Malinovska Liliana, Alberti Simon
Max-Planck Institute for Molecular Cell Biology and Genetics.
Max-Planck Institute for Molecular Cell Biology and Genetics;
J Vis Exp. 2016 Dec 2(118):54730. doi: 10.3791/54730.
The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.
社会变形虫盘基网柄菌复杂的生活方式使其成为研究各种生物过程的宝贵模型。最近，我们发现盘基网柄菌对蛋白质聚集具有显著的耐受性，可用于深入了解细胞蛋白质质量控制系统。然而，将盘基网柄菌用作模型系统对基于显微镜的实验方法提出了若干挑战，例如细胞的高运动性及其对光毒性的敏感性。在研究蛋白质稳态时，后者被证明尤其具有挑战性，因为光毒性效应可诱导细胞应激反应，从而改变蛋白质质量控制系统的行为。温度升高是诱导细胞应激的常用方法。在此，我们描述了一种温度可控的成像方案，该方案可用于观察盘基网柄菌中温度诱导的扰动。此外，当在正常生长温度下应用时，该成像方案还可显著降低光毒性，从而允许以更高强度进行成像。这在对表达水平极低的蛋白质进行成像时可能特别有用。此外，细胞的高运动性通常需要获取多个视野以跟踪单个细胞，并且视野数量需要与所需的时间间隔和曝光时间相平衡。
