Systematic Characterization of Cytochalasan Activities on Actin Dynamics in Mammalian Cells

已知细胞松弛素抑制细胞分裂和各种类型的基于肌动蛋白的运动，如迁移和血小板聚集。此外，据报道，它们影响与肌动蛋白动力学不太直接或明显相关的过程，如葡萄糖转运，并具有抗生素，抗生物膜和抗肿瘤活性。尽管在真菌提取物中已经检测到大量的细胞松弛素，但迄今为止只有少数以足够的量分离出以探测其生物活性，并且少于15种可商购获得。其中大多数被认为是直接阻止肌动蛋白丝的快速生长末端的聚合，但到目前为止，它们的活动之间的生物化学和细胞生物学共性和差异的系统表征缺失。在这个项目中，我们将填补这一知识的空白，在一个新的研究单位（FOR 5170）与几个小组合作，提供新的，主要是真菌细胞松弛素分离物或衍生物的个别细胞松弛素来自半合成，甚至全合成库。所有这些cytochalasans将系统地探讨在各种检测，探测其对肌动蛋白细胞骨架和运动过程的影响，通过各种手段和方法。这些将包括从化合物库的高通量筛选与直接的鬼笔环肽染色处理的样品和低mag视频显微镜的全细胞群体的更详细的，单细胞表征的影响，选定的细胞松弛素或细胞松弛素衍生物对亚细胞肌动蛋白结构和营业额的细丝内。在初步工作中，我们已经建立了复杂的实验方法，在各种规模的细胞松弛素分析。使用这些，我们已经发现了不同的细胞松弛素变体之间令人兴奋的和特定的差异，随后将进行更深入的研究。除了这些努力，我们将结合联合收割机上述筛选和更详细的表征方法，采用我们不断增长的基因组编辑的细胞系携带特定肌动蛋白结合蛋白的破坏面板。这有可能明确地证明特定的细胞松弛素或细胞松弛素家族的细胞肌动蛋白丝的子集的不同的选择性。这些知识将有助于开发具有特定生物活性的细胞松弛素。在工作方案期间，将沿着对各种宿主-病原体相互作用的研究，利用后者。最后，有趣的cytochalasan活动将立即遵循他们的调谐与帮助的cytochalasan衍生或合成组内FOR 5071，并辅以生化和结构分析在校外合作。

Cytochalasans are known to inhibit cell division and various types of actin-based motility such as migration and platelet aggregation. Moreover, they were reported to affect processes less directly or evidently connected to actin dynamics, such as glucose transport, and to harbor antibiotic, anti-biofilm and antitumor activities. Whereas a large number of cytochalasans has been detected in fungal extracts, only few were hitherto isolated in sufficient amounts to probe their biological activities, and less than 15 are commercially available. Most of these are thought to directly block the polymerization of the fast growing ends of actin filaments, but a systematic characterization of the biochemical and cell biological commonalities and differences between their activities are so far missing. In this project, we will fill this gap of knowledge by teaming up within a novel research unit (FOR 5170) with several groups providing libraries of novel, mostly fungal cytochalasan isolates or derivatives of individual cytochalasans derived from semisynthesis or even total synthesis. All these cytochalasans will be systematically explored in various assays, probing their effects on the actin cytoskeleton and motility processes by various means and approaches. These will range from high-throughput screening of compound libraries with straight-forward phalloidin stainings of treated samples and low mag video microscopy of whole cell populations to more detailed, single cell characterization of the impact of selected cytochalasans or cytochalasan derivatives on subcellular actin structures and the turnover of filaments within them. In preliminary work, we have established sophisticated experimental approaches for cytochalasan analyses at various scales. Using these, we have already found exciting and specific differences between distinct cytochalasan variants, which will be followed by more in depth studies. Aside from these efforts, we will combine aforementioned screening and more detailed characterization methods with employing our continuously growing panels of genome-edited cell lines carrying disruptions of specific actin-binding proteins. This harbors the potential to unambiguously demonstrate distinct selectivities of specific cytochalasans or cytochalasan families for subsets of cellular actin filaments. Such knowledge would be instrumental for developing cytochalasans with specified biological activities. The latter will be exploited during the course of the work program along with studies on, for instance, various host-pathogen interactions. Finally, interesting cytochalasan activities will be immediately followed by their tuning with the help of the cytochalasan-derivatizing or -synthesizing groups within FOR5071, and complemented by biochemical and structural analyses in extramural collaborations.