Quantification of mechanical forces and physiological processes involved in pollen tube growth using microfluidics and microrobotics

Pollen tubes face many obstacles on their way to the ovule. They have to decide whether to navigate around cells or penetrate the cell wall and grow through it or even within it. Besides chemical sensing, which directs the pollen tubes on their path to the ovule, this involves mechanosensing to determine the optimal strategy in specific situations. Mechanical cues then need to be translated into physiological signals, which eventually lead to changes in the growth behavior of the pollen tube. To study these events, we have developed a system to directly quantify the forces involved in pollen tube navigation. We combined a lab-on-a-chip device with a microelectromechanical systems-based force sensor to mimic the pollen tube's journey from stigma to ovary in vitro. A force-sensing plate creates a mechanical obstacle for the pollen tube to either circumvent or attempt to penetrate while measuring the involved forces in real time. The change of growth behavior and intracellular signaling activities can be observed with a fluorescence microscope

Feeling the force: how pollen tubes deal with obstacles

Physical forces are involved in the regulation of plant development and morphogenesis by translating mechanical stress into the modification of physiological processes, which, in turn, can affect cellular growth. Pollen tubes respond rapidly to external stimuli and provide an ideal system to study the effect of mechanical cues at the single‐cell level. Here, pollen tubes were exposed to mechanical stress while monitoring the reconfiguration of their growth and recording the generated forces in real‐time. We combined a lab‐on‐a‐chip device with a microelectromechanical systems (MEMS)‐based capacitive force sensor to mimic and quantify the forces that are involved in pollen tube navigation upon confronting mechanical obstacles. Several stages of obstacle avoidance were identified, including force perception, growth adjustment and penetration. We have experimentally determined the perceptive force threshold, which is the force threshold at which the pollen tube reacts to an obstacle, for Lilium longiflorum and Arabidopsis thaliana. In addition, the method we developed provides a way to calculate turgor pressure based on force and optical data. Pollen tubes sense physical barriers and actively adjust their growth behavior to overcome them. Furthermore, our system offers an ideal platform to investigate intracellular activity during force perception and growth adaption in tip growing cells

Mechanical factors contributing to the Venus flytrap's rate-dependent response to stimuli.

Funder: schweizerischer nationalfonds zur förderung der wissenschaftlichen forschung; doi: http://dx.doi.org/10.13039/501100001711Funder: ETH ZurichThe sensory hairs of the Venus flytrap (Dionaea muscipula Ellis) detect mechanical stimuli imparted by their prey and fire bursts of electrical signals called action potentials (APs). APs are elicited when the hairs are sufficiently stimulated and two consecutive APs can trigger closure of the trap. Earlier experiments have identified thresholds for the relevant stimulus parameters, namely the angular displacement [Formula: see text] and angular velocity [Formula: see text]. However, these experiments could not trace the deformation of the trigger hair's sensory cells, which are known to transduce the mechanical stimulus. To understand the kinematics at the cellular level, we investigate the role of two relevant mechanical phenomena: viscoelasticity and intercellular fluid transport using a multi-scale numerical model of the sensory hair. We hypothesize that the combined influence of these two phenomena and [Formula: see text] contribute to the flytrap's rate-dependent response to stimuli. In this study, we firstly perform sustained deflection tests on the hair to estimate the viscoelastic material properties of the tissue. Thereafter, through simulations of hair deflection tests at different loading rates, we were able to establish a multi-scale kinematic link between [Formula: see text] and the cell wall stretch [Formula: see text]. Furthermore, we find that the rate at which [Formula: see text] evolves during a stimulus is also proportional to [Formula: see text]. This suggests that mechanosensitive ion channels, expected to be stretch-activated and localized in the plasma membrane of the sensory cells, could be additionally sensitive to the rate at which stretch is applied

The Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica

Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Microrobotic tools for mechanical characterization and stimulation: from single cells to organisms

Physical forces regulate the behavior of cells and tissues and are essential in cell organization and morphogenesis. Especially in plants, where pressurized cells are surrounded by a rigid cell wall that constricts cell expansion, growth is a highly coordinated process of stress and strain. With the advent of new biomechanical tools, forces at the cellular and multicellular level can finally be directly measured. This spatial force resolution is crucial to study how cells’ perception of mechanical cues leads to the cascade of biochemical and electrical signals that ultimately regulates the behavior, functions, and mechanical properties of cells. This thesis investigates biomechanical processes with an interdisciplinary approach that combines engineering sciences, biology, and physics. Newly engineered microrobotic tools for mechanical characterization and stimulation of cells and tissues, together with analytical and numerical models, allow us to assess the intricate interplay between extracellular forces, intracellular pressure, and cell wall elasticity. Biochemical and genetic techniques enable us to monitor the physiological processes controlling the cellular response to mechanical cues. In the first chapter, we explore the theoretical background of mechanobiology, the study of mechanics in biology at the cellular level, encompassing the highly integrated genetic, biochemical, and biomechanical processes involved in growth and morphogenesis. In chapter two, we introduce the cellular force microscope (CFM), an existing microrobotic platform to study the mechanobiology of cells. The chapter presents this system and the improvements made to it over the course of this project. We integrated the CFM with an inverted microscope for fluorescence imaging, which is essential to correlate cytomechanics with biochemical signaling. A newly modular CFM-structure can be rearranged to fit the requirements of different biological samples, and the operational mode (e.g., microindentation, relaxation test) can be programmed as needed. The third chapter documents mechanical characterizations at the microscale using different adaptations of the CFM, each tailored to a specific task. We present a configuration for non-perpendicular indentation using a dualforce read-out, and a multiple-CFM configuration to separately measure turgor pressure and cell wall elasticity in growing pollen tubes (PTs). We measure the mechanical properties of plants at the single- and multi-cellular level, using PTs and roots, respectively. Additionally, we demonstrate the applicability of the system for use in animal tissues with nematode embryos. In the fourth chapter, we investigate the effect of external mechanical cues on plants at the cell level using PTs, and at the organ level using Venus flytraps. We show that PTs literally feel their way through their environment to avoid obstacles as they deliver male gametes to the ovule. We measure their force sensitivity to understand this remarkable behavior. In Venus flytraps, we show for the first time a direct correlation between the magnitude of the mechanical stimulus in vivo and the electrical response that leads to trap closure. In contrast to the generally accepted idea that two hair deflections are needed to initiate trap closure, we show that under certain conditions, a single deflection is sufficient. Major conclusions and research contributions provided by this work are highlighted in the fifth chapter