RESUMEN

Depletion interactions are thought to significantly contribute to the organization of intracellular structures in the crowded cytosol. The strength of depletion interactions depends on physical parameters such as the depletant number density and the depletant size ratio. Cells are known to dynamically regulate these two parameters by varying the copy number of proteins of a wide distribution of sizes. However, mammalian cells are also known to keep the total protein mass density remarkably constant, to within 0.5% throughout the cell cycle. We thus ask how the strength of depletion interactions varies when the total depletant mass is held fixed, a.k.a. fixed-mass depletion. We answer this question via scaling arguments, as well as by studying depletion effects on networks of reconstituted semiflexible actin in silico and in vitro. We examine the maximum strength of the depletion interaction potential U∗ as a function of q, the size ratio between the depletant and the matter being depleted. We uncover a scaling relation U∗ ∼ qζ for two cases: fixed volume fraction φ and fixed mass density ρ. For fixed volume fraction, we report ζ < 0. For the fixed mass density case, we report ζ > 0, which suggests that the depletion interaction strength increases as the depletant size ratio is increased. To test this prediction, we prepared our filament networks at fixed mass concentrations with varying sizes of the depletant molecule poly(ethylene glycol) (PEG). We characterize the depletion interaction strength in our simulations via the mesh size. In experiments, we observe two distinct actin network morphologies, which we call weakly bundled and strongly bundled. We identify a mass concentration where different PEG depletant sizes lead to weakly bundled or strongly bundled morphologies. For these conditions, we find that the mesh size and intra-bundle spacing between filaments across the different morphologies do not show significant differences, while the dynamic light scattering relaxation time and storage modulus between the two states do show significant differences. Our results demonstrate the ability to tune actin network morphology and mechanics by controlling depletant size and give insights into depletion interaction mechanisms under the fixed-depletant-mass constraint relevant to living cells.

Asunto(s)

Actinas , Actinas/química , Actinas/metabolismo , Polietilenglicoles/química , Animales , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo

RESUMEN

Active biological molecules present a powerful, yet largely untapped, opportunity to impart autonomous regulation to materials. Because these systems can function robustly to regulate when and where chemical reactions occur, they have the ability to bring complex, life-like behavior to synthetic materials. Here, we achieve this design feat by using functionalized circadian clock proteins, KaiB and KaiC, to engineer time-dependent crosslinking of colloids. The resulting material self-assembles with programmable kinetics, producing macroscopic changes in material properties, via molecular assembly of KaiB-KaiC complexes. We show that colloid crosslinking depends strictly on the phosphorylation state of KaiC, with kinetics that are synced with KaiB-KaiC complexing. Our microscopic image analyses and computational models indicate that the stability of colloidal super-structures depends sensitively on the number of Kai complexes per colloid connection. Consistent with our model predictions, a high concentration stabilizes the material against dissolution after a robust self-assembly phase, while a low concentration allows circadian oscillation of material structure. This work introduces the concept of harnessing biological timers to control synthetic materials; and, more generally, opens the door to using protein-based reaction networks to endow synthetic systems with life-like functional properties.

RESUMEN

Biological systems have the unique ability to self-organize and generate autonomous motion and work. Motivated by this, we investigate a 2D model colloidal network that can repeatedly transition between disordered states of low connectivity and ordered states of high connectivity via rhythmic binding and unbinding of biomimetic crosslinkers. We use Langevin dynamics to investigate the time-dependent changes in structure and collective properties of this system as a function of colloidal packing fractions and crosslinker oscillation periods and characterize the degree of order in the system by using network connectivity, bond length distributions, and collective motion. Our simulations suggest that we can achieve distinct states of this colloidal system with pronounced differences in microstructural order and large residence times in the ordered state when crosslinker kinetics and lifetimes depend directly on the oscillation period and this oscillation period is much larger than the colloidal diffusion time. Our results will provide insights into the rational design of smart active materials that can independently cycle between ordered and disordered states with desired material properties on a programmed schedule.

Asunto(s)

Modelos Biológicos , Movimiento (Física)

RESUMEN

Controlling mechanical properties of polymeric biomaterials, including the elastic modulus, is critical to direct cell behavior, such as proliferation and differentiation. Dityrosine photocrosslinking is an attractive and simple method to prepare materials that exhibit a wide range of elastic moduli by rapidly crosslinking tyrosyl-containing polymers. However, high concentrations of commonly used oxidative crosslinking reagents, such as ruthenium-based photoinitiators and persulfates, present cytotoxicity concerns. We found the elastic moduli of materials prepared by crosslinking an artificial protein with tightly controlled tyrosine molarity can be modulated up to 40 kPa by adjusting photoinitiator and persulfate concentrations. Formulations with various concentrations of the crosslinking reagents were able to target a similar material elastic modulus, but excess unreacted persulfate resulted in cytotoxic materials. Therefore, we identified a systematic method to prepare non-cytotoxic photocrosslinked polymeric materials with targeted elastic moduli for potential biomaterials applications in diverse fields, including tissue engineering and 3D bioprinting.