激光调控细胞钙信号的技术与机制

Technique and Mechanism of Modulating Cellular Ca^(2+) Signaling Using Laser

钙离子是细胞内重要的第二信使,调节基因转录、能量合成及细胞增殖和凋亡等功能。细胞膜与细胞器上钙相关蛋白协同作用,形成复杂而有序的钙信号网络。在亚细胞结构上特异性激活与抑制某个钙相关蛋白而不影响其他蛋白及其他细胞器能够极大促进亚细胞结构钙信号调节机制及相关功能研究。然而,由于药物在细胞内的自由扩散及蛋白在细胞内的广泛表达,药物的分子特异性及空间特异性有限,因此基于激光的钙信号调节方法得到发展。主要讨论了光解锁笼、光遗传以及全光调控三种基于激光的高空间分辨率的细胞内钙信号调控技术的优点及局限性。理论上,它们对细胞的刺激可以局限在亚微米区域。特别地,分析阐述了基于多光子激发的低功率近红外飞秒激光调控细胞内钙信号的新型技术与机制。

Significance Ca^(2+),an intracellular second messenger,is crucial for cell proliferation,differentiation,metabolism,and programmed cell death by participating in gene transcription,protein modification,adenosine triphosphate (ATP) generation,and the initiation of numerous signaling pathway cascades.Intracellular Ca^(2+) is first arrested by the plasma membrane,the first barrier that maintains an approximately 20000-fold Ca2+concentration gradient from the extracellular environment to the cytosol,by controlling Ca^(2+) influx and efflux through a series of Ca2+channels.Cytoplasmic Ca2+concentration is maintained at approximately 100 nmol/L in most non-excitable cells.Excess cytosolic Ca2+can be chelated by Ca2+-binding proteins,compartmentalized into intracellular Ca2+stores,or extruded into the extracellular environment.Organelles typically play the role of subcellular Ca2+stores (or temporary Ca2+buffers) and further synergize with each other to form a Ca2+network.As nodes on the network,they coordinate with the plasma membrane (PM) to regulate intracellular Ca2+signaling.The endoplasmic reticulum (ER) is the primary Ca2+store,with a resting Ca2+concentration of approximately 200μmol/L,releasing free Ca2+into the cytoplasm.Mitochondria are temporary calcium buffers that transiently accommodate excess Ca2+in the cytosol.Lysosomes and Golgi are small,but important in Ca2+signaling.Among these subcellular structures,the nucleus appears to be a mist for cell calcium research.However,little is known about the existence of Ca2+stores in the nucleus,how nuclear Ca2+is regulated,or how Ca2+is transported through the nuclear membrane.There are lots of membrane contact sites (MCSs) between organelles,such as ER-mitochondria,ER-PM,and ER-nucleus contacts.Local Ca2+interactions at the MCSs also perform vital cellular functions.A ubiquitous broad group of Ca2+sensors,channels,pumps,exchangers,and binding proteins (receptors) in subcellular structures regulate intracellular Ca2+for dynamic hemostasis.Ca2+signaling modulation techniques with high spatial and temporal resolutions can facilitate the study of subcellular Ca2+modulation mechanisms.To the best of our knowledge,owing to the difficulty of isolating Ca2+solely in the submicron domain,some parts of the intracellular Ca2+network,for example,Ca2+regulation in some MCSs and nuclei,remain unclear.Thus,the conductive method of precisely regulating Ca2+in subcellular organelles may provide further insights into the mechanisms of Ca2+hemostasis and Ca2+-involved cell processes.Pharmacological reagents have been extensively used to control intracellular Ca2+signaling in Ca2+research.Ideally,subcellular Ca2+stores can be precisely modulated by these reagents if they activate the target calcium channels/receptors,or organelles with high specificity.However,in practice,owing to poor specificity,most reagents simultaneously bind with a broad spectrum of molecules and perturb the entire cell,finally leading to a significant calcium increase or Ca2+oscillations that smear all interactions between organelles under physiological conditions.In another dimension,the targeting of pharmacological drugs to organelles deteriorates significantly owing to the global distribution of Ca2+channels/receptors in the entire cell.To improve spatial resolution,Ca2+modulation technologies based on lasers have been developed,which can theoretically work in the submicron region by utilizing lasers and microscopic systems.Progress has been made,but their applications vary and are limited because of their different methodological properties.Therefore,it is important and necessary to discuss the methodological advances and limitations of laser-based techniques to provide a systematic reference for researchers in bio-photonics and related biological fields.Progress In this review,three laser-based modulation methods of Ca2+signaling are summarized.It is sensible to take advantage of lasers to precisely control cellular Ca2+at a submicron resolution if the photon energy can be transformed to modulate biochemical processes for Ca2+release.This concept is realized by uncaging (Fig.1) and optogenetics (Fig.3),which utilize lasers to excite Ca2+-caged compounds or optogenetic proteins that have been introduced into cells and are sensitive to lasers for Ca2+modulation.Photouncaging and optogenetics are effective and powerful tools for studying neuroscience.Theoretically,lasers can achieve diffractionlimited spatial resolution,but the specificity and efficiency of these two methods are limited by the intracellular distribution of Ca2+caging compounds or the expression of optogenetic genes.Recently,all-optical technology using a laser to control cellular Ca2+with high resolution and specificity has been reported (Fig.4).Specifically,we provide a deep insight into low-power near-infrared femtosecond laser modulation technologies for precise control of intracellular calcium and derive possible mechanisms for subcellular calcium regulation.Conclusions and Prospects Laser-based Ca2+modulation techniques are powerful tools for cellular calcium research.In particular,the multiphoton excitation of cells with a near-infrared femtosecond laser contributes to high spatial resolution and facilitates the study of subcellular Ca2+modulation mechanisms in organelle contacts and the nucleus.We propose that lasers are capable of bringing breakthroughs in the calcium theory.The interaction mechanism of lasers with cells requires in-depth and detailed exploration to promote the development of subcellular Ca2+signaling.