The microtubule (MT) cytoskeleton plays critically important roles in numerous cellular functions in eukaryotes, and it does so across a functionally diverse and morphologically disparate range of cell types [1]. In these roles, MT assemblies must adopt distinct morphologies and physical dimensions to perform specific functions [2-5]. As such, these macromolecular assemblies-as well as the dynamics of the individual MT polymers from which they are made-must scale and change in accordance with cell size and geometry. As first shown by Inoue using polarization microscopy, microtubules assemble to a steady state in mass, leaving enough of their subunits soluble to allow rapid growth and turnover. This suggests some negative feedback that limits the extent of assembly, for example decrease in growth rate, or increase in catastrophe rate, as the soluble subunit pool decreases. Such feedbacks might be global or local. Although these ideas have informed the field for decades, they have not been observed experimentally. Here we describe an experimental system designed to examine these long-standing ideas and determine a role for MT plus-end density in regulating MT growth rates. MT based assemblies, specifically interphase MT asters and mitotic spindles, have been confined to and characterized in discrete droplets of cell-free Xenopus egg extract [6-9] to study MT selforganization and scaling phenomena. In these droplets, however, it is difficult to collect images with the sufficiently high spatial and temporal resolution needed to characterize dynamic molecular-scale phenomena such as MT growth. This is partly due to the propensity of droplets to fuse, and the unpredictable movements of the droplets within the imaging plane. In addition, the spherical geometry of the droplets presents a unique imaging challenge, as light emitted from the specimen must travel through multiple refractive indices before being collected by the objective. Additionally, the spherical shape of the droplets means that cellular events of interest are not always confined to a region near the coverslip. To circumvent these limitations in a way that still allows for precise control of extract volume, we used well-characterized hydrogel photolithography methods [10] and developed an approach that enabled us to confine cell-free extracts and MT asters within biologically inert hydrogel enclosures of precise geometrical shape and size (Figure 1a). To photo-pattern structures on the coverslip surface, we placed a digital micro-mirror array in the light path of a microscope and projected light patterns onto a pre-polymer solution of poly(ethylene glycol) diacrylate (PEGDA) contained within a microfluidic flow chamber (Figure 1a, b; leftmost and center-left panels). The positions of these enclosures within the device were dictated by the random spatial arrangement of artificial microtubule organizing centers (aMTOCs) present within .

The microtubule (MT) cytoskeleton plays critically important roles in numerous cellular functions in eukaryotes, and it does so across a functionally diverse and morphologically disparate range of cell types [1]. In these roles, MT assemblies must adopt distinct morphologies and physical dimensions to perform specific functions [2][3][4][5]. As such, these macromolecular assemblies-as well as the dynamics of the individual MT polymers from which they are made-must scale and change in accordance with cell size and geometry. As first shown by Inoue using polarization microscopy, microtubules assemble to a steady state in mass, leaving enough of their subunits soluble to allow rapid growth and turnover. This suggests some negative feedback that limits the extent of assembly, for example decrease in growth rate, or increase in catastrophe rate, as the soluble subunit pool decreases. Such feedbacks might be global or local. Although these ideas have informed the field for decades, they have not been observed experimentally. Here we describe an experimental system designed to examine these long-standing ideas and determine a role for MT plus-end density in regulating MT growth rates.

Summary Cell-free extract derived from the eggs of the African clawed frog Xenopus laevis is a well-established model system that has been used historically in bulk aliquots. Here, we describe a microfluidic approach for isolating discrete, biologically relevant volumes of cell-free extract, with more expansive and precise control of extract shape compared with extract-oil emulsions. This approach is useful for investigating the mechanics of intracellular processes affected by cell geometry or cytoplasmic volume, including organelle scaling and positioning mechanisms. For complete details on the use and execution of this protocol, please refer to Geisterfer et al. (2020) .

Self-organization of and by the cytoskeleton is central to the biology of the cell. Since their introduction in the early 1980s, cytoplasmic extracts derived from the eggs of the African clawed-frog, Xenopus laevis, have flourished as a major experimental system to study the various facets of cytoskeleton-dependent self-organization. Over the years, the many investigations that have used these extracts uniquely benefited from their simplified cell cycle, large experimental volumes, biochemical tractability and cell-free nature. Here, we review the contributions of egg extracts to our understanding of the cytoplasmic aspects of self-organization by the microtubule and the actomyosin cytoskeletons as well as the importance of cytoskeletal filaments in organizing nuclear structure and function.

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