Modular Design of Micropattern Geometry Achieves Combinatorial Enhancements in Cell Motility

Basic micropattern shapes, such as stripes and teardrops, affect individual facets of cell motility, such as migration speed and directional bias, respectively. Here, we test the idea that these individual effects on cell motility can be brought together to achieve multidimensional improvements in cell behavior through the modular reconstruction of the simpler “building block” micropatterns. While a modular design strategy is conceptually appealing, current evidence suggests that combining environmental cues, especially molecular cues, such as growth factors and matrix proteins, elicits a highly nonlinear, synergistic cell response. Here, we show that, unlike molecular cues, combining stripe and teardrop geometric cues into a hybrid, spear-shaped micropattern yields combinatorial benefits in cell speed, persistence, and directional bias. Furthermore, cell migration speed and persistence are enhanced in a predictable, additive manner on the modular spear-shaped design. Meanwhile, the spear micropattern also improved the directional bias of cell movement compared to the standard teardrop geometry, revealing that combining geometric features can also lead to unexpected synergistic effects in certain aspects of cell motility. Our findings demonstrate that the modular design of hybrid micropatterns from simpler building block shapes achieves combinatorial improvements in cell motility. These findings have implications for engineering biomaterials that effectively mix and match micropatterns to modulate and direct cell motility in applications, such as tissue engineering and lab-on-a-chip devices

Slope-Dependent Cell Motility Enhancements at the Walls of PEG-Hydrogel Microgroove Structures

In recent years, research utilizing micro- and nanoscale geometries and structures on biomaterials to manipulate cellular behaviors, such as differentiation, proliferation, survival, and motility, have gained much popularity; however, how the surface microtopography of 3D objects, such as implantable devices, can affect these various cell behaviors still remains largely unknown. In this study, we discuss how the walls of microgroove topography can influence the morphology and the motility of unrestrained cells, in a different fashion from 2D line micropatterns. Here adhesive substrates made of tetra­(polyethylene glycol) (tetra-PEG) hydrogels with microgroove structures or 2D line micropatterns were fabricated, and cell motility on these substrates was evaluated. Interestingly, despite being unconstrained, the cells exhibited drastically different migration behaviors at the edges of the 2D micropatterns and the walls of microgroove structures. In addition to acquiring a unilamellar morphology, the cells increased their motility by roughly 3-fold on the microgroove structures, compared with the 2D counterpart or the nonpatterned surface. Immunostaining revealed that this behavior was dependent on the alignment and the aggregation of the actin filaments, and by varying the slope of the microgroove walls, it was found that relatively upright walls are necessary for this cell morphology alterations. Further progress in this research will not only deepen our understanding of topography-assisted biological phenomena like cancer metastasis but also enable precise, topography-guided manipulation of cell motility for applications such as cancer diagnosis and cell sorting

Lectin-Tagged Fluorescent Polymeric Nanoparticles for Targeting of Sialic Acid on Living Cells

In this study, we fabricated lectin-tagged fluorescent polymeric nanoparticles approximately 35 nm in diameter using biocompatible polymers conjugated with lectins for the purpose of detecting sialic acid on a living cell surface, which is one of the most important biomarkers for cancer diagnosis. Through cellular experiments, we successfully detected sialic acid overexpression on cancerous cells with high specificity. These fluorescent polymeric nanoparticles can be useful as a potential bioimaging probe for detecting diseased cells

Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy

The conformation and dynamics of the unfolded state of ubiquitin doubly labeled regiospecifically with Alexa488 and Alexa647 were investigated using single-molecule fluorescence spectroscopy. The line confocal fluorescence detection system combined with the rapid sample flow enabled the characterization of unfolded proteins at the improved structural and temporal resolutions compared to the conventional single-molecule methods. In the initial stage of the current investigation, however, the single-molecule Förster resonance energy transfer (sm-FRET) data of the labeled ubiquitin were flawed by artifacts caused by the adsorption of samples to the surfaces of the fused-silica flow chip and the sample delivery system. The covalent coating of 2-methacryloyloxyethyl phosphorylcholine polymer to the flow chip surface was found to suppress the artifacts. The sm-FRET measurements based on the coated flow chip demonstrated that the histogram of the sm-FRET efficiencies of ubiquitin at the native condition were narrowly distributed, which is comparable to the probability density function (PDF) expected from the shot noise, demonstrating the structural homogeneity of the native state. In contrast, the histogram of the sm-FRET efficiencies of the unfolded ubiquitin obtained at a time resolution of 100 μs was distributed significantly more broadly than the PDF expected from the shot noise, demonstrating the heterogeneity of the unfolded state conformation. The variety of the sm-FRET efficiencies of the unfolded state remained even after evaluating the moving average of traces with a window size of 1 ms, suggesting that conformational averaging of the heterogeneous conformations mostly occurs in the time domain slower than 1 ms. Local structural heterogeneity around the labeled fluorophores was inferred as the cause of the structural heterogeneity. The heterogeneity and slow dynamics revealed by the line confocal tracking of sm-FRET might be common properties of the unfolded proteins