In vitro assays to study force generation at dynamic microtubule ends

Biopolymers are essential for cellular organization. They bridge the cell interior, forming a framework that is used as a reference for different cellular organelles. Interestingly, this framework called the cytoskeleton is not static but constantly reorganizes. The dynamics of the cytoskeleton allows the cell to rearrange its interior for various processes such as cell division. This dynamic reorganization relies at least partly on forces that arise from assembly and disassembly of the cytoskeletal polymers. In many cases, these forces are generated when cytoskeletal polymers interact with the cell boundary. This chapter focuses on force generation by and regulation of microtubules (MTs) that interact with opposing barriers. In this chapter we describe four in vitro assays to study how MT interactions with the cell boundary play a role in cellular organization. In our minimal systems, (functionalized) microfabricated barriers mimic cell boundaries. We carefully design experiments where we grow MTs against these microfabricated structures to study a specific cellular process. Furthermore in this chapter different methods and assays necessary to realize these in vitro experiments are described. Section II describes the materials used, and Section III elaborates on the microfabrication. In Section III.C we explain how we specifically label our microfabricated structures, and in Section III.D we present how these functionalized microfabricated structures are incorporated into assays, with a discussion of the details of the assays themselves. Finally in Section IV we give examples of data obtained with these assays, and in Section V we discuss the assays in a general context.

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