Kennedy Omondi Okeyo

Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells

Coupling interactions among mechanical and biochemical factors are important for the realization of various cellular processes that determine cell migration. Although F-actin network dynamics has been the focus of many studies, it is not yet clear how mechanical forces generated by actomyosin contractility spatiotemporally regulate this fundamental aspect of cell migration. In this study, using a combination of fluorescent speckle microscopy and particle imaging velocimetry techniques, we perturbed the actomyosin system and examined quantitatively the consequence of actomyosin contractility on F-actin network flow and deformation in the lamellipodia of actively migrating fish keratocytes. F-actin flow fields were characterized by retrograde flow at the front and anterograde flow at the back of the lamellipodia, and the two flows merged to form a convergence zone of reduced flow intensity. Interestingly, activating or inhibiting actomyosin contractility altered network flow intensity and convergence, suggesting that network dynamics is directly regulated by actomyosin contractility. Moreover, quantitative analysis of F-actin network deformation revealed that the deformation was significantly negative and predominant in the direction of cell migration. Furthermore, perturbation experiments revealed that the deformation was a function of actomyosin contractility. Based on these results, we suggest that the actin cytoskeletal structure is a mechanically self-regulating system, and we propose an elaborate pathway for the spatiotemporal self-regulation of the actin cytoskeletal structure during cell migration. In the proposed pathway, mechanical forces generated by actomyosin interactions are considered central to the realization of the various mechanochemical processes that determine cell motility.

Strain field in actin filament network in lamellipodia of migrating cells: Implication for network reorganization

Cell motility is spatiotemporally regulated by interactions among mechanical and biochemical factors involved in the regulation of cytoskeletal actin structure reorganization. Although the molecular mechanisms underlying cell motility have been well investigated, the contributions of mechanical factors such as strain in the network reorganization remain unclear. In this study, we have quantitatively evaluated the strain field in the actin filament network forming the lamellipodia of migrating fish keratocytes to elucidate the mechanism by which actin filament network reorganization is regulated by biomechanical factors. The results highlight the existence of a negative (compressive) strain in the lamellipodia whose direction is parallel to that of cell movement. A close correlation was found between the distributions of the strain and the actin filament density in the lamellipodia, suggesting that negative strain may be involved in filament depolymerization. Based on this result, we propose a selective depolymerization model which suggests that negative strain may couple with biomechanical factors such as ADF/cofilin to promote selective depolymerization of filaments oriented in the direction of the deformation because such filaments experience relatively higher levels of the deformation. This model, in conjunction with others, may explain the observed reduction in filament density and the reorganization of actin filament network at the back of the lamellipodia of migrating fish keratocytes. Thus, we suggest that by coupling with biochemical factors, mechanical factors are involved in the regulation of actin filament depolymerization, thereby contributing to the regulation of cell motility.

Dynamic coupling between actin network flow and turnover revealed by flow mapping in the lamella of crawling fragments

Dynamic turnover and transport of actin filament network is essential for protrusive force generation and traction force development during cell migration. To elucidate the dynamic coupling between actin network flow and turnover, we focused on flow dynamics in the lamella of one of the simplest but elegant motility systems; crawling fragments derived from fish keratocytes. Interestingly, we show that actin network in the lamella of fragments is not stationary as earlier reported, but exhibits a flow dynamics that is strikingly similar to that reported for higher order cells, suggesting that network flow is an intrinsic property of the actin cytoskeleton that is fundamental to cell migration. We also demonstrate that whereas polymerization mediates network assembly at the front, surprisingly, network flow convergence modulates network disassembly toward the rear of the lamella, suggesting that flow and turnover are coupled during migration. These results obtained using simple motility systems are significant to the understanding of actin network dynamics in migrating cells, and they will be found useful for developing biophysical models for elucidating the fundamental mechanisms of cell migration.

Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells.

Mechanical methods for inducing differentiation and directing lineage specification will be instrumental in the application of pluripotent stem cells. Here, we demonstrate that minimization of cell-substrate adhesion can initiate and direct the differentiation of human pluripotent stem cells (hiPSCs) into cyst-forming trophoblast lineage cells (TLCs) without stimulation with cytokines or small molecules. To precisely control cell-substrate adhesion area, we developed a novel culture method where cells are cultured on microstructured mesh sheets suspended in a culture medium such that cells on mesh are completely out of contact with the culture dish. We used microfabricated mesh sheets that consisted of open meshes (100∼200 μm in pitch) with narrow mesh strands (3-5 μm in width) to provide support for initial cell attachment and growth. We demonstrate that minimization of cell adhesion area achieved by this culture method can trigger a sequence of morphogenetic transformations that begin with individual hiPSCs attached on the mesh strands proliferating to form cell sheets by self-assembly organization and ultimately differentiating after 10-15 days of mesh culture to generate spherical cysts that secreted human chorionic gonadotropin (hCG) hormone and expressed caudal-related homeobox 2 factor (CDX2), a specific marker of trophoblast lineage. Thus, this study demonstrates a simple and direct mechanical approach to induce trophoblast differentiation and generate cysts for application in the study of early human embryogenesis and drug development and screening.

Nucleosomes Exhibit Non-uniform Unwrapping Along Native Chromatin Fibers with Increasing Salt Concentration as Revealed by Direct Imaging in a Microfluidic Channel

Identifying the distribution of the higher‐order structure of chromatin – a complex of DNA and proteins – along genomic DNA can clarify the mechanisms underlying cell development and differentiation, including gene regulation. However, genome‐wide analysis of this distribution at the single‐cell level remains an outstanding challenge. Here, the authors report a new method for investigating changes in and the distribution of higher‐order structures along native chromatin fibers – ranging over 100 µm in length – relative to changes in salt concentration. To this end, the authors developed a microfluidic platform that enabled us to isolate chromatin fibers from single cells and tether them to microstructures in a microfluidic channel without fragmentation. The fibers were then exposed to varying concentrations of salt solution under microscopic observation. As a result, the fibers are non‐uniformly elongated by up to 2–3 times along the fiber axis as salt concentration was increased from 0 to 3 M, suggesting that chromosome structural stability is non‐uniformly distributed along chromatin fibers in their native form. Thus, our system enables direct microscopic analysis of individual chromatin fibers from single cells, which can provide insights into epigenetic mechanisms of cell development, cell differentiation, and carcinogenesis.

Minimization of cell-substrate interaction using suspended microstructured meshes initiates cell sheet formation by self-assembly organization

There is growing interest to achieve tissue fabrication by designing the architecture of the substrate. Here we present a mesh culture approach which enables fabrication of standalone cell sheets by cell–cell contact mediated self-assembly organization on highly porous and microstructured SU-8 polymer meshes (apertures size >100 μm in length, mesh strands 3–5 μm in width), which are set suspended in a culture medium to limit cell-substrate interaction only to the fine mesh strands. We demonstrate that ingression of cells into the wide mesh openings proceeds solely by cell–cell adhesion, resulting in the formation of a uniform cell sheet over the highly porous mesh scaffolds. A uniformly spread actin meshwork dotted with reinforcement structures including transverse actin cables was present in cell sheets of fibroblasts while ring-like cables of actin/E-cadherin architecture was confirmed in epithelial cell sheets, suggesting the requirement for mechanically stable cell–cell adhesion architecture to overcome substrate adhesion limitations. Expression of type I collagen suggested the deposition of collagen and other extracellular matrices. Moreover, cell sheets could be maintained for an extended period of time (>100 d) without noticeable loss of cell viability, owing to improved exposure of the cell layer to medium in a suspension setup. Thus, the mesh culture offers an alternative method for extended cell culture and fabrication of standalone cell sheets for easy integration into microfluidics platforms for organ-on-chip studies.

Adhesion patterning by a novel air-lock technique enables localization and in-situ real-time imaging of reprogramming events in one-to-one electrofused hybrids.

Although fusion of somatic cells with embryonic stem (ES) cells has been shown to induce reprogramming, single-cell level details of the transitory phenotypic changes that occur during fusion-based reprogramming are still lacking. Our group previously reported on the technique of one-to-one electrofusion via micro-slits in a microfluidic platform. In this study, we focused on developing a novel air-lock patterning technique for creating localized adhesion zones around the micro-slits for cell localization and real-time imaging of post fusion events with a single-cell resolution. Mouse embryonic fibroblasts (MEF) were fused individually with mouse ES cells using a polydimethylsiloxane (PDMS) fusion chip consisting of two feeder channels with a separating wall containing an array of micro-slits (slit width ∼3 μm) at a regular spacing. ES cells and MEFs were introduced separately into the channels, juxtaposed on the micro-slits by dielectrophoresis and fused one-to-one by a pulse voltage. To localize fused cells for on-chip culture and time-lapse microscopy, we implemented a two-step approach of air-lock bovine serum albumin patterning and Matrigel coating to create localized adhesion areas around the micro-slits. As a result of time-lapse imaging, we could determine that cell division occurs within 24 h after fusion, much earlier than the 2–3 days reported by earlier studies. Remarkably, Oct4-GFP (Green Fluorescent Protein) was confirmed after 25 h of fusion and thereafter stably expressed by daughter cells of fused cells. Thus, integrated into our high-yield electrofusion platform, the technique of air-lock assisted adhesion patterning enables a single-cell level tracking of fused cells to highlight cell-level dynamics during fusion-based reprogramming.

Innovative approaches to cell biomechanics

Actin Cytoskeletal Structure in Migrating Cells.- Actin Cytoskeleton Generates Mechanical Forces for Cell Migration.- Multi-scale Mechanochemical Interactions between Cell Membrane and Actin Filaments.- Actin Network Flow and Turnover are coupled in Migrating Cells.- Mechanical Strain is involved in Actin Network Reorganization.- Actin Network Dynamics is Regulated by Actomyosin Interactions.- Biophysical Interactions between Cells and Extracellular Matrix.- Cell Migration in Engineered Micro-/Nano-environments with Controlled Physical Properties.- Engineered Biomaterial for Cell Manipulation.

A microfluidic device for isolating intact chromosomes from single mammalian cells and probing their folding stability by controlling solution conditions

Chromatin folding shows spatio-temporal fluctuations in living undifferentiated cells, but fixed spatial heterogeneity in differentiated cells. However, little is known about variation in folding stability along the chromatin fibres during differentiation. In addition, effective methods to investigate folding stability at the single cell level are lacking. In the present study, we developed a microfluidic device that enables non-destructive isolation of chromosomes from single mammalian cells as well as real-time microscopic monitoring of the partial unfolding and stretching of individual chromosomes with increasing salt concentrations under a gentle flow. Using this device, we compared the folding stability of chromosomes between non-differentiated and differentiated cells and found that the salt concentration which induces the chromosome unfolding was lower (≤500 mM NaCl) for chromosomes derived from undifferentiated cells, suggesting that the chromatin folding stability of these cells is lower than that of differentiated cells. In addition, individual unfolded chromosomes, i.e., chromatin fibres, were stretched to 150–800 µm non-destructively under 750 mM NaCl and showed distributions of highly/less folded regions along the fibres. Thus, our technique can provide insights into the aspects of chromatin folding that influence the epigenetic control of cell differentiation.

Self-organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self‐organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self‐organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm‐mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro‐structured meshes with limited surface area for cell adhesion. We show that this adhesion‐restriction strategy can trigger a two‐stage self‐organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8–10, then into spherical cysts following differentiation and self‐organization of the sheet‐forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two‐photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E‐cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late‐stage blastocyst. Thus, the mesh culture microenvironment can initiate self‐organization of hiPSCs into trophectoderm‐mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development.