Capturing the dynamics of inter-organelle associations

体内细胞通过将质膜（PM）接收到的信号转导到一系列事件来响应外部信号，这些事件控制着从膜兴奋性到细胞核DNA调节的功能。为了做到这一点，细胞已经发展出一个复杂的内部细胞器系统，从PM到细胞核。在PM的水平上，一个小的天线状纤毛对外部环境中的信号做出反应。PM中的离子通道被激活，允许钙进入细胞并触发生化事件。在PM以下不到20 nm处是一个复杂的内质网合胞体（ER），它作为细胞核周围膜的延伸，通过细胞内部深处延伸到其起源（1,2）。线粒体的第二个管状系统（Mito）缠绕在内质网中，控制能量产生和钙浓度（3,4）。所有这些结构的定位都是由结构蛋白肌动蛋白和微管蛋白的骨架框架指导的(5)。现在很清楚，这些细胞器不是孤立地起作用，而是保持仅10-20纳米的物理距离，以促进“ER-PM”或“Mito-ER”连接等专门区域的相互作用（1,3,4,6,7）。这些元素进一步在几秒钟内动态变化，以响应外部刺激，改变相互作用的性质，直至核水平(2)。为了捕捉这些元素之间的动态相互作用，我们迫切需要升级现有的2D刺激发射损耗（STED）显微镜，以快速获取从PM水平到活细胞细胞核的超分辨率图像。***这些升级将立即使RW Turner博士基于NSERC dg的项目受益，该项目旨在确定ER-PM连接如何影响PM钙通道控制大脑中神经元尖峰输出。它还将使一个由7个实验室组成的多元化小组受益，这些实验室基于nserc的研究项目，研究细胞骨架、内质网、水户和核水平上的细胞器间相互作用。这组有一个共同的需要捕捉动态的相互作用，需要快速超分辨率成像从PM水平到细胞核的活细胞随着时间的推移。我们可以通过升级现有的STED显微镜作为一个单一的操作单元来实现我们所有的目标。该系统将安装在一个资源设施中，以最大限度地提高访问、维护、可持续性和我们培训HQP最新显微镜技术的能力。我们需要一个STEADYFOCUS系统和一个STEDyCon Plus反卷积软件包来实现动态细胞器间相互作用的最佳聚焦。显微镜上需要一个培养系统来维持培养的细胞和交换培养基，以刺激细胞器间的在线重排。因此，所要求的设备升级将通过实现能够捕获内部细胞器之间动态变化的超分辨率成像，显著增强NSERC博士资助的多个研究项目的影响和我们的培训能力

Cells in the body respond to external cues by transducing signals received at a plasma membrane (PM) to a series of events that control functions ranging from membrane excitability to DNA regulation in the nucleus. To manage this cells have developed an intricate system of internal organelles that span from the PM to the nucleus. At the level of the PM, a small antenna-like cilium responds to signals in the external environment. Ion channels in the PM are activated to allow calcium to enter a cell and trigger biochemical events. Less than 20 nm below the PM is a complex syncytium of endoplasmic reticulum (ER) that extends through the internal depths of the cell to its origin as an extension of the membrane around the nucleus (1,2). Winding throughout the ER is a second tubular system of mitochondria (Mito) that controls energy production and calcium concentration (3,4). The localization of all these structures is guided by a skeletal framework of the structural proteins actin and tubulin (5). It is now clear that these organelles do not function in isolation, but rather maintain physical distances of only 10-20 nm to facilitate interactions at specialized regions such as “ER-PM” or “Mito-ER” junctions (1,3,4,6,7). These elements further change dynamically in only secs in response to external stimuli that alter the nature of interactions down to the nuclear level (2). To capture the dynamic interactions between these elements we have a critical need to upgrade an existing 2D Stimulus Emission Depletion (STED) microscope to rapidly acquire super-resolution images from the level of PM to the nucleus in live cells.*** These upgrades will immediately benefit the NSERC DG-based program of Dr. RW Turner in defining how ER-PM junctions influence PM calcium channels control neuronal spike output in the brain. It will also benefit a diverse group of 7 labs with NSERC-based research programs that study inter-organelle interactions at the cytoskeletal, ER, Mito, and nuclear levels. This group has a common need to capture dynamic interactions that require fast super-resolution imaging from the level of PM to nucleus in live cells over time. We can achieve all our goals by upgrading an existing STED microscope as a single operational unit. This system will be installed in a resource facility to maximize access, up-keep, sustainability, and our ability to train HQP in the latest microscopy techniques. We request a STEADYFOCUS system and a STEDyCon Plus deconvolution software package to enable optimal focus of dynamic inter-organelle interactions. An incubator system on the microscope is needed to maintain cultured cells and exchange medium that will stimulate inter-organelle rearrangements on-line. The requested equipment upgrades will thus significantly enhance the impact of multiple NSERC DG-funded research programs and our training capacity by enabling super-resolution imaging capable of capturing dynamic changes between internal organelles.**