Kinetics of Nisin-Induced Pore Formation in Giant Unilamellar Vesicles.

We have studied the kinetics of pore formation in giant unilamellar vesicles (GUV) with the antimicrobial peptide nisin. The role of charged lipid composition in the rate of pore formation by nisin in the vesicle membrane is investigated using fluorescence microscopy. We propose a model and obtain an analytical expression for the variation in the fluorescence intensity of a GUV as a function of time. We find that the analytical equation fits well to the experimental data, and the membrane surface potential can be estimated from the fit parameters. We further show that the formation of multiple pores on the vesicle membrane is affected by the charged lipid composition of the membrane.

Rapid Detection of Ag(I) via Size-Induced Photoluminescence Quenching of Biocompatible Green-Emitting, l-Tryptophan-Scaffolded Copper Nanoclusters.

Atomically precise metal nanoclusters capped with small molecules like amino acids are highly favored due to their specific interactions and easy incorporation into biological systems. However, they are rarely explored due to the challenge of surface functionalization of nanoclusters with small molecules. Herein, we report the synthesis of a green-emitting (λex = 380 nm, λem = 500 nm), single-amino-acid (l-tryptophan)-scaffolded copper nanocluster (Trp-Cu NC) via a one-pot route under mild reaction conditions. The synthesized nanocluster can be used for the rapid detection of a heavy metal, silver (Ag(I)), in the nanomolar concentration range in real environmental and biological samples. The strong green photoluminescence intensity of the nanocluster quenched significantly upon the addition of Ag(I) due to the formation of bigger nanoparticles, thereby losing its energy quantization. A notable color change from light yellow to reddish-brown can also be observed in the presence of Ag(I), allowing its visual colorimetric detection. Portable paper strips fabricated with the Trp-Cu NC can be reliably used for on-site visual detection of Ag(I) in the micromolar concentration range. The Trp-Cu NC possesses excellent biocompatibility, making it a suitable nanoprobe for cell imaging; thus, it can act as an in vivo biomarker. The nanocluster showed a significant spectral overlap with anticancer drug doxorubicin and thus can be used as an effective fluorescence resonance energy transfer (FRET) pair. FRET results can reveal important information regarding the attachment of the drug to the nanocluster and hence its role as a potential drug carrier for targeted drug delivery within the human body.

Coordination between Intra- and Extracellular Forces Regulates Focal Adhesion Dynamics.

Focal adhesions (FAs) are important mediators of cell-substrate interactions. One of their key functions is the transmission of forces between the intracellular acto-myosin network and the substrate. However, the relationships between cell traction forces, FA architecture, and molecular forces within FAs are poorly understood. Here, by combining Förster resonance energy transfer (FRET)-based molecular force biosensors with micropillar-based traction force sensors and high-resolution fluorescence microscopy, we simultaneously map molecular tension across vinculin, a key protein in FAs, and traction forces at FAs. Our results reveal strong spatiotemporal correlations between vinculin tension and cell traction forces at FAs throughout a wide range of substrate stiffnesses. Furthermore, we find that molecular tension within individual FAs follows a biphasic distribution from the proximal (toward the cell nucleus) to distal end (toward the cell edge). Using super-resolution imaging, we show that such a distribution relates to that of FA proteins. On the basis of our experimental data, we propose a model in which FA dynamics results from tension changes along the FAs.

Adaptive rheology and ordering of cell cytoskeleton govern matrix rigidity sensing.

Matrix rigidity sensing regulates a large variety of cellular processes and has important implications for tissue development and disease. However, how cells probe matrix rigidity, and hence respond to it, remains unclear. Here, we show that rigidity sensing and adaptation emerge naturally from actin cytoskeleton remodelling. Our in vitro experiments and theoretical modelling demonstrate a biphasic rheology of the actin cytoskeleton, which transitions from fluid on soft substrates to solid on stiffer ones. Furthermore, we find that increasing substrate stiffness correlates with the emergence of an orientational order in actin stress fibres, which exhibit an isotropic to nematic transition that we characterize quantitatively in the framework of active matter theory. These findings imply mechanisms mediated by a large-scale reinforcement of actin structures under stress, which could be the mechanical drivers of substrate stiffness-dependent cell shape changes and cell polarity.

Micropillar substrates: a tool for studying cell mechanobiology.

Increasing evidence has shown that mechanical cues from the environment play an important role in cell biology. Mechanotransduction or the study of how cells can sense these mechanical cues, and respond to them, is an active field of research. However, it is still not clear how cells sense and respond to mechanical cues. Thus, new tools are being rapidly developed to quantitatively study cell mechanobiology. Particularly, force measurement tools such as micropillar substrates have provided new insights into the underlying mechanisms of mechanosensing. In this chapter, we provide detailed protocol for fabrication, characterization, functionalization, and use of the micropillar substrates.

Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro.

Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell-cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.