Background Actin stress fibers are abundant in cultured cells, but little is known about them in vivo. In podocytes, much evidence suggests that mechanobiologic mechanisms underlie podocyte shape and adhesion in health and in injury, with structural changes to actin stress fibers potentially responsible for pathologic changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. A technology to image the actin cytoskeleton at high resolution is needed to better understand the role of structures such as actin stress fibers in podocytes. Methods We developed the first visualization technique capable of resolving the three-dimensional cytoskeletal network in mouse podocytes in detail while definitively identifying the proteins that comprise this network. This technique integrates membrane extraction, focused ion beam scanning electron microscopy, and machine learning image segmentation. Results Using isolated mouse glomeruli from healthy animals, we observed actin cables and intermediate filaments linking the interdigitated podocyte foot processes to newly described contractile actin structures located at the periphery of the podocyte cell body. Actin cables within foot processes formed a continuous, mesh-like, electron-dense sheet that incorporated the slit diaphragms. Conclusions Our new technique revealed, for the first time, the detailed three-dimensional organization of actin networks in healthy podocytes. In addition to being consistent with the gel compression hypothesis, which posits that foot processes connected by slit diaphragms act together to counterbalance the hydrodynamic forces across the glomerular filtration barrier, our data provide insight into how podocytes respond to mechanical cues from their surrounding environment.

Chronic kidney diseases are widespread and incurable. The biophysical mechanisms underlying them are unclear, in part because material systems for reconstituting the microenvironment of relevant kidney cells are limited. A critical question is how kidney podocytes (glomerular epithelial cells) regenerate foot processes of the filtration apparatus following injury. Recently identified sarcomere-like structures (SLSs) with periodically spaced myosin IIA and synaptopodin appear in injured podocytes in vivo. We hypothesized that SLSs template synaptopodin in the initial stages of recovery in response to microenvironmental stimuli and tested this hypothesis by developing an ex vivo culture system that allows control of the podocyte microenvironment. Results supported our hypothesis. SLSs in podocytes that migrated from isolated kidney glomeruli presented periodic synaptopodin-positive clusters that nucleated peripheral, foot process–like extensions. SLSs were mechanoresponsive to actomyosin inhibitors and substrate stiffness. Results suggest SLSs as mechanobiological mediators of podocyte recovery and as potential targets for therapeutic intervention.

Surgical repair of chronic wounds often requires split-thickness skin grafts in which epidermis and a thin part of the dermis from one site are harvested, cut into a meshwork, and stretched to cover a larger wound area. The main drawback of split-thickness skin grafting is excessive contracture of the skin graft in the wound site which is caused by the activation and transition of low-contractile fibroblasts to hyper-contractile myofibroblast cells. While a low amount of cellular contractile forces is required for wound healing, excessive contractile forces generated by hyperactivated cells can cause tightening of the skin graft, pain, scarring, and even graft failure. To test whether is a relationship between graft preparation and graft contracture, we hypothesized that the strain field that cells experience within a meshed graft regulates the activation of fibroblasts, and that the strain field and therefore cell activation level can be tailored by adjusting the meshing configurations to control the graft contracture. To test the hypothesis, we estimated strain fields of grafts with a wide range of clinically relevant meshing configurations, and evaluated the responses of human dermal fibroblasts to these strain levels within tissue constructs. We first showed that the choice of meshing configuration can induce a significant change in the graft strain level ranging from less than 5% to more than 30%. We then found that cells within tissue constructs statically stretched to 30% had significantly lower contractile force than cells within tissue constructs stretched to 5%. Data were used to fit a model of fibroblast activation, with activation peaking at a critical strain before decreasing due to cytoskeletal fluidization. The model was applied to predict conditions of meshing that tune graft contracture to desired levels. Results suggest a potential pathway for harnessing the memory of mechanical loading in fibroblasts to define and predict outcomes of skin graft surgeries.

In ferroelectric heterostructures, the interaction between intrinsic polarization and the electric field generates a rich set of localized electrical properties. The local electric field is determined by several connected factors, including the charge distribution of individual unit cells, the interfacial electromechanical boundary conditions, and chemical composition of the interfaces. However, especially in ferroelectric perovskites, a complete description of the local electric field across micro-, nano-, and atomic-length scales is missing. Here, by applying four-dimensional scanning transmission electron microscopy (4D STEM) with multiple probe sizes matching the size of structural features, we directly image the electric field of polarization vortices in (PbTiO3)16/(SrTiO3)16 superlattices and reveal different electric field configurations corresponding to the atomic scale electronic ordering and the nanoscale boundary conditions. The separability of two different fields probed by 4D STEM offers the possibility to reveal how each contributes to the electronic properties of the film.