Systematic Characterization of Cytochalasan Activities on Actin Dynamics in Mammalian Cells

众所周知，细胞松弛体可以抑制细胞分裂和各种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，如葡萄糖运输，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chalasans，但迄今为止，只有很少的细胞chalasans被分离到足以探测其生物活性的量，而且不到15种是可商用的。它们中的大多数被认为直接阻断了肌动蛋白丝快速生长末端的聚合，但迄今为止，对它们的生物化学和细胞生物学共性和差异的系统表征尚不清楚。在这个项目中，我们将通过在一个新的研究单位（FOR 5170）内与几个小组合作来填补这一知识空白，这些小组提供了新的，主要是真菌的细胞chalasan分离物或从半合成甚至全合成中获得的单个细胞chalasan衍生物的文库。所有这些细胞chalasans将在各种实验中系统地探索，通过各种手段和方法探索它们对肌动蛋白细胞骨架和运动过程的影响。这些将包括高通量筛选化合物文库，直接对处理过的样品进行phalloidin染色和全细胞群体的低磁视频显微镜，以及更详细的单细胞表征所选细胞chalasan或细胞chalasan衍生物对亚细胞肌动蛋白结构和其中的细丝的转换的影响。在初步工作中，我们已经建立了复杂的实验方法，用于各种尺度的细胞chalasan分析。利用这些，我们已经发现了不同的细胞查拉桑变体之间令人兴奋的和特定的差异，接下来将进行更深入的研究。除了这些努力之外，我们将结合上述筛选和更详细的表征方法，使用我们不断增长的基因组编辑细胞系面板，这些细胞系携带特定肌动蛋白结合蛋白的破坏。这有可能明确地证明细胞肌动蛋白丝亚群的特定细胞查喇桑或细胞查喇桑家族的不同选择性。这些知识将有助于开发具有特定生物活性的细胞链。后者将在工作计划的过程中与诸如各种宿主-病原体相互作用的研究一起加以利用。最后，在FOR5071内的细胞chalasan衍生化或-合成基团的帮助下，将立即进行有趣的细胞chalasan活性调整，并辅以外部合作的生化和结构分析。

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.