Automated DNA Fragments Recognition and Sizing through AFM Image Processing

This paper presents an automated algorithm to determine DNA fragment size from atomic force microscope images and to extract the molecular profiles. The sizing of DNA fragments is a widely used procedure for investigating the physical properties of individual or protein-bound DNA molecules. Several atomic force microscope (AFM) real and computer-generated images were tested for different pixel and fragment sizes and for different background noises. The automated approach minimizes processing time with respect to manual and semi-automated DNA sizing. Moreover, the DNA molecule profile recognition can be used to perform further structural analysis. For computer-generated images, the root mean square error incurred by the automated algorithm in the length estimation is 0.6% for a 7.8 nm image pixel size and 0.34% for a 3.9 nm image pixel size. For AFM real images we obtain a distribution of lengths with a standard deviation of 2.3% of mean and a measured average length very close to the real one, with an error around 0.33%

Label-free, atomic force microscopy-based mapping of DNA intrinsic curvature for the nanoscale comparative analysis of bent duplexes

We propose a method for the characterization of the local intrinsic curvature of adsorbed DNA molecules. It relies on a novel statistical chain descriptor, namely the ensemble averaged product of curvatures for two nanosized segments, symmetrically placed on the contour of atomic force microscopy imaged chains. We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature. The centrosymmetric character of the chain descriptor enables targetting strands with unknown orientation. This overcomes a remarkable limitation of the current experimental strategies that estimate curvature maps solely from the trajectories of end-labeled molecules or palindromes. As a consequence our approach paves the way for a reliable, unbiased, label-free comparative analysis of bent duplexes, aimed to detect local conformational changes of physical or biological relevance in large sample numbers. Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far. We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes

Analysis of intrinsic DNA curvature in the TP53 tumour suppressor gene using atomic force microscopy.

The research described in this thesis aimed to evaluate the intrinsic DNA curvature ofthe region of the TP53 tumour suppressor gene that codes for the sequence-specific DNA-binding domain of the p53 protein, a key protein that protects the cell from chemical insultsand tumourogenesis. There have been no previous attempts to experimentally investigate theintrinsic DNA curvature within TP53 or its relation to the functional or structural properties ofthe gene, such as DNA repair and nucleosomal architecture. The present study usedtheoretical models of TP53 in concert with an atomic force microscopy based experimentalinvestigation of TP53 DNA molecules to analyse intrinsic DNA curvature within the gene. Thiswas achieved by developing a novel software platform for the atomic force microscopy basedinvestigation of DNA curvature, named ADIPAS. Dinucleotide wedge models of DNA curvaturewere used to model TP53 in order to investigate the relationship between intrinsic DNAcurvature and the structure and function of the gene. ADIPAS was applied to atomic forcemicroscopy images of TP53 DNA molecules immobilised on a mica surface in order toexperimentally measure intrinsic DNA curvature. The experimental findings were compared totheoretical models of intrinsic curvature in TP53. The resulting intrinsic curvature profilesshowed that exons exhibited significantly lower intrinsic DNA curvature than introns withinTP53, this was also shown to be true for regions of slow DNA repair. This indicated that DNAcurvature may play a role in TP53 as a controlling factor for nucleosomal architecture tofacilitate open chromatin and active DNA transcription. The evolutionary selection for intrinsiccurvature may have played a role in the development of exons with low intrinsic DNAcurvature. Low intrinsic curvature in exon position has also been implicated in the reducedefficiency of DNA repair in a number of cancer specific mutation hotspots

Algorithmic approaches to high speed atomic force microscopy

Thesis (Ph.D.)--Boston UniversityThe atomic force microscope (AFM) has a unique set of capabilities for investigating biological systems, including sub-nanometer spatial resolution and the ability to image in liquid and to measure mechanical properties. Acquiring a high quality image, however, can take from minutes to hours. Despite this limited frame rate, researchers use the instrument to investigate dynamics via time-lapse imaging, driven by the need to understand biomolecular activities at the molecular level. Studies of processes such as DNA digestion with DNase, DNA-RNA polymerase binding and RNA transcription from DNA by RNA polymerase redefined the potential of AFM in biology. As a result of the need for better temporal resolution, advanced AFMs have been developed. The current state of the art in high-speed AFM (HS-AFM) for biological studies is an instrument developed by Toshio Ando at Kanazawa University in Japan. This instrument can achieve 12 frames/sec and has successfully visualized the motion of protein motors at the molecular level. This impressive instrument as well as other advanced AFMs, however, comes with tradeoffs that include a small scan size, limited imaging modes and very high cost. As a result, most AFM users still rely on standard commercial AFMs. The work in this thesis develops algorithmic approaches that can be implemented on existing instruments, from standard commercial systems to cutting edge HS-AFM units, to enhance their capabilities. There are four primary contributions in this thesis. The first is an analysis of the signals available in an AFM with respect to the information they carry and their suitability for imaging at different scan speeds. The next two are algorithmic approaches to HS-AFM that take advantage of these signals in different ways. The first algorithm involves a new sample profile estimator that yields accurate topology at speeds beyond the bandwidth of the limiting actuator. The second involves more efficient sampling, using the data in real time to steer the tip. Both algorithms yield at least an order of magnitude improvement in imaging rate but with different tradeoffs. The first operates beyond the bandwidth of the controller managing the tip-sample interaction and therefore the applied force is not well-regulated. The second keeps this control intact but is effective only on a limited set of samples, namely biopolymers or other string-like samples. Experiments on calibration samples and λ-DNA show that both of the algorithms improve the imaging rate by an order of magnitude. In the fourth contribution, extended applications of AFMs equipped with the algorithmic approaches are the tracking of a macromolecule moving along a string-like sample and a time optimal path for repetitive non-raster scans along string-like samples

Computational Design and Study of Structural and Dynamic Nucleic Acid Systems

abstract: DNA and RNA are generally regarded as one of the central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology witnessed the success of usage of DNA/RNA as programmable molecules to construct nano-objects with predefined shapes and dynamic molecular machines for various functions. From the perspective of structural design with nucleic acid, there are basically two types of assembly method, DNA tile based assembly and DNA origami based assembly, used to construct infinite-sized crystal structures and finite-sized molecular structures. The assembled structure can be used for arrangement of other molecules or nanoparticles with the resolution of nanometers to create new type of materials. The dynamic nucleic acid machine is based on the DNA strand displacement, which allows two nucleic acid strands to hybridize with each other to displace one or more prehybridized strands in the process. Strand displacement reaction has been implemented to construct a variety of dynamic molecular systems, such as molecular computer, oscillators, in vivo devices for gene expression control. This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.Dissertation/ThesisDoctoral Dissertation Chemistry 201

Unraveling the mechanisms of alpha-synuclein aggregation and toxicity

Parkinson’s disease is the second most common neurodegenerative disorder after Alzheimer’s disease and affects about 1% of the population over 65 years old. This disorder can be both sporadic and familial and some genetic forms are due to mutations in SNCA gene, encoding for the protein alpha-synuclein (aS). PD pathological hallmarks are the prominent death of the dopaminergic neurons in the substantia nigra pars compacta and the presence of proteins and lipid inclusions, termed Lewy’s body (LBs), in the surviving neurons in parkinsonian brains. The main constituent of LBs is an aggregated fibrillar beta-sheet rich form of aS. aS aggregation process was widely studied in the past years: the protein is unfolded in its native state, but in pathological conditions it tends to aggregate forming oligomeric species. These oligomers constitute a heterogeneous and transient ensemble and rapidly convert into amyloid fibrils when they reach a critical concentration. Amyloid fibrils then deposit in LBs along with several other proteins and lipids. aS aggregation was mainly studied in vitro, but recently more efforts were put into the study of this process in cell and animal models, to identify not only aS aggregation intermediates, but also the associated toxic mechanism(s) that lead to neurons cell death in PD. In this thesis two main issues were faced: the study of aS aggregation in cells using unconventional methods and the characterization of the effects of the family of chaperone-like proteins 14-3-3, on aS aggregation. In the first part, two cellular models for the study of aS aggregation were set and characterized: the first one is obtained just overexpressing aS and allowed the characterization of an ensemble of heterogeneous oligomeric species (about 6±4 monomers per oligomer) using a new fluorescence microscopy method termed Number and Brightness analysis. These oligomeric species induced autophagic lysosomal pathway activation and mitochondrial fragmentation in this model. The second cellular model provides a method to study aS fibrils and larger aggregates in a physiological environment: aS was overexpressed in cells and aggregation was triggered by introducing in cell cytoplasm recombinant aS fibrils fragments, termed seeds. In both cases aS overexpression and aggregation cause cellular death, in good agreement with what was previously published by others groups. The characterization of aS aggregation in cells went further looking at the variation in cellular metabolism, possibly induced by mitochondrial damage. These changes were quantified measuring NADH fluorescence properties in the two models with respect to the control. These results showed that in cells presenting aS oligomer or aggregates, NADH fluorescence lifetime and emission spectra change, suggesting that these measurements may be used to detect aS aggregates in live cells and in vivo using a non-invasive dye-free method. The second part of the thesis concerns the ability of 14-3-3 chaperone-like proteins of interacting with aS and of interfering with aS aggregation process rescuing the induced toxicity in cells. Among the seven 14-3-3 isoforms, 14-3-3 eta can re-route aS amyloidogenic process in vitro, leading to the formation of curved objects rather than aS fibrils. These curved objects have diameters and curvatures that depend on 14-3-3 eta amount in the aggregation assays; moreover, 14-3-3 eta molecules were found in these aggregates, suggesting the formation of a stable complex between the two proteins. When aS amount is too large or seeds are used to trigger the aggregation process in vitro, 14-3-3 eta is not able any more to affect aS aggregation and is sequestered into aS fibrils. In cell models, 14-3-3 eta overexpression leads to a rescue when aS was only overexpressed, but not when aggregation in cell cytoplasm was triggered by seeds. Overexpressed 14-3-3 eta was found to interact with overexpressed aS using image correlation spectroscopy methods (cross raster image correlation spectroscopy and cross Number and Brightness analysis), mainly at plasma membrane. Moreover, 14-3-3 eta is sequestered into aggregates when aS aggregation is triggered by seeds, highlighting another possible toxic mechanism due to aS aggregation. All the results obtained in cells are in good agreement with the in vitro results previously reported, further suggesting that 14-3-3 proteins and eta isoform in particular are interesting in aS aggregation frame and may be used to interfere in the process to rescue its toxic effects

Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.We thank R. Parton (Institute for Molecular Biosciences, Queensland), P. Pilch (Boston University School of Medicine) and L. Liu (Boston University School of Medicine) for kindly providing PTRFKO cells and reagents, S. Casas Tintó for kindly providing SH-Sy5y cells, P. Bassereau (Curie Institute, Paris) for kindly providing OT setup, V. Labrador Cantarero from CNIC microscopy Unit for helping with ImageJ analysis, O. Otto and M. Herbig for providing help with RTDC experiments, S. Berr and K. Gluth for technical assistance in cell culture, F. Steiniger for support in electron tomography, and A. Norczyk Simón for providing pCMV-FLAG-PTRF construct. This project received funding from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 641639; grants from the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033): SAF2014-51876-R, SAF2017-83130-R co-funded by ‘ERDF A way of making Europe’, PID2020-118658RB-I00, PDC2021-121572-100 co-funded by ‘European Union NextGenerationEU/PRTR’, CSD2009- 0016 and BFU2016-81912-REDC; and the Asociación Española Contra el Cáncer foundation (PROYE20089DELP) all to M.A.d.P. M.A.d.P. is member of the Tec4Bio consortium (ref. S2018/NMT¬4443; Comunidad Autónoma de Madrid/FEDER, Spain), co-recipient with P.R.-C. of grants from Fundació La Marató de TV3 (674/C/2013 and 201936- 30-31), and coordinator of a Health Research consortium grant from Fundación Obra Social La Caixa (AtheroConvergence, HR20-00075). M.S.-A. is recipient of a Ramón y Cajal research contract from MCIN (RYC2020-029690-I). The CNIC Unit of Microscopy and Dynamic Imaging is supported by FEDER ‘Una manera de hacer Europa’ (ReDIB ICTS infrastructure TRIMA@CNIC, MCIN). We acknowledge the support from Deutsche Forschungsgemeinschaft through grants to M.M.K. (KE685/7-1) and B.Q. (QU116/6-2 and QU116/9-1). Work in D.N. laboratory was supported by grants from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 812772 and MCIN (DPI2017-83721-P). Work in C.L. laboratory was supported by grants from Curie, INSERM, CNRS, Agence Nationale de la Recherche (ANR-17-CE13-0020-01) and Fondation ARC pour la Recherche (PGA1-RF20170205456). Work in P.R.-C. lab is funded by the MCIN (PID2019-110298GB-I00), the EC (H20 20-FETPROACT-01-2016-731957). Work in X.T. lab is funded by the MICIN (PID2021-128635NB-I00), ERC (Adv-883739) and La Caixa Foundation (LCF/PR/HR20/52400004; co-recipient with P.R.-C.). IBEC is recipient of a Severo Ochoa Award of Excellence from the MINECO. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033).S

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc1–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities

AFM applications to native cell membranes and membrane proteins

The cell membrane is a special casing necessary to keep homeostasis for cells survival. It is a two-dimensional agglomeration of lipids that holds a large number of membrane proteins with diverse vital functions. The fluid nature of the membrane makes it difficult to be handled, and requires the development of ad hoc techniques to investigate its properties and composition. In this PhD thesis, I developed a method to isolate the apical cell membrane of single cells. Taking advantage of the Atomic Force Microscope (AFM), I imaged and probed the mechanical properties of these isolated patches of membranes. I also extensively performed AFM-based single-molecule force spectroscopy to unfold the membrane proteins from the native membranes, collecting hundreds of thousands of unfolding curves. I analyzed these data with a custom software able to find the recurrent pattern of unfolding in the data, and I developed a Bayesian inference method to assign these unfolding curves to a limited number of membrane proteins. The underlying motivation of these experiments is to bring AFM technologies a step closer to an application in biomedicine. This work demonstrates that i) the cell membrane can be reliably isolated from single cells; ii) AFM can be used to characterize the membrane topography and mechanical properties of the cells of interest (e.g. I found that neural cell membranes are thicker and stiffer than membranes of brain cancer cells); iii) it is possible to record the unfolding pathways of the membrane proteins contained in the cell membranes and to identify them with the cross-matching of proteomic databases, and iv) the population of unfolding curves obtained with SMFS reflects the actual population of membrane proteins obtained with Mass Spectrometry