Microfluidic and micromechanical in vivo studies of Caenorhabditis elegans

Caenorhabditis elegans is one of the most attractive model organisms in biomedical research for understanding human diseases and for drug testing at a whole-organism level, since many biological pathways and genes have been conserved between itself and humans. The nematode is sufficiently complex so that complex biological questions can be addressed, yet the organism is still simple enough to be used in high-content in vivo assays. In this work, we present new microfluidic and MEMS-based tools enabling the characterization of C. elegans worms and embryos in vivo.

In a first part of this thesis, we measured the metabolic heat production of small C. elegans larval populations using a custom nanocalorimetric system. Our system is based on microfabricated Si thermopile sensors which are integrated with PDMS microfluidic chips. In addition to versatile fluidic manipulation of samples on-chip, our microfluidic approach allows confining worm populations close to the Si sensor surface, thus enabling high sensitivity of the assays. Our results indicate an increase of the heat production per worm body volume from the L2 to the L3 larval stages, and a significant decrease from the L3 to the L4 stages. Additionally, we investigated the metabolic heat production of the larval populations during maximal respiratory capacity after treatment with a mitochondrial uncoupling agent. Depending on the larval stage, inducing uncoupled respiration causes an increase of the metabolic heat production ranging from 55% up to 95% with respect to untreated worms. Our experiments demonstrated that nanocalorimetry is a direct and highly-sensitive technique that can be combined with other analytical methods to open the way to a more holistic understanding of fundamental biological processes.

In a second study, we present a new microfluidic worm culture system with integrated luminescence-based sensing of the on-chip oxygen concentration for measuring the oxygen uptake of C. elegans worms. The microfluidic chip was fabricated in OSTE+ to allow reliable measurement of the dissolved oxygen and the fabrication of high-resolution microstructures down to the &m-range. Our microfluidic approach allows confining a single nematode from the L4 stage in a culture chamber over a time span of up to 7 days. An automated protocol for feeding and for performing oxygen consumption rate (OCR) measurements during the worm development was applied. We found an increase of OCR values from the L4 stage to the adult worm, and a continuous decrease as the worm ages. In addition, we performed a C. elegans metabolic assay using a mitochondrial uncoupling agent, which increased OCR by a factor of ¿ 2 when compared to basal respiration rates. Treatment with sodium azide inhibited mitochondrial respiration and returned OCR values to zero.

The biomechanical properties of the C. elegans embryonic eggshell are largely unknown. In a third study, we developed a new method to study its mechanical properties. In our work, we use cellular force microscopy (CFM), a technique that combines micro-indentation with high-resolution force sensing approaching that of atomic force microscopy, to quantitatively study the mechanical properties of the eggshell of living C. elegans embryos.

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AFM Based Single Cell Microinjection

Joanna Katarzyna Bitterli

The development of atomic force microscopy (AFM) has enabled a major breakthrough in the study of individual biological objects, such as nucleic acids, proteins and protein complexes. More recently the use of AFM to investigate eukaryotic cells has been explored. In one approach, the AFM probe can be used as a needle that delivers material into a single living cell while the AFM microscope controls precisely the interactions between the probe and the biological sample. The work presented here was dedicated to the development of a microinjection system for single cells based on atomic force microscopy. Demonstration experiments of liquid delivery into cells were also performed in order to characterize the system, its potential and its limits. As the injection of liquid into a cell requires the insertion of the tip into a cell, a detailed study of AFM probe-cell interactions was carried out. In the introduction microinjection into adherent cells, its applications and limitations are described. The main limitation of this method is lack of control over the cell penetration. Since atomic force microscope (AFM) offers this possibility, a novel microinjection tool for liquid delivery into single adherent cells based on the AFM is proposed in this work. A case study examines the specifications of an AFM-based microinjection system, such as control of delivered volume and control of AFM-probe cell interactions. Given the specifications, a detail design of the system is proposed with an AFM probe with microfluidic channels (NADIS) as a core component. In next two chapters, the fabrication and characterization of the system is presented including the flow of liquids through the NADIS probes. Some limitations of the system are discussed together with possible approaches to improvement. Further, an in depth analysis of cell indentation is undertaken. Aspects such as determination of tip insertion and factors influencing the probability of cell membrane penetration by an AFM tip are discussed. Cell membrane rupture with an AFM probe is described with a simple mechanical model. Biophysical analysis of the tip insertion is presented followed by development of a five parameter analysis of force-separation curves. In addition the effect of tip penetration on cell viability is addressed. Finally, the AFM-based microinjection system is used to deliver liquids into individual adherent cells. Microinjection into the cytoplasm, but not into the nucleus is demonstrated. The experiments study possible system leakage, clogging of the tip opening with cell residues and injection parameters. Finally the probe-cell interactions during the injections are analysed.

EPFL2012

DNA Topology

Guillaume Witz

This thesis explores different aspects of DNA topology through experimental and numerical techniques. Topology is a vast mathematical field, that deals with the spatial properties of objects undergoing continuous deformations, but here it is restricted to the description of closed space curves such as rings, knots and catenanes. Topology is connected to practical problems in different domains including polymer physics, because a polymer chain is a type of space curve, whose statistical properties are affected by the closure of its ends; and biology, because the double-helical interwinding of the two strands formingDNA is described by topological concepts, and also because a closed DNA molecule can adopt complex topologies like knotted or catenated conformations in vivo. Both physical and biological aspects of topology are investigated in the three chapters of this thesis. Chapter 1 introduces on a general level the experimental techniques, numerical methods and topology concepts that are at the core of the presented research. Chapter 2 is an experimental study of the statistical properties of DNA rings adsorbed on surfaces, and imaged by atomic force microscopy (AFM). DNA rings of different sizes are first studied in a dilute state, allowing us to investigate the consequences of the circular topology on polymer physics of two-dimensional chains. We confirm certain theoretical results, e.g. the topological invariance of the scaling exponent in two dimensions; but we also propose new models taking into account the specificities of the problem, in particular the interplay between excluded volume effects and topology, for the description of standard polymer physics measures like the asphericity or the bond correlation function. In addition to isolated DNA rings, we study DNA rings in a semi-dilute state. Comparing the statistical properties of the DNA rings imaged by AFM with a numerical model, we formulate a simplified description of this system in terms of two dimensional pressurized vesicles. Finally, we study a last case, in which DNA rings are confined within circular boundaries in two dimensions. We show how confinement strongly affects the statistical properties of the DNA chains, and verify that our experimental system is a good model for polymer confinement by comparing the data with numerical simulations. In chapter 3, we use numerical simulations to study two other topological forms taken by closed DNA in vivo: catenanes and knots. In both cases, we analyze the conformations of these objects, in particular as a function of their torsional state, and find complex behaviors proper to these topologies: for example, the large scale supercoiling of toroidal catenanes, or the different response of left- and right-handed knots to torsion. We then focus on the activity of a class of enzymes called topoisomerases, that are able to change the topology of DNA molecules (e.g. by unknotting a DNA knot). In particular, we try to deduce how the structural properties of DNA knots and catenanes affect the activity of one of these enzymes called topo IV, and also simulate its activity. We show that our numerical model provides results that compare well with experimental data, and could therefore provide a basis for the design of new experiments. Finally, we present a new model for the simulation of the activity of another topoisomerase called gyrase, that is responsible for introducing supercoiling into DNA. This model includes a few essential features of the interaction between gyrase and DNA, which permit to efficiently reproduce experimental measurements.

EPFL2010

Statistical Study of the Unfolding of Multimodular Proteins and their Energy Landscape by Atomic Force Microscopy

Fabrizio Benedetti

The aim of the present thesis is to investigate several aspects of: the proteins mechanics, interprotein interactions and to study also new techniques, theoretical and technical, to obtain and analyze the force spectroscopy experiments. The first section is dedicated to the statistical properties of the unfolding forces in a chain of homomeric multimodular proteins. The basic idea of this kind of statistic is to divide the peaks observed in a force extension curve in separate groups and then analyze these groups considering their position in the force curves. In fact in a multimodular homomeric protein the unfolding force is related to the number of not yet unfolded modules (we call it "N"). Such effect yields to a linear dependence of the most probable unfolding force of a peak on ln(N). We demonstrate how such dependence can be used to extract the kinetic parameters and how, ignoring it, could lead to significant errors. Following this topic we continue with non kinetic methods that, using the resampling from the rupture forces of any peak, could reconstruct the rupture forces for all the other peaks in a chain. Then a discussion about the Monte Carlo simulation for protein pulling is present. In fact a theoretical framework for such methodology has to be introduced to understand the various simulations done. In this chapter we also introduce a methodology to study the ligand receptor interactions when we directly functionalize the AFM tip and the substrate. In fact, in many of our experiments, we see a "cloud of points" in the force vs loading rate graph. We have modeled a system composed by "N" parallel springs, and studying the distribution of forces obtained in the force vs loading rate graph we have establish a procedure to restore the kinetic parameters used. Such procedure has then been used to discuss real experiments similar to biotin-avidin interaction. In the following chapter we discuss a first order approximation of the Bell-Evans model where a more explicit form of the potential is considered. In particular the dependence of the curvature of the potential on the applied force at the minimum and at the metastable state is considered. In the well known Bell-Evans model the prefactors of the transition rate are fixed at any force, however this is not what happen in nature, where the prefactors (that are the second local derivative of the interacting energy with respect to the reaction coordinate in its minimum and maximum) depend on the force applied. The results obtained with the force spectroscopy of the Laminin-binding-protein are discussed, in particular this protein showed a phase transition when the pH was changed. The behavior of this protein changes, from a normal WLC behavior to a plateau behavior. The analysis of the force spectroscopy curves shows a distribution of length where the maximum of the first prominent peak correspond to the full length of the protein. However, length that could be associated with dimers and trymers are also present in this distribution. Later a new approach to study the lock and key mechanism, using "handles" with a specific force extension pattern, is introduced. In particular handles of (I27)3 and (I27–SNase)3 were biochemically attached to: strept-actin molecules, biotin molecules, RNase and Angiogenin. The main idea is to have a system composed by "handle-(molecule A)-(molecule B)-handle" where the handles are covalently attached to the respective molecules and the two molecules "A and B" are attached by secondary bonds. This approach allows a better recognition of the protein-protein interaction enabling us to filter out spurious events. Doing a statistic on the rupture forces and comparing this with the statistic of the detachments of the system of the bare handles, we are able to extract the information of the interaction between the molecule A and B. The two last chapters are of more preliminary character that the previous part of the thesis. A section is dedicated to the estimation of effective mass and viscous drag of the cantilevers studied by autocorrelation and noise power spectrum. Usually the noise power spectrum method is the most used, however the autocorrelation should give approximately the same information. The parameters obtained are important in high frequency modulation techniques. In fact, they are needed to interpret the results. The results of these two methods show a good agreement in the estimation of the mass and the viscous drag of the various cantilever used. Afterwards a chapter is dedicated to the discussion of the force spectroscopy experiments using a low frequency modulation of the cantilever base. Such experiments allow us to record the phase and the amplitude shift of the modulation signal used. Using the amplitude channel we managed to restore the static force signal with a lower level of noise. Moreover these signals give us direct information about the dynamic stiffness and the lose of energy in the system, information that, using the standard technique would be difficult (or even impossible) to obtain.

EPFL2012

AFM Based Single Cell Microinjection

Joanna Katarzyna Bitterli

EPFL2012

Statistical Study of the Unfolding of Multimodular Proteins and their Energy Landscape by Atomic Force Microscopy

Fabrizio Benedetti

EPFL2012

DNA Topology

Guillaume Witz

EPFL2010