Cellular communication via directed protrusion growth: Critical length-scales and membrane morphology

AbstractWe investigated the growth of cell protrusions from adherent HEK 293 cells and their capability to bridge cytophobic Teflon®  AF microgaps, establishing a critical length scale, beyond which cells cannot probe free space. For this purpose, we employed a photolithography-based surface fabrication strategy for producing micropatterned substrates composed of glass and the amorphous polymer Teflon®  AF. Cell protrusions growing from HEK 293 cells on these substrates were confined to extend on 2 μm wide glass lanes, intersected by Teflon®  AF microgaps of various lengths between 2 and 16 μm. After 24 hours of incubation, the frequency of cell protrusions crossing the gap was found to be strongly dependent on the gap size. Gaps which are greater than 4 μm were found to be increasingly difficult to cross. Cell extensions crossing the microgaps either appeared as nanosized connections, in approximately 30% of all observed cases, or as microsized connections. Molecular transport in the established cell-to-cell connection across the microgap was investigated by activation of TRPM8 ion channels followed by supply of Ca2+ to one of the connected cells. The diffusion of the Ca2+ ions was visualized by means of a cell-permeant pre-fluorescent dye. We observed both open- and closed-ended intercellular connections in both nano- and microsized cell protrusions

Intercellular Transport of Oct4 in Mammalian Cells: A Basic Principle to Expand a Stem Cell Niche?

Background: The octamer-binding transcription factor 4 (Oct4) was originally described as a marker of embryonic stem cells. Recently, the role of Oct4 as a key regulator in pluripotency was shown by its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. While artificial induction of pluripotency using transcription factors is possible in mammalian cell culture, it remains unknown whether a potential natural transfer mechanism might be of functional relevance in vivo. The stem cell based regeneration of deer antlers is a unique model for rapid and complete tissue regeneration in mammals and therefore most suitable to study such mechanisms. Here, the transfer of pluripotency factors from resident stem cell niche cells to differentiated cells could recruit more stem cells and start rapid tissue regeneration. Methodology/Principal Findings: We report on the ability of STRO-1 + deer antlerogenic mesenchymal stem cells (DaMSCs) to transport Oct4 via direct cell-to-cell connections. Upon cultivation in stem cell expansion medium, we observed nuclear Oct4 expression in nearly all cells. A number of these cells exhibit Oct4 expression not only in the nucleus, but also with perinuclear localisation and within far-ranging intercellular connections. Furthermore, many cells showed intercellular connections containing both F-actin and a-tubulin and through which transport could be observed. To proof that intercellular Oct4-transfer has functional consequences in recipient cells we used a co-culture approach with STRO-1 + DaMSCs and a murine embryonic fibroblast indicator cell line (Oct4-GFP MEF). In this cell line a reporter gene (GFP) unde

UNDERSTANDING NANOPARTICLE-CELL INTERACTION

Nanotechnology has revolutionalized the landscape of modern science and technology, including materials, electronics, therapeutics, bioimaging, sensing, and the environment. Along with these technological advancements, there arises a concern that engineered nanomaterials, owing to their high surface area and high reactivity, may exert adverse effects upon discharge to compromise biological and ecological systems. Research in the past decade has examined the fate of nanomaterials in vitro and in vivo, as well as the interactions between nanoparticles and biological and ecosystems using primarily toxicological and ecotoxicological approaches. However, due to the versatility in the physical and physicochemical properties of nanoparticles, and due to the vast complexity of their hosting systems, the solubility, transformation, and biocompatibility of nanomaterials are still poorly understood. Accordingly, this dissertation offers a mechanistic study on the differential translocation of pristine and water-soluble fullerene nanoparticles in mammalian and plant cells (Chapter 2), an investigation on membrane fluidity upon exocytosis of gold nanoparticles by the cell (Chapter 3), and an in-depth examination of the formation of an array of nanoparticle-protein coronas and their interactions with lipid vesicles and the cell (Chapters 4 and 5). The organization of this dissertation is as follows. Chapter 1 presents a review on the general applications (gene and drug delivery, imaging, sensing, nanotherapy) and implications (toxicity) of nanomaterials, mostly within the context of biological systems. In addition, this chapter documents theendocytotic and exocytotic pathways of the cell, and reviews the state-of-the-art of our understanding of nanoparticle-protein corona formation and nanoparticle-cell interactions, two precursors of nanotoxicity. Chapter 2 offers, for the first time, a parallel study on the differential uptake of hydrophobic and amphiphilic fullerene nanoparticles by Allium cepa plant cells and HT- 29 mammalian cells, two model systems representing ecological and biological systems. Methodologically, this study was conducted using a plant cell viability assay, bright field and fluorescence imaging, and, extensively, electron microscopy imaging. Chapter 3 examines an important but rarely documented aspect of cellular response to nanoparticles - exocytosis. A lipophilic Laurdan dye was used to partition into HT-29 mammalian cell membranes. Membrane fluidity as a result of the discharge of gold nanoparticles was inferred from UV-vis absorbance as well as by calculating the general polarization value of the dye -- hereby treated an electric dipole in a lipid bilayer continuum -- based on its fluorescence emissions at two characteristic wavelengths. Chapter 4 concerns protein adsorption on carbon nanotubes (CNTs) to form protein coronas in cell culture media, an environment relevant to both in vitro and in vivo studies. A label-free mass spectrometry-based proteomic approach was employed, and the compositions of the protein forming coronas on a set of CNTs were examined. The physicochemical properties of the CNTs were also extensively characterized in order to establish a correlation between protein adsorption and CNT surface properties. Chapter 5 characterizes the formation of a serum albumin corona on silver nanoparticles and evaluates the impact of silver nanoparticle-albumin corona on thefluidity of an artificial lipid vesicle. The reason of adopting a lipid vesicle in this study is to eliminate endo- and exocytosis and pinpoint the roles of physical forces in nanoparticle-cell interactions. In this chapter we also show the formation and conformational changes of fibrinogen corona in HT-29 cell lines. Fibrinogen is one of the most abundant types of plasma proteins in the bloodstream. Chapter 6 summarizes the major findings in this dissertation and presents future work inspired by this Doctor of Philosophy (PhD) research

Intercellular crosstalk mediated by tunneling nanotubes between central nervous system cells. What we need to advance

Long-range intercellular communication between Central Nervous System (CNS) cells is an essential process for preserving CNS homeostasis. Paracrine signaling, extracellular vesicles, neurotransmitters and synapses are well-known mechanisms involved. A new form of intercellular crosstalk mechanism based on Tunneling Nanotubes (TNTs), suggests a new way to understand how neural cells interact with each other in controlling CNS functions. TNTs are long intercellular bridges that allow the intercellular transfer of cargoes and signals from one cell to another contributing to the control of tissue functionality. CNS cells communicate with each other via TNTs, through which ions, organelles and other signals are exchanged. Unfortunately, almost all these results were obtained through 2D in-vitro models, and fundamental mechanisms underlying TNTs-formation still remain elusive. Consequently, many questions remain open, and TNTs role in CNS remains largely unknown. In this review, we briefly discuss the state of the art regarding TNTs identification and function. We highlight the gaps in the knowledge of TNTs and discuss what is needed to accelerate TNTs-research in CNS-physiology. To this end, it is necessary to: 1) Develop an ad-hoc TNTs-imaging and software-assisted processing tool to improve TNTs-identification and quantification, 2) Identify specific molecular pathways involved into TNTs-formation, 3) Use in-vitro 3D-CNS and animal models to investigate TNTs-role in a more physiological context pushing the limit of live-microscopy techniques. Although there are still many steps to be taken, we believe that the study of TNTs is a new and fascinating frontier that could significantly contribute to deciphering CNS physiology

Creating complex protocells and prototissues using simple DNA building blocks

Building synthetic protocells and prototissues hinges on the formation of biomimetic skeletal frameworks. Recreating the complexity of cytoskeletal and exoskeletal fibers, with their widely varying dimensions, cellular locations and functions, represents a major material hurdle and intellectual challenge which is compounded by the additional demand of using simple building blocks to ease fabrication and control. Here we harness simplicity to create complexity by assembling structural frameworks from subunits that can support membrane-based protocells and prototissues. We show that five oligonucleotides can anneal into nanotubes or fibers whose tunable thicknesses and lengths spans four orders of magnitude. We demonstrate that the assemblies' location inside protocells is controllable to enhance their mechanical, functional and osmolar stability. Furthermore, the macrostructures can coat the outside of protocells to mimic exoskeletons and support the formation of millimeter-scale prototissues. Our strategy could be exploited in the bottom-up design of synthetic cells and tissues, to the generation of smart material devices in medicine

Determining the molecular mechanisms mediating cytoplasmic material transfer between photoreceptors in the transplantation paradigm

Retinal degenerations are a complex group of disorders that all culminate in the same final common path, the loss of the light sensing cells of the eye, the photoreceptors. Photoreceptor replacement strategies aim to reverse the loss of vision by transplanting healthy cells to replace those lost through degeneration. Over the past decade, research has shown that transplanting photoreceptor precursors into models of retinal dysfunction results in restoration of visual function. Until recently, this was thought to be attributed solely to donor photoreceptor cells integrating into the retina. However, we have recently demonstrated that the observed rescue was instead largely due to exchange of RNA and/or protein between donor and remaining host photoreceptor cells, a mechanism we named material transfer. Since this process appears to render host cells functional, the mechanisms by which this occurs are of significant interest. In this PhD thesis, I sought to determine the molecular mechanism underlying material transfer. I hypothesized that this may involve direct physical contacts or indirect shedding and uptake of information packaged in extracellular vesicles (EVs). I first developed a robust protocol to maintain primary rod precursors in an isolated culture system to enable the study of both molecular mechanisms. I established that cultured photoreceptors release vesicles bearing the phenotypical and molecular characteristics of EVs, accompanied with the molecular signature of the cell of origin. By employing the Cre-loxP system I confirmed that photoreceptor-derived EVs can alter gene expression in glia cells, both in vitro and in vivo, but not in other photoreceptors, strongly indicating that EVs are not the primary mediators of material transfer in the transplantation paradigm. However, a combination of imaging methods, alongside pharmacological inhibition of the actin cytoskeleton of photoreceptor cultures, revealed transient tubulovesicular processes between photoreceptors, that are capable of transferring fluorescent reporters, organelles, and lipids. These fine structures are typically destroyed during fixation, impeding comprehensive assessment in vivo. Finally, I demonstrated and characterized a few examples of donor-host contacts in vivo, when fluorescent reporters were tagged to the membrane of donor cells. Taken together the above findings support that physical connections are most likely the mechanism underlying photoreceptor communication during material transfer

Carbon Nanotube Arrays for Intracellular Delivery and Biological Applications

Introducing nucleic acids into mammalian cells is a crucial step to elucidate biochemical pathways, modify gene expression in immortalized cells, primary cells, and stem cells, and intoduces new approaches for clinical diagnostics and therapeutics. Current gene transfer technologies, including lipofection, electroporation, and viral delivery, have enabled break-through advances in basic and translational science to enable derivation and programming of embryonic stem cells, advanced gene editing using CRISPR (Clustered regularly interspaced short palindromic repeats), and development of targeted anti-tumor therapy using chimeric antigen receptors in T-cells (CAR-T). Despite these successes, current transfection technologies are time consuming and limited by the inefficient introduction of test molecules into large populations of target cells, and the cytotoxicity of the techniques. Moreover, many cell types cannot be consistently transfected by lipofection or electroporation (stem cells, T-cells) and viral delivery has limitations to the size of experimental DNA that can be packaged. In this dissertation, a novel coverslip-like platform consisting of an array of aligned hollow carbon nanotubes (CNTs) embedded in a sacrificial template is developed that enhances gene transfer capabilities, including high efficiency, low toxicity, in an expanded range of target cells, with the potential to transfer mixed combinations of protein and nucleic acids. The CNT array devices are fabricated by a scalable template-based manufacturing method using commercially available membranes, eliminating the need for nano-assembly. High efficient transfection has been demonstrated by delivering various cargos (nanoparticles, dye and plasmid DNA) into populations of cells, achieving 85% efficiency of plasmid DNA delivery into immortalized cells. Moreover, the CNT-mediated transfection of stem cells shows 3 times higher efficiency compared to current lipofection methods. Evaluating the cell-CNT interaction elucidates the importance of the geometrical properties of CNT arrays (CNT exposed length and surface morphology) on transfection efficiency. The results indicate that densely-packed and shortly-exposed CNT arrays with planar surface will enhance gene delivery using this new platform. This technology offers a significant increase in efficiency and cell viability, along with the ease of use compared to current standard methods, which demonstrates its potential to accelerate the development of new cell models to study intractable diseases, decoding the signaling pathways, and drug discovery

The 2018 biomembrane curvature and remodeling roadmap

The importance of curvature as a structural feature of biological membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one hand, changes in membrane morphology are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo-and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of Journal of Physics D: Applied Physics addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area

Carbon Nanomaterials: Applications in Physico-chemical Systemsand Biosystems

In the present article, various forms of carbon and carbon nanomaterials (CNMs) and a new approach to classify them on the basis of sp2-sp3 configuration are presented. Utilising the concept of junction formation (like p:n junction) a concept is developed to explain the special reactivity of nanosized carbon materials. Geometric consideration of chiral and achiral symmetry of single-walled carbon nanotubes is presented which is also responsible for manifesting special propertiesof carbon nanotubes. A brief introduction to various common synthesis techniques of CNMs is given. These is increased chemical and biological activities have resulted in many engineer ednanoparticles, which are being designed for specific purposes, including diagnostic or the rapeuticmedical uses and environmental remediation.Defence Science Journal, 2008, 58(4), pp.460-485, DOI:http://dx.doi.org/10.14429/dsj.58.166