Taberner Núria, Lof Andries, Roth Sophie, Lamers Dimitry, Zeijlemaker Hans, Dogterom Marileen
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.
FOM Institute AMOLF, Amsterdam, The Netherlands.
Methods Cell Biol. 2015;128:1-22. doi: 10.1016/bs.mcb.2015.02.008. Epub 2015 Apr 8.
Establishment of cell polarity is essential for processes such as growth and division. In fission yeast, as well as other species, polarity factors travel at the ends of microtubules to cortical sites where they associate with the membrane and subsequently maintain a polarized activity pattern despite their ability to diffuse in the membrane. In this chapter we present methods to establish an in vitro system that captures the essential features of this process. This bottom-up approach allows us to identify the minimal molecular requirements for microtubule-based cell polarity. We employ microfabrication techniques combined with surface functionalization to create rigid chambers with affinity for proteins, as well as microfluidic techniques to create and shape emulsion droplets with functionalized lipid boundaries. Preliminary results are shown demonstrating that a properly organized microtubule cytoskeleton can be confined to these confined spaces, and proteins traveling at the ends of growing microtubules can be delivered to their boundaries.
细胞极性的建立对于生长和分裂等过程至关重要。在裂殖酵母以及其他物种中，极性因子沿着微管末端移动到皮质位点，在那里它们与膜结合，尽管它们能够在膜中扩散，但随后仍能维持极化的活动模式。在本章中，我们介绍了建立体外系统的方法，该系统能够捕捉这一过程的基本特征。这种自下而上的方法使我们能够确定基于微管的细胞极性的最小分子要求。我们采用微制造技术结合表面功能化来创建对蛋白质具有亲和力的刚性腔室，以及微流控技术来创建和塑造具有功能化脂质边界的乳液滴。初步结果表明，适当组织的微管细胞骨架可以被限制在这些受限空间内，并且沿着生长中的微管末端移动的蛋白质可以被递送到它们的边界。
