Fabrication of microcompartments with controlled size and shape for encapsulating active matter

Abstract

In all living systems, the cytoplasm is separated from the external environment by membranes. This confinement imposes spatial constraints on the self-organization of internal components, filaments and organelles. While reconstituted systems are instrumental for understanding fundamental biological principles, traditional experiments often utilize volumes vastly larger than actual cells. In recent studies, water-in-oil droplets or giant unilamellar vesicles have been widely used to impose confinement. However, these compartments present imaging challenges and make precise protein content control difficult. To address these limitations, we have developed versatile microwells that are straightforward to implement, compatible with different types of imaging and suitable for long-term experiments. These microwells are compatible with several surface treatments and a wide range of experimental techniques making them a powerful tool for answering key questions in cell biology. We present here a detailed protocol of the fabrication of the microwells as well as characterization of the method to ensure quality throughout the manufacturing process. These microwells support various cytoskeleton-based processes including actin polymerization, dynamic steady-state actin networks, and composite actin-microtubule networks. More broadly, they can be used to encapsulate and study over time any kind of active matter.

Keywords: active matter; compartments; cytoskeleton; lipids; microchambers; microfabrication; microwells.

FIGURE 1

Fabrication of NOA microwells and characterization of their height. (A) Schemes for the fabrication of SU8 mold. (B) Schemes for the fabrication of PDMS pillars. (C) Schemes for the microwells fabrication. (D) Left: snapshots of NOA microwells with different heights. Right: fluorescence profiles (taken on the yellow lines in the images) of the microwells. Scale bar is 200 µm. (E) Resliced z-stacks of microwells to measure microwell height. Scale bars are 50 µm.

FIGURE 2

Filling and closing of the microwells. (A) Schemes for the passivation, filling and closing of microwells. (B) Images showing microwells first filled with a green dye, then replaced with a red dye and closed with mineral oil. Scale bars are 50 µm. (C) Estimation of the optimal volume of reaction mix to introduce in microwells.

FIGURE 3

Control of microwell closure. (A) Top: images of open and closed microwells where the fluorescence was bleached and monitored over time (FRAP experiment). Bottom: Curves showing fluorescence recovery as a function of time for open and closed microwells. (B) Examples of resliced z-stacks for microwells with diameters of 50 or 100 µm and heights of 25 µm. Curvature radius (r = 110 µm) was measured on those images and was similar for both diameters. Scale bars are 25 µm. (C) Scheme of the microwell with the meniscus. (D) Left: Estimation of meniscus height as a function of microwell diameter. Colored circles represent maximum possible microwell diameter for various microwell heights. Middle: Theoretical and corrected microwell volume as a function of microwell diameter. Volume was corrected with meniscus height. Right: Corrected volume as a function of microwell diameter for various microwell heights.

FIGURE 4

Examples of imaging and functionalization of microwells in order to study actin-related processes. (A) Bright-field images of microwells with different sizes (from 50 to 200 µm in diameter) and with different shapes (circle, square or ellipse). (B) Imaging. Left: images of microwells with branched actin network observed with TIRF microscopy. Right: array of microwells imaged with epifluorescence microscopy (×20 objective). (C) Introduction of polystyrene beads (2 µm diameter) in microwells for the reconstitution of actin structures without or with turnover. (D) Reconstruction of confocal z-stacks showing the growth of actin network from microwells walls from biotin-lipids functionalized with a streptavidin nucleation promoting factor for branched actin networks. (E) Micropatterning at bottom of microwells. 3D reconstructions of various micropatterns designs leading to various actin network shapes.