Exploring cell surface-nanopillar interactions with 3D super-resolution microscopy

Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used 3D nanostructures such as nanopillars to imprint well-defined membrane curvatures (the "nano-bio interface"). In these studies, proteins and their interactions were probed by 2D fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone oil immersion objective and 3D double-helix point-spread function microscopy. We carefully optimize the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.

Curvature sensing steers actin-driven cell migration

Cell migration is fundamental for the immune response, development, and morphogenesis. For navigation through complex and ever-changing environments, migrating cells require a balance between a stable leading-edge, which is necessary for directional migration, and some unstable features to enable the required dynamic behaviors. The leading edge is often composed of actin-driven protrusions including lamellipodia and ruffles with continuously changing membrane curvature. Whether their membrane topography affects the cell's leading edge and motion persistence in complex environments remains unknown. To study this, we combined a theoretical analysis with machine learning-based segmentation for time-resolved TIRF microscopy, membrane topography analysis from electron microscopy images and microfluidics. We discovered that cell motion persistence and directionality, in both freely moving and environmentally-constrained cells, strongly depend on the curvature-sensing protein Snx33. Specifically, Snx33 promotes leading edge instabilities by locally inhibiting WAVE2- driven actin polymerization in a curvature-dependent manner. Snx33 knockout cells migrate faster and are more persistent during unobstructed migration, but fail when a change in direction is required. Thus, Snx33 is key for steering cell motility in complex environments by facilitating contact inhibition of locomotion and promoting efficient turning. These results identify cell surface topography as an organizing principle at the cell periphery that directs cell migration.

A 3D-Printed, Modular, and Parallelized Microfluidic System with Customizable ECM Integration to Investigate the Roles of Basement Membrane Topography on Endothelial Cells

<p>Because dysfunctions of endothelial cells are involved in many pathologies, <i>in vitro </i>endothelial cell models for pathophysiological and pharmaceutical studies have been a valuable research tool. Although numerous microfluidic-based endothelial models have been reported, they had the cells cultured on a flat surface without considering the possible 3D structure of the native ECM. Endothelial cells rest on the basement membrane <i>in vivo</i>, which contains an aligned microfibrous topography. To better understand and model the cells, it is necessary to know if and how the fibrous topography can affect endothelial functions. With conventional fully integrated microfluidic apparatus, it is difficult to include additional topographies in a microchannel. Therefore, we developed a modular microfluidic system by 3D-printing and electrospinning, which enabled easy integration and switching of desired ECM topographies. Also, with standardized designs, the system allowed for high flow rates up to 4000 µL/min, which covered the full shear stress range for endothelial studies. We found that the aligned fibrous topography on the ECM altered arginine metabolism in endothelial cells, and thus increased nitric oxide production. To the best of our knowledge, this is the most versatile endothelial model that has been reported, and the new knowledge generated thereby lays a groundwork for future endothelial research and modeling. </p>

A 3D-Printed, Modular, and Parallelized Microfluidic System with Customizable ECM Integration to Investigate the Roles of Basement Membrane Topography on Endothelial Cells

<p>Because dysfunctions of endothelial cells are involved in many pathologies, <i>in vitro </i>endothelial cell models for pathophysiological and pharmaceutical studies have been a valuable research tool. Although numerous microfluidic-based endothelial models have been reported, they had the cells cultured on a flat surface without considering the possible 3D structure of the native ECM. Endothelial cells rest on the basement membrane <i>in vivo</i>, which contains an aligned microfibrous topography. To better understand and model the cells, it is necessary to know if and how the fibrous topography can affect endothelial functions. With conventional fully integrated microfluidic apparatus, it is difficult to include additional topographies in a microchannel. Therefore, we developed a modular microfluidic system by 3D-printing and electrospinning, which enabled easy integration and switching of desired ECM topographies. Also, with standardized designs, the system allowed for high flow rates up to 4000 µL/min, which covered the full shear stress range for endothelial studies. We found that the aligned fibrous topography on the ECM altered arginine metabolism in endothelial cells, and thus increased nitric oxide production. To the best of our knowledge, this is the most versatile endothelial model that has been reported, and the new knowledge generated thereby lays a groundwork for future endothelial research and modeling. </p>

Microvillar cartography: a super-resolution single-molecule imaging method to map the positions of membrane proteins with respect to cellular surface topography

We introduce Microvillar Cartography (MC), a method to map proteins on cellular surfaces with respect to the membrane topography. The surfaces of many cells are not smooth, but are rather covered with various protrusions such as microvilli. These protrusions may play key roles in multiple cellular functions, due to their ability to control the distribution of specific protein assemblies on the cell surface. Thus, for example, we have shown that the T-cell receptor and several of its proximal signaling proteins reside on microvilli, while others are excluded from these projections. These results have indicated that microvilli can function as key signaling hubs for the initiation of the immune response. MC has facilitated our observations of particular surface proteins and their specialized distribution on microvillar and non-microvillar compartments. MC combines membrane topography imaging, using variable-angle total internal microscopy, with stochastic localization nanoscopy, which generates deep sub-diffraction maps of protein distribution. Since the method is based on light microscopy, it avoids some of the pitfalls inherent to electron-microscopy-based techniques, such as dehydration, carbon coating and immunogold clustering, and is amenable to future developments involving e.g. live-cell imaging. This Protocol details the procedures we developed for MC, which can be readily adopted to study a broad range of cell surface molecules and dissect their distribution within distinct surface assemblies under multiple cell activation states.

Engineering Biomimetic Gelatin Based Nanostructures as Synthetic Substrates for Cell Culture

There is a need for synthetic substrates that replicate the natural environment for in vitro intestinal models. Electrospinning is one of the most versatile and cost-effective techniques to produce nanofibrous scaffolds mimicking the basement membrane topography. In this study, three different novel electrospun nanofibrous scaffolds made of a polycaprolactone (PCL), gelatin, and poloxamer 188 (P188) blend were produced and compared with PCL and PCL/gelatin fibers produced using the same solvent system and electrospinning parameters. Each polymer solution used in this experiment was electrospun at four different voltages to study its influence on fiber diameter. The morphology and physical characteristics of the fibers were studied using scanning electron microscopy and atomic force microscopy. The average fiber diameter of all scaffolds was within 200–600 nm and no significant decrease in diameter with an increase in voltage was observed. Attenuated total reflection Fourier transform infrared spectroscopy was used to determine the chemical characteristics of the nanofibrous scaffold. The conductivity of the polymer solutions was also analyzed. Biocompatibility of the scaffolds was determined by a cell proliferation study performed using colorectal carcinoma (Caco-2) cells. PCL/gelatin/P188 scaffolds exhibited higher cell proliferation compared to PCL, PCL/gelatin scaffolds, and the control (tissue culture multi-well plate) with PCL/gelatin/P188 80:10:10 sample showing the highest cell proliferation.