质膜、液泡膜和内质网膜的Ca2 通道内流进入胞质,导致胞质Ca2 浓度迅速增加,产生钙瞬变和钙振荡,传递到钙信号靶蛋白,如钙调素、钙依赖型蛋白激酶及钙调磷酸酶B类蛋白,引起特异的生理生化反应。本文综述了植物钙素吸收、转运以及代谢研究的最新进展,包括植物对钙的需求和作物缺钙的原因,根系维管束组织及果实钙素吸收机理,Ca2 跨膜运输特性,钙的信使作用以及钙信号靶蛋白等方面内容。     

Structural basis of the ultrasensitive calcium indicator GCaMP6

GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology.The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change.In this study,we determined crystal structures of Ca2+-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its parent version GCaMP5G.Our analyses reveal the structural basis for the outperformance of this newly developed Ca2+indicator.Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and increased fluorescence intensity common to all three versions of GCaMP6.Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity,maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling.Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.     

活细胞钙动态的共聚焦扫描显微镜检测技术

共聚焦激光扫描显微镜（Confocal Laser Scarming Microscope,CLSM）广泛应用于活细胞内钙敏感探针标记的钙水平的动态测量。较之传统的显微镜CLSM在钙成像分析上有着不可比拟的优越性,但也存在一些缺陷,近些年陆续出现了一些针对这些缺陷的改善措施,如比率法、葡聚糖探针及其他一些新技术与共聚焦显微镜的联合应用等,并且出现了诸如双光子显微镜等新型激光共聚焦显微镜。随着共聚焦钙成像技术的不断发展进步,其今后的应用前景将会越越广阔。     

L-type calcium channels as drug targets in CNS disorders

L-type calcium channels are present in most electrically excitable cells and are needed for proper brain, muscle, endocrine and sensory function. There is accumulating evidence for their involvement in brain diseases such as Parkinson disease, febrile seizures and neuropsychiatric disorders. Pharmacological inhibition of brain L-type channel isoforms, Cav1.2 and Cav1.3, may therefore be of therapeutic value. Organic calcium channels blockers are clinically used since decades for the treatment of hypertension, cardiac ischemia, and arrhythmias with a well-known and excellent safety profile. This pharmacological benefit is mainly mediated by the inhibition of Cav1.2 channels in the cardiovascular system. Despite their different biophysical properties and physiological functions, both brain channel isoforms are similarly inhibited by existing calcium channel blockers. In this review we will discuss evidence for altered L-type channel activity in human brain pathologies, new therapeutic implications of existing blockers and the rationale and current efforts to develop Cav1.3-selective compounds.     

Calcium homeostasis and signaling in yeast cells and cardiac myocytes

Calcium ions are the most ubiquitous and versatile signaling molecules in eukaryotic cells. Calcium homeostasis and signaling systems are crucial for both the normal growth of the budding yeast Saccharomyces cerevisiae and the intricate working of the mammalian heart. In this paper, we make a detailed comparison between the calcium homeostasis/signaling networks in yeast cells and those in mammalian cardiac myocytes. This comparison covers not only the components, structure and function of the networks but also includes existing knowledge on the measured and simulated network dynamics using mathematical models. Surprisingly, most of the factors known in the yeast calcium homeostasis/signaling network are conserved and operate similarly in mammalian cells, including cardiac myocytes. Moreover, the budding yeast S. cerevisiae is a simple organism that affords powerful genetic and genomic tools. Thus, exploring and understanding the calcium homeostasis/signaling system in yeast can provide a shortcut to help understand calcium homeostasis/signaling systems in mammalian cardiac myocytes. In turn, this knowledge can be used to help treat relevant human diseases such as pathological cardiac hypertrophy and heart failure.     

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz & Shatz, 1996; Khaziphov & Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic & Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.     

Enlightenment on the aequorin-based platform for screening Arabidopsis stress sensory channels related to calcium signaling

ProspectLacking an efficient method to isolate mutants in Ca²⁺ signal generation process may limit Ca²⁺ signaling research in rice. Typical forward genetic screening is always useful to find genes involved in Ca²⁺ signaling. Looking back at existing research in rice, rice calcium signal research has only just begun. Following the Arabidopsis mature research methods and techniques, especially the mutant screening system, we expect to find several important Ca²⁺ related calcium sensors which have important agronomic traits in the near future. We are looking forward to great advances in rice calcium signaling research.     

Biomineralizations: insights and prospects from crustaceans

For growing, crustaceans have to molt cyclically because of the presence of a rigid exoskeleton. Most of the crustaceans harden their cuticle not only by sclerotization, like all the arthropods, but also by calcification. All the physiology of crustaceans, including the calcification process, is then linked to molting cycles. This means for these animals to find regularly a source of calcium ions quickly available just after ecdysis. The sources of calcium used are diverse, ranging from the environment where the animals live to endogenous calcium deposits cyclically elaborated by some of them. As a result, crustaceans are submitted to an important and energetically demanding calcium turnover throughout their life. The mineralization process occurs by precipitation of calcium carbonate within an organic matrix network of chitin-proteins fibers. Both crystalline and stabilized amorphous polymorphs of calcium carbonate are found in crustacean biominerals. Furthermore, Crustacea is the only phylum of animals able to elaborate and resorb periodically calcified structures. Notably for these two previous reasons, crustaceans are more and more extensively studied and considered as models of choice in the biomineralization research area.     

Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS*

Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca²⁺ oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca²⁺ enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca²⁺ changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca²⁺ oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca²⁺ influx. This is the first study showing, in real time, Ca²⁺ signals preceding egress and their direct link with motility, an essential virulence trait.     

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis.