Surface Topography Modulates Cell Mechanosensing of Extracellular Matrix

The interaction between the cell membrane and the extracellular matrix is crucial for many cellular functions by modulating mechanosensitive signaling pathways.Physical properties of the extracellular matrix such as stiffness and topography affect such interactions.Our recent work reveals that surface topography of tens to hundreds of nanometer scale modulates cell signaling by activating intracellular curvature-sensitive proteins.We use vertical nanostructures protruding from a flat surface as a platform to induce precise curvatures on the cell membrane and to probe biological processes in live cells.Vertical nanopillars deform the plasma membrane inwards and induce membrane curvature when the cell engulfs them,leading to a reduction of the membrane-substrate gap distance.We found that the high membrane curvature induced by vertical nanopillars significantly affects the distribution of curvature-sensitive proteins and stimulates several cellular processes in live cells including cellular endocytosis and cytoskeleton dynamics.Our studies show a strong interplay between biological cells and nano-featured surfaces,which is an essential consideration for future development of interfacing devices.

Mechanosensing via Immunereceptors

The immune response is orchestrated by a variety of immune cells,the function of which then is determined by the collective signals from different immunoreceptors.Recent studies have highlighted the presence of mechanical force on these receptor-ligand pairs and its important role in regulating antigen recognition/discrimination and function.In this perspective,we use the T cell receptor as an example to review the current understanding of the mechanosensing properties of immunoreceptors.We discuss the types of forces that immunoreceptors may encounter,the effects on ligand recognition,conformational changes and mechanosensing mechanisms,as well as the consequences in downstream signal transduction and function.

Mechanical force drives the polarization and orientation of cells

Collective cells are organized to form specific patterns which play important roles in various physiological and pathological processes, such as tissue morphogenesis, wound healing, and cancer invasion. Compared to single cell behaviors, which has been intensively studied from many aspects (cell migration, adhesion, polarization, proliferation, etc.) and at various scales (molecular, subcellular, and cellular), the multiple cell behaviors are relatively less understood, particularly in a quantitative manner. In this paper, we will present our recent studies of collective polarization and orientation of multiple cells through both experimental measurement and theoretical modeling, including those cell behaviors on/in 2D and 3D substrate/tissue. We find that the collective cell behaviors, including polarization, alignment and migration are closely related to local stress states in cell layer or tissue, which demonstrate the crucial roles of mechanical forces in the living organisms. Specifically, the cells prefer to polarize and align along the maximum principal stress in the cell layer, and the aspect ratio of cell increases with the in-plane maximum shear stress, suggesting that the maximum shear stress is the underlying driving force of cell polarization and orientation. This theory of stress-driven cell behaviors of polarization and orientation provides a new perspective for understanding cell behaviors in living organisms and the guideline for tissue engineering in biomedical applications.

Mineral Fabrication and Golgi Apparatus Activity in <i>Spirostomum ambiguum</i>: A Primordial Paradigm of the Stressed Bone Cell?

The histological basis for acute osteocyte mechanosensitivity remains uncertain. A novel bone cell model of mechanotransduction and inorganic trafficking may be the powerful, silt-burrowing protozoan Spirostomum ambiguum which when being physically challenged fabricates within vesicles populations of bone-like calcium phosphate microspheres, about 1 μm in diameter. These not only attribute considerable compression-resilience but also resemble the Golgi-directed mineral assemblies we recently reported in osteocytes. Advantageously, calcification in the protozoan (confirmed by ultramicroscopy with EDX elemental microanalysis) enabled Golgi comparison under overt, natural phases of both high (i.e. silt-tunnelling) and low (i.e free-swimming) stress. Established hard-tissue microscopy techniques previously positive in bone cells included quantitative fluorescent tetracycline labelling for bone salt together with the same metazoan Golgi body marker (Green Fluorescent Protein-tagged mannosidase II construct). Organellar modulation was monitored by transfection of live organisms in situ (some post-stained with red nuclear fluorochrome TOPRO-3). Results showed that GFP-tagged Golgi fluorescence increased from swimmers (mean 74.5 ± SD 6.7 AU) to burrowers (mean 104.6 ± SD 2.7;p < 0.0001) synchronous with juxtanuclear tetracycline-labelled mineral fluorescence (swimmers, mean 89.7 ± SD 3.3 AU;burrowers, mean 138.0 ± SD 4.0;p < 0.0001). Intracellular dense microspheres, single or bridged, were harvested as pellets rich in Ca, P (Ca:P 0.98) and Si, their polarised alignment moving from transaxial in swimmers to axial in burrowers. It was concluded that Golgi-directed mineral fabrication in the large, accessible, silt-enclosed ciliate resembles that in the smaller, less-accessible bone cell and may be a conserved early mechanobiological intracellular development predicating force translation into compression-resistant mineral fabrication in loaded segments of the osteocyte syncitium.

Loss of Aβ-nerve endings associated with the Merkel cell-neurite complex in the lesional oral mucosa epithelium of lichen planus and hyperkeratosis

The Merkel cell-neurite complex initiates the perception of touch and mediates Ap slowly adapting type I responses. Lichen planus is a chronic inflammatory autoimmune disease with T-cell-mediated inflammation, whereas hyperkeratosis is characterized with or without epithelial dysplasia in the oral mucosa. To determine the effects of lichen planus and hyperkeratosis on the Merkel cell-neurite complex, healthy oral mucosal epithelium and lesional oral mucosal epithelium of lichen planus and hyperkeratosis patients were stained by immunohistochemistry （the avidin-biotin-peroxidase complex and double immunofluorescence methods）using pan cytokeratin, cytokeratin 20 （K20, a Merkel cell marker）, and neurofilament 200 （NF200, a myelinated Aβ- and Aδ-nerve fibre marker） antibodies. N F200-immunoreactive （ir） nerve fibres in healthy tissues and in the lesional oral mucosa epithelium of lichen planus and hyperkeratosis were counted and statistically analysed. In the healthy oral mucosa, K20-positive Merkel cells with and without close association to the intraepithelial NF200-ir nerve fibres were detected. In the lesional oral mucosa of lichen planus and hyperkeratosis patients, extremely rare NF200-ir nerve fibres were detected only in the lamina propria. Compared with healthy tissues, lichen planus and hyperkeratosis tissues had significantly decreased numbers of NF200-ir nerve fibres in the oral mucosal epithelium. Lichen planus and hyperkeratosis were associated with the absence of Aβ-nerve endings in the oral mucosal epithelium. Thus, we conclude that mechanosensation mediated by the Merkel cell-neurite complex in the oral mucosal epithelium is impaired in lichen planus and hvperkeratosis.

Analysis of Freely Swimming C. elegans Using Laser Diffraction

Soil and aquatic multicellular microorganisms play a critical role in the nutrient-cycling and organismal ecology of soil and aquatic ecosystems. These organisms live and behave in a complex three-dimensional environment. Most studies of microorganismal behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field. We report on a novel analytical approach that provides real-time analysis of freely swimming C elegans without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light onto nematodes in a cuvette. We measured oscillation frequencies for freely swimming nematodes in cuvettes of different sizes to provide different physical constraints on their swimming. We compared these frequencies with those obtained for nematodes swimming within a small droplet of water on a microscope slide, a strategy used by microscope-based locomotion analysis systems. We collected data from diffraction patterns using two methods: video analysis and real time data acquisition using a fast photodiode. Swimming frequencies of nematodes in a droplet of ionic solution on a microscope slide was confirmed to be 2.00 Hz with a variance of 0.05 Hz for the video analysis method and 0.03 Hz for the real time data acquisition using a photodiode;this result agrees with previously published estimates using microscope-based analytical techniques. We find the swimming frequency of unconstrained worms within larger cuvettes to be 2.37 Hz with a variance of 0.02 Hz. As the cuvette size decreased, so did the oscillation frequency, indicating a change in locomotion when physical constraints are introduced.

Biomechanical control of lymphatic vessel physiology and functions

The ever-growing research on lymphatic biology has clearly identified lymphatic vessels as key players that maintain human health through their functional roles in tissue fluid homeostasis,immunosurveillance,lipid metabolism and inflammation.It is therefore not surprising that the list of human diseases associated with lymphatic malfunctions has grown larger,including issues beyond lymphedema,a pathology traditionally associated with lymphatic drainage insufficiency.Thus,the discovery of factors and pathways that can promote optimal lymphatic functions may offer new therapeutic options.Accumulating evidence indicates that aside from biochemical factors,biomechanical signals also regulate lymphatic vessel expansion and functions postnatally.Here,we review how mechanical forces induced by fluid shear stress affect the behavior and functions of lymphatic vessels and the mechanisms lymphatic vessels employ to sense and transduce these mechanical cues into biological signals.

Tunable nano-engineered anisotropic surface for enhanced mechanotransduction and soft-tissue integration

Electrochemically engineered titania(TiO_(2))nanopores enable tailored cellular function;however,the cellular mechanosensing mechanisms dictating the cell response and soft tissue integration are yet to be elucidated.Here,we report the fabrication of anisotropic TiO_(2)nanopores with diameters of 46 and 66 nm on microrough titanium(Ti)via electrochemical anodization,towards short-and long-term guidance of human primary gingival fibroblasts(hGFs).Cells on tissue culture plates and bare Ti substrates were used as controls.Notably,we show that nanopores with a diameter of 66 nm induced more mature focal adhesions of vinculin and paxillin at the membrane,encouraged the development of actin fibers at focal adhesion sites,led to elongated cell and nuclear shape.These topographical-driven changes were attributed to the Ras-related C3 botulinum toxin substrate 1(Rac 1)GTPase pathway and nuclear localisation of LAMIN A/C and yes-associated protein(YAP)and associated with increased ligament differentiation with elevated expression of the ligament marker Mohawk homeobox(MKX).Study findings reveal that minor tuning of nanopore diameter is a powerful tool to explore intracellular and nuclear mechanotransduction and gain insight into the relationships between nanomaterials and mechanoresponsive cellular elements.

Functions and clinical significance of mechanical tumor microenvironment:cancer cell sensing,mechanobiology and metastasis

Dynamic and heterogeneous interaction between tumor cells and the surrounding microenvironment fuels the occurrence,progression,invasion,and metastasis of solid tumors.In this process,the tumormicroenvironment(TME)fractures cellular and matrix architecture normality through biochemical and mechanical means,abetting tumorigenesis and treatment resistance.Tumor cells sense and respond to the strength,direction,and duration of mechanical cues in the TME by various mechanotransduction pathways.However,far less understood is the comprehensive perspective of the functions and mechanisms of mechanotransduction.Due to the great therapeutic difficulties brought by the mechanical changes in the TME,emerging studies have focused on targeting the adverse mechanical factors in the TME to attenuate disease rather than conventionally targeting tumor cells themselves,which has been proven to be a potential therapeutic approach.In this review,we discussed the origins and roles ofmechanical factors in the TME,cell sensing,mechano-biological coupling and signal transduction,in vitro construction of the tumormechanicalmicroenvironment,applications and clinical significance in the TME.

A novel electrospinning method for self-assembled tree-like fibrous scaffolds:Microenvironment-associated regulation of MSC behavior and bone regeneration

Numerous studies highlight advantages of electrospun scaffolds in bone tissue engineering,in which cellular behavior is tightly affected by fiber topographical cues of scaffolds.However,the classic electrospinning setup limits a desired presentation of biomimetic fibrous microenvironments that sense mechanosignaling and regulate stem cell behavior.The aims of this study were to fabricate advanced asspun scaffolds presenting tree-like microfiber/nanonet networks and to evaluate their regulatory potentials on behavior of human mesenchymal stem cells(h MSCs)and bone regeneration.Here we developed a novel electrospinning setup that allowed the presentation of patterned Trunk microfibers(TMF)and/or branched nanonet fibers(BNn Fs)in biomimetic fibrous scaffolds.As the cellular mechanisms,anisotropichierarchical topography of TMF controlled behavior of h MSCs through focal adhesion formation and Yesassociated protein(YAP)induction,whereas BNn F disturbed such mechanosensing responses in the cells.The fiber microenvironment-related expression and nuclear localization of YAP were.also correlated with the potentials of as-spun scaffolds to enhance osteogenic differentiation of the h MSCs and alveolar bone defect healing in an animal model.Collectively,this study provides an advanced approach of the modified electrospinning setup for presentation of biomimetic fibrillar microenvironments in as-spun scaffolds along with their application in stem cell behavior regulation and regenerative tissue engineering.

Joining actions: crosstalk between intermediate filaments and actin orchestrates cellular physical dynamics and signaling

Many key cellular functions are regulated by the interplay of three distinct cytoskeletal networks, made of actin filaments,microtubules, and intermediate filaments(IFs), which is a hitherto poorly investigated area of research. However, there are growing evidence in the last few years showing that the IFs cooperate with actin filaments to exhibit strongly coupled functions.This review recapitulates our current knowledge on how the crosstalk between IFs and actin filaments modulates the migration properties, mechano-responsiveness and signaling transduction of cells, from both biophysical and biochemical point of view.

Demystifying Mechanosensitive Piezo Ion Channels

Mechanosensitive channels mediate touch,hearing,proprioception,and blood pressure regulation.Piezo proteins,including Piezo1 and Piezo2,represent a new class of mechanosensitive channels that have been reported to play key roles in most,if not all,of these modalities.The structural architecture and molecular mechanisms by which Piezos act as mechanosensitive channels,however,remain mysterious.Two new studies have now provided critical insights into the atomic structure and molecular basis of the ion permeation and mechano-gating properties of the Piezo1 channel.

Polymodal Functionality of C. elegans OLL Neurons in Mechanosensation and Thermosensation

Sensory modalities are important for survival but the molecular mechanisms remain challenging due to the polymodal functionality of sensory neurons. Here, we report the C. elegans outer labial lateral(OLL) sensilla sensory neurons respond to touch and cold. Mechanosensation of OLL neurons resulted in cell-autonomous mechanically-evoked Ca~(2+) transients and rapidly-adapting mechanoreceptor currents with a very short latency.Mechanotransduction of OLL neurons might be carried by a novel Na~+ conductance channel, which is insensitive to amiloride. The bona fide mechano-gated Na~+ -selective degenerin/epithelial Na~+ channels, TRP-4, TMC, and Piezo proteins are not involved in this mechanosensation.Interestingly, OLL neurons also mediated cold but not warm responses in a cell-autonomous manner. We further showed that the cold response of OLL neurons is not mediated by the cold receptor TRPA-1 or the temperaturesensitive glutamate receptor GLR-3. Thus, we propose the polymodal functionality of OLL neurons in mechanosensation and cold sensation.