Visualizing the molecular timing of a physiological decision at the nanoscale.
Visualizing the molecular timing of a physiological decision at the nanoscale.
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在纳米尺度上可视化生理决定的分子时间。
DOI：
10.1016/j.bpj.2013.11.019
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发表时间：
2013
影响因子：
3.4
通讯作者：
Gosse,JulieA
中科院分区：
文献类型：
--
作者：
Hess,SamuelT;Gosse,JulieA
肥大细胞是许多疾病的关键参与者，包括过敏，哮喘，传染病，癌症，甚至许多中枢神经系统疾病，如自闭症，焦虑症和多发性硬化症（1，2）。与这些众多的角色相结合，肥大细胞几乎存在于人体的每个组织中。许多对肥大细胞功能至关重要的分子元件，如细胞骨架参与和钙信号传导，也是多种细胞类型（包括神经元和T细胞）中信号传导所必需的。Shelby et al.的文章(3)在这一期的《生物物理杂志》上发表的文章展示了我们认为是第一个活肥大细胞的超分辨率成像，其中一个重要的细胞决定（与生理反应有关）是通过高亲和力IgE受体FcεRI的横向组织在分子水平上做出的。这种分子决定决定了细胞是否会去免疫，释放组胺、血清素和其他效应物，导致下游的一系列后果。抗原诱导IgE结合FcεRI受体聚集后，肥大细胞信号传导导致酪氨酸磷酸化级联反应，导致磷脂酶C活化。继而，产生肌醇1，4，5-三磷酸并与内质网中的其受体结合，这激活Ca 2+流入细胞质，然后导致脱粒（4）。(3)显示了使用随机光学重建显微镜对活细胞中IgE受体进行突破性的超分辨率成像（5，6），并发现了刺激后以纳米分辨率可视化的受体分子的戏剧性重组。他们的工作是超分辨率在膜横向组织中的出色应用：它包括对膜受体聚集和扩散的仔细量化，并强调了如何在活细胞中使用基于单分子定位的超分辨率方法获得空间和动态信息。此外，Shelby et al. (3)证明了我们对一个重要生物系统的理解的进步。重要的是，这项工作不仅证明了膜簇的性质，而且还证明了分子动力学、纳米级簇（以前无法获得的长度尺度）和（至关重要的）细胞功能之间的关系。作者能够区分刺激对受体迁移率和聚集的影响。有些令人惊讶的是，受体迁移率的降低和受体组装成簇并不同时发生。相反，在短时间尺度上（刺激后2分钟内），受体迁移率降低而没有强烈的聚集，这发生在Ca 2+动员的功能反应之前。然后，在刺激后约5分钟，受体重组成约70 nm的簇，每个簇有约100个受体。如果不使用定位显微镜，进行这些研究将是极其困难的：电子显微镜提供了出色的分辨率，但通常与活细胞不兼容，而传统的荧光成像将无法达到必要的分辨率（即< 70 nm）或能够计数分子。因此，这项工作是一个重大的生物学进步，
Mast cells are critical players in numerous diseases, including allergy, asthma, infectious disease, cancer, and even many central nervous system disorders such as autism, anxiety, and multiple sclerosis (1, 2). In conjunction with these numerous roles, mast cells are found in almost every tissue in the human body. Many of the molecular elements crucial to mast cell function, such as cytoskeletal involvement and calcium signaling, are also essential to signaling in a variety of cell types, including neurons and T cells. The article by Shelby et al.(3) in this issue of the Biophysical Journal demonstrates what we believe to be the first superresolution imaging of living mast cells, where an important cellular decision (related to a physiological response) is made at the molecular level through the lateral organization of the high-affinity IgE receptor FcεRI. This molecular decision determines whether the cell will degranulate, releasing histamine, serotonin, and other effectors that lead to a host of downstream consequences. Upon antigen-induced aggregation of IgE-bound FcεRI receptors, mast cell signaling results in a tyrosine phosphorylation cascade, leading to the activation of phospholipase C. In turn, inositol 1, 4, 5-triphosphate is produced and binds to its receptor in the endoplasmic reticulum, which activates Ca2+ influx into the cytosol, which then leads to degranulation (4).Shelby et al.(3) show groundbreaking superresolution imaging of IgE receptors in living cells using stochastic optical reconstruction microscopy (5, 6), and find a dramatic reorganization of receptor molecules visualized with nanometer resolution as a function of time after stimulation. Their work is an excellent application of superresolution to membrane lateral organization: it includes careful quantification of membrane receptor clustering and diffusion, and it highlights how both spatial and dynamic information can be obtained with single-molecule localization-based superresolution methods in live cells. Furthermore, Shelby et al.(3) demonstrate an advance in our understanding of an important biological system. Importantly, this work demonstrates not just the properties of membrane clustering, but the relationships among molecular dynamics, nanoscale clustering (at length scales not previously accessible), and (crucially) cellular function. The authors are able to separate the effects of stimulation on both receptor mobility and clustering. Somewhat surprisingly, reduction in receptor mobility and assembly of receptors into clusters do not occur simultaneously. Rather, on short timescales (within 2 min of stimulation), receptor mobility reduces without strong clustering, and this occurs before the functional response of Ca2+ mobilization. Then, at~ 5 min after stimulation, the receptors reorganize into~ 70 nm clusters with~ 100 receptors each. It would be extremely difficult to carry out these studies without the use of localization microscopy: electron microscopy provides outstanding resolution, but is generally incompatible with living cells, and conventional fluorescence imaging would not reach the necessary resolution (ie,< 70 nm) or be able to count molecules. Thus, this work is a substantial biological advance that