Intracellular calmodulin availability accessed with two-photon cross-correlation
Intracellular calmodulin availability accessed with two-photon cross-correlation
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DOI：
10.1073/pnas.2436461100
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发表时间：
2004-01-06
影响因子：
11.1
通讯作者：
Schwille, P
中科院分区：
文献类型：
--
作者：
Kim, SA;Heinze, KG;Schwille, P
信号蛋白的可用性和相互作用在时间和空间上受到严格调控，以产生特定和局部的效应。钙调素(CaM)是细胞内钙信号的关键转导分子，与其多种靶点结合可启动信号转导通路并调节其亚细胞定位，从而使其不能用于后续的结合作用。在CaM的众多靶点中，钙/钙M依赖的蛋白激酶11是最引人注目的靶点之一，因为它具有独特的能力，通过自身磷酸化增加与CaM的亲和力，并在与Ca2+/CaM结合时进行移位。利用双光子荧光相关光谱和互相关光谱比较了CaM和钙/CaM依赖的蛋白激酶11在溶液和活细胞中的迁移率和分子相互作用。这些技术表明，细胞内CaM的利用率可以通过改变细胞内的条件来改变。双光子荧光互相关光谱是研究活细胞中蛋白质-蛋白质相互作用的一种普遍适用的方法，它允许访问信号分子在其自然环境中的行为，以探测不同细胞间隔中信号通路的异质性。
The availability and interactions of signaling proteins are tightly regulated in time and space to produce specific and localized effects. For calmodulin (CaM), a key transducer of intracellular Ca2+ signaling, binding to its variety of targets initiates signaling cascades and regulates its subcellular localization, thereby making it unavailable for subsequent binding interactions. Among CaM's numerous targets, Ca2+ /Ca M-dependent protein kinase 11 is one of the most striking due to its unique ability to increase its affinity for CaM by autophosphorylation and to translocate when bound to Ca2+/CaM. Two-photon fluorescence correlation spectroscopy and cross-correlation spectroscopy were utilized to compare mobility and molecular interactions between CaM and Ca2+/CaM-dependent protein kinase 11 in solution and in living cells. These techniques revealed that CaM availability in cells could be altered by a change in intracellular conditions. Two-photon fluorescence cross-correlation spectroscopy exemplifies a generally applicable approach for studying protein-protein interactions in living cells that allows access to the behavior of signaling molecules within their native environment to probe for heterogeneities in signaling pathways in different cellular compartments.