Bottom-up biology is an expanding research field that aims to understand the mechanisms underlying biological processes via in vitro assembly of their essential components in synthetic cells. As encapsulation and controlled manipulation of these elements is a crucial step in the recreation of such cell-like objects, microfluidics is increasingly used for the production of minimal artificial containers such as single-emulsion droplets, double-emulsion droplets, and liposomes. Despite the importance of cell morphology on cellular dynamics, current synthetic-cell studies mainly use spherical containers, and methods to actively shape manipulate these have been lacking. In this paper, we describe a microfluidic platform to deform the shape of artificial cells into a variety of shapes (rods and discs) with adjustable cell-like dimensions below 5 μm, thereby mimicking realistic cell morphologies. To illustrate the potential of our method, we reconstitute three biologically relevant protein systems (FtsZ, microtubules, collagen) inside rod-shaped containers and study the arrangement of the protein networks inside these synthetic containers with physiologically relevant morphologies resembling those found in living cells.

Although cytoskeletal networks are interpenetrating and interacting in living cells, very little is understood as to the effect their interaction has on their properties. Here, as a step towards elucidating the synergistic cellular role of these structural proteins, we investigate isolated keratin and actin composites and show how the in vitro network formation of keratin influences the properties of actin networks and vice versa. By encapsulating purified composite networks into vesicles and separating the time scales of network formation we are able to demonstrate that the actin network stabilizes keratin networks by providing an elastic resistance to their collapse in vitro.

Changes in cell-substrate adhesion are believed to signal the onset of cancer metastasis, but such changes must be quantified against background levels of intrinsic heterogeneity between cells. Variations in cell-substrate adhesion strengths can be probed through biophysical measurements of cell detachment from substrates upon the application of an external force. Here, we investigate, theoretically and experimentally, the detachment of cells adhered to substrates when these cells are subjected to fluid shear. We present a theoretical framework within which we calculate the fraction of detached cells as a function of shear stress for fast ramps as well as the decay in this fraction at fixed shear stress as a function of time. Using HEK and 3T3 fibroblast cells as experimental model systems, we extract characteristic force scales for cell adhesion as well as characteristic detachment times. We estimate force-scales of  ∼500 pN associated to a single focal contact, and characteristic time-scales of [Formula: see text] s representing cell-spread-area dependent mean first passage times to the detached state at intermediate values of the shear stress. Variations in adhesion across cell types are especially prominent when cell detachment is probed by applying a time-varying shear stress. These methods can be applied to characterizing changes in cell adhesion in a variety of contexts, including metastasis.

Regulation of adhesion is a ubiquitous feature of living cells, observed during processes such as motility, antigen recognition, or rigidity sensing. At the molecular scale, a myriad of mechanisms are necessary to recruit and activate the essential proteins, whereas at the cellular scale, efficient regulation of adhesion relies on the cell’s ability to adapt its global shape. To understand the role of shape remodeling during adhesion, we use a synthetic biology approach to design a minimal experimental model, starting with a limited number of building blocks. We assemble cytoskeletal vesicles whose size, reduced volume, and cytoskeletal contractility can be independently tuned. We show that these cytoskeletal vesicles can sustain strong adhesion to solid substrates only if the actin cortex is actively remodeled significantly. When the cytoskeletal vesicles are deformed under hypertonic osmotic pressure, they develop a crumpled geometry with deformations. In the presence of molecular motors, these deformations are dynamic in nature, and the excess membrane area generated thereby can be used to gain adhesion energy. The cytoskeletal vesicles are able to attach to the rigid glass surfaces even under strong adhesive forces just like the cortex-free vesicles. The balance of deformability and adhesion strength is identified to be key to enable cytoskeletal vesicles to adhere to solid substrates.