1. Gene profile of zebrafish fin regeneration offers clues to kinetics, organization and biomechanics of basement membrane.
Nauroy P, Guiraud A, Chlasta J, Malbouyres M, Gillet B, Hughes S, Lambert E, Ruggiero F. Matrix Biology
How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.
2. Changes in nano-mechanical properties of human epidermal cornified cells depending on their proximity to the skin surface
Milani P, Chlasta J, Abdayem R, Kezic S, Haftek M. J Mol Recognit. 22 mai 2018;e2722.
During formation of the stratum corneum (SC) barrier, terminally differentiated keratinocytes continue their maturation process within the dead superficial epidermal layer. Morphological studies of isolated human corneocytes have revealed differences between cornified envelopes purified from the deep and superficial SC. We used atomic force microscopy to measure the mechanical properties of native human corneocytes harvested by tape‐stripping from different SC depths. Various conditions of data acquisition have been tested and optimized, in order to obtain exploitable and reproducible results. Probing at 200 nN allowed us to investigate the total stiffness of the cells (at 50 nm indentation) and that of the cornified envelopes (at 10 to15 nm), and lipid envelopes (at 5 to 10 nm). The obtained data indicated statistically significant differences between the superficial (more rigid) and deep (softer) corneocytes, thus confirming the existence of depth and maturation‐related morphological changes within the SC. The proposed approach can be potentially used for minimally invasive evaluation of various skin conditions such as aging, skin hydration, and pathologies linked to SC.
3. Variations in basement membrane mechanics are linked to epithelial morphogenesis
Chlasta J, Milani P, Runel G, Duteyrat JL, Arias L, Lamiré LA, Boudaoud A, Grammont M. Development 2017 : doi: 10.1242/dev.152652
The regulation of morphogenesis by the basement membrane (BM) may rely on changes in its mechanical properties. To test this, we developed an atomic force microscopy-based method to measure BM mechanical stiffness during two key processes in Drosophila ovarian follicle development. First, follicle elongation depends on epithelial cells that collectively migrate, secreting BM fibrils perpendicularly to the anteroposterior axis. Our data show that BM stiffness increases during this migration and that fibril incorporation enhances BM stiffness. In addition, stiffness heterogeneity, due to oriented fibrils, is important for egg elongation. Second, epithelial cells change their shape from cuboidal to either squamous or columnar. We prove that BM softens around the squamous cells and that this softening depends on the TGFβ pathway. We also demonstrate that interactions between BM constituents are necessary for cell flattening. Altogether, these results show that BM mechanical properties are modified during development and that, in turn, such mechanical modifications influence both cell and tissue shapes.
4. An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis
Sassi M, Ali O, Boudon F, Cloarec G, Abad U, Cellier C, Chen X, Gilles B, Milani P, Friml J, Vernoux T, Godin C, Hamant O, Traas J. Curr Biol. 2014 Oct 6;24(19):2335-42.
To control morphogenesis, molecular regulatory networks have to interfere with the mechanical properties of the individual cells of developing organs and tissues, but how this is achieved is not well known. We study this issue here in the shoot meristem of higher plants, a group of undifferentiated cells where complex changes in growth rates and directions lead to the continuous formation of new organs. Here, we show that the plant hormone auxin plays an important role in this process via a dual, local effect on the extracellular matrix, the cell wall, which determines cell shape. Our study reveals that auxin not only causes a limited reduction in wall stiffness but also directly interferes with wall anisotropy via the regulation of cortical microtubule dynamics. We further show that to induce growth isotropy and organ outgrowth, auxin somehow interferes with the cortical microtubule-ordering activity of a network of proteins, including AUXIN BINDING PROTEIN 1 and KATANIN 1. Numerical simulations further indicate that the induced isotropy is sufficient to amplify the effects of the relatively minor changes in wall stiffness to promote organogenesis and the establishment of new growth axes in a robust manner.
5. Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells
Sampathkumar A, Krupinski P, Wightman R, Milani P, Jönsson H, Boudaoud A, Hamant O, Meyerowitz EM. Elife. 2014 Apr 16;3:e01967.
Although it is a central question in biology, how cell shape controls intracellular dynamics largely remains an open question. Here, we show that the shape of Arabidopsis pavement cells creates a stress pattern that controls microtubule orientation, which then guides cell wall reinforcement. Live-imaging, combined with modeling of cell mechanics, shows that microtubules align along the maximal tensile stress direction within the cells, and atomic force microscopy demonstrates that this leads to reinforcement of the cell wall parallel to the microtubules. This feedback loop is regulated: cell-shape derived stresses could be overridden by imposed tissue level stresses, showing how competition between subcellular and supracellular cues control microtubule behavior. Furthermore, at the microtubule level, we identified an amplification mechanism in which mechanical stress promotes the microtubule response to stress by increasing severing activity. These multiscale feedbacks likely contribute to the robustness of microtubule behavior in plant epidermis.
6. Cell identities correlate with mechanical identities in plant meristems
Milani P, Mirabet Vincent, Cellier Coralie, Hamant Olivier, Pradeep Das, Arezki Boudaoud. Plant Physiol. 2014 Jun 12;165(4):1399-1408 4.
Cell differentiation has been associated with changes in mechanical stiffness in single-cell systems, yet it is unknown whether this association remains true in a multicellular context, particularly in developing tissues. In order to address such questions, we have developed a methodology, termed quantitative tandem epifluorescence and nanoindentation, wherein we sequentially determine cellular genetic identity with confocal microscopy and mechanical properties with atomic force microscopy. We have applied this approach to examine cellular stiffness at the shoot apices of Arabidopsis (Arabidopsis thaliana) plants carrying a fluorescent reporter for the CLAVATA3 (CLV3) gene, which encodes a secreted glycopeptide involved in the regulation of the centrally located stem cell zone in inflorescence and floral meristems. We found that these CLV3-expressing cells are characterized by an enhanced stiffness. Additionally, by tracking cells in young flowers before and after the onset of GREEN FLUORESCENT PROTEIN expression, we observed that an increase in stiffness coincides with this onset. This work illustrates how quantitative tandem epifluorescence and nanoindentation can reveal the spatial and temporal dynamics of both gene expression and cell mechanics at the shoot apex and, by extension, in the epidermis of any thick tissue.
7. Investigating Cell Mechanics with Atomic Force Microscopy
Slade A, Milani P, Boudaoud A, Hamant O, Berquand A, Pittenger B. Microscopy & Analysis. Avril 2013.
8. Toward Quantitative Nanomechanical Measurements on Live Cells with PeakForce QNM
Pittenger B, Slade A, Berquand A (Bruker Nano Surfaces) and Milani P, Boudaoud A, Hamant O (Ecole Normale Superieure de Lyon, France). Bruker Application Note #141.
Measuring and mapping mechanical properties of live cells is of high importance in today’s biological research. Atomic force microscopy 1 has been recognized since the mid-eighties as an excellent technique to image a wide range of samples in their near-natural environment. Although the primary function of atomic force microscopy is to generate a three-dimensional (3D) profile of th scanned surface, much more information is available through the technique. TappinMode™, which was developed in 1993, 2 prevents tip and sample damage from friction and shear forces, and allows qualitative mechanical property mapping through phase imaging. Around the same time, force spectroscopy and force volume are the most commonly used modes to quantitatively measure mechanical forces at the nanometer scale. Unfortunately, both techniques have suffered from slow acquisition speed and a lack of automated tools to analyze the hundreds of thousands of curves required for good statistics.
9. Shrinking the hammer: micromechanical approaches to morphogenesis
Milani P, Braybrook S, Boudaoud A. J. Exp.Bot. 2013 Nov;64(15):4651-62.
Morphogenesis, the remarkable process by which a developing organism achieves its shape, relies on the coordinated growth of cells, tissues, and organs. While the molecular and genetic basis of morphogenesis is starting to be unravelled, understanding shape changes is lagging behind. Actually, shape is imposed by the structural elements of the organism, and the translation of cellular activity into morphogenesis must go through these elements. Therefore, many methods have been developed recently to quantify, at cellular resolution, the properties of the main structural element in plants, the cell wall. As plant cell growth is restrained by the cell wall and powered by turgor pressure, such methods also address the quantification of turgor. These different micromechanical approaches are reviewed here, with a critical assessment of their strengths and weaknesses, and a discussion of how they can help us understand the regulation of growth and morphogenesis.
10. Structure and dynamics of the IL-2RA gene promoter revealed by modeling and Atomic Force Microscopy
Milani P, Marilley M, Sanchez-Sevilla A, Imbert J, Vaillant C, Argoul F, Egly JM, Rocca-Serra J, A. Arneodo. PLoS ONE. 2011;6(4) :e18811.
Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static.
11. In vivo analysis of local wall stiffness at the Shoot Apical Meristem in Arabidopsis using atomic force microscopy
Milani P, Gholamirad M, Traas J, Arneodo A, Boudaoud A, Argoul F, Hamant O. Plant J, 2011;67(6):1116-1123.
Whereas the morphogenesis of developing organisms is relatively well understood at the molecular level, the contribution of the mechanical properties of the cells to shape changes remains largely unknown, mainly because of the lack of quantified biophysical parameters at cellular or subcellular resolution. Here we designed an atomic force microscopy approach to investigate the elastic modulus of the outer cell wall in living shoot apical meristems (SAMs). SAMs are highly organized structures that contain the plant stem cells, and generate all of the aerial organs of the plant. Building on modeling and experimental data, we designed a protocol that is able to measure very local properties, i.e. within 40-100 nm deep into the wall of living meristematic cells. We identified three levels of complexity at the meristem surface, with significant heterogeneity in stiffness at regional, cellular and even subcellular levels. Strikingly, we found that the outer cell wall was much stiffer at the tip of the meristem (5 ± 2 MPa on average), covering the stem cell pool, than on the flanks of the meristem (1.5 ± 0.7 MPa on average). Altogether, these results demonstrate the existence of a multiscale spatialization of the mechanical properties of the meristem surface, in addition to the previously established molecular and cytological zonation of the SAM, correlating with regional growth rate distribution.
12. Atomic force microscopy experiment confirm that genomic long-range correlations reduce DNA persistence length
Milani P*, Moukhtar J*, Faivre-Moskalenko C*, Audit B, Vaillant C, Fontaine E, Mongelard F, Lavorel G, Saint-Jean P, Bouvet P, Argoul F, Arneodo A. J. Phys. Chem. 2010;114(15):5125-5143. (*co-first-authors)
Sequence dependency of DNA intrinsic bending properties has been emphasized as a possible key ingredient to in vivo chromatin organization. We use atomic force microscopy (AFM) in air and liquid to image intrinsically straight (synthetic), uncorrelated (hepatitis C RNA virus) and persistent long-range correlated (human) DNA fragments in various ionic conditions such that the molecules freely equilibrate on the mica surface before being captured in a particular conformation. 2D thermodynamic equilibrium is experimentally verified by a detailed statistical analysis of the Gaussian nature of the DNA bend angle fluctuations. We show that the worm-like chain (WLC) model, commonly used to describe the average conformation of long semiflexible polymers, reproduces remarkably well the persistence length estimates for the first two molecules as consistently obtained from (i) mean square end-to-end distance measurement and (ii) mean projection of the end-to-end vector on the initial orientation. Whatever the operating conditions (air or liquid, concentration of metal cations Mg(2+) and/or Ni(2+)), the persistence length found for the uncorrelated viral DNA underestimates the value obtained for the straight DNA. We show that this systematic difference is the signature of the presence of an uncorrelated structural intrinsic disorder in the hepatitis C virus (HCV) DNA fragment that superimposes on local curvatures induced by thermal fluctuations and that only the entropic disorder depends upon experimental conditions. In contrast, the WLC model fails to describe the human DNA conformations. We use a mean-field extension of the WLC model to account for the presence of long-range correlations (LRC) in the intrinsic curvature disorder of human genomic DNA: the stronger the LRC, the smaller the persistence length. The comparison of AFM imaging of human DNA with LRC DNA simulations confirms that the rather small mean square end-to-end distance observed, particularly for G+C-rich human DNA molecules, more likely results from a large-scale intrinsic curvature due to a persistent distribution of DNA curvature sites than from some increased flexibility.
13. Nucleosome positioning by excluding genomic energy barriers
Milani P, Chevereau G, Vaillant C, Audit B, Haftek-Terreau Z, Marilley M, Bouvet P, Argoul F, Arneodo A. Proc. Natl. Acad. Sci. USA. 2009;106(52):22257-22262.
Recent genome-wide nucleosome mappings along with bioinformatics studies have confirmed that the DNA sequence plays a more important role in the collective organization of nucleosomes in vivo than previously thought. Yet in living cells, this organization also results from the action of various external factors like DNA-binding proteins and chromatin remodelers. To decipher the code for intrinsic chromatin organization, there is thus a need for in vitro experiments to bridge the gap between computational models of nucleosome sequence preferences and in vivo nucleosome occupancy data. Here we combine atomic force microscopy in liquid and theoretical modeling to demonstrate that a major sequence signaling in vivo are high-energy barriers that locally inhibit nucleosome formation rather than favorable positioning motifs. We show that these genomic excluding-energy barriers condition the collective assembly of neighboring nucleosomes consistently with equilibrium statistical ordering principles. The analysis of two gene promoter regions in Saccharomyces cerevisiae and the human genome indicates that these genomic barriers direct the intrinsic nucleosome occupancy of regulatory sites, thereby contributing to gene expression regulation.
14. TBP binding capacity of the TATA box is associated with specific structural properties: AFM study of the IL2RA gene promoter
Milani P, Marilley M, Rocca-Serra J. Biochimie. 2007;89(4):528-533.
DNA is not only a nucleotide sequence which allows the binding of regulators but its intrinsic structural properties such as curvature and flexibility are also viewed as playing an active role in the regulation of transcription. Our combination of computer modelling and AFM imaging allow direct access to DNA curvature and flexibility. We have searched for these DNA structural features involved in transcription regulation within the IL-2Ralpha gene promoter. Investigation of these structural characteristics shows concordant results. First, in the core promoter, the region containing the functional TATA box shows intrinsic curvature associated with a peculiar distribution of flexibility. Both these inherent properties are characteristic of this region as compared with the other parts of the promoter. Second, the proximal promoter exhibits two important regions: a first one flexible and curved, followed by a segment of rigid linear DNA, each localised within one of the two Positive Regulatory Regions PRRI and PRRII respectively. Based on these observations, we propose different roles for DNA curvature and/or flexibility in promoter sequences.
15. Gradual melting of a replication origin (Schizosaccharomyces pombe ars1): in situ atomic force microscopy (AFM) analysis
Marilley M, Milani P, Rocca-Serra J, Baldacci G. Biochimie. 2007;89(4):534-541 13.
Local DNA melting is integral to fundamental processes such as replication or transcription. In vivo, these two processes do not occur on molecules free in solution but, instead, involve DNA molecules which are organized into DNA/proteins complexes. Atomic force microscopy imaging offers a possibility to look at individual molecules. It allowed us to follow the progress of local denaturation in liquid, but with the added constraints of DNA lying on a surface. We present a kinetic analysis of the mapping of the temperature-driven melting seen at a replication origin (Schizosaccharomyces pombe ars1). The results indicate an expected base composition dependency, but also a strong extremity effect. Noteworthy, a “structural” effect is clearly occurring – which is shown by the greater susceptibility of the strongly curved region present in the sequence to unwind. DNA melting, at this place, is seen to occur after an increase in the curvature amplitude and a simultaneous shift of the nucleotide sequence positioned at the apex. Because this may determine the position of the Replication Initiation (R.I.) site, the result suggests that eukaryotic replication origins, although described as possessing no consensus sequences, may well have their mechanics sustained by the properties of common structural features. Our analysis may, therefore, provide new information that will give genuine insights on how DNA molecules behave when organized into primosomes, replisomes, promoter initiation complexes, etc. and thus, be essential to better understanding the way genes function.
16. DNA structure in replication initiation: Atomic Force Microscopy in liquid and DNA modelling of the Schizosaccharomyces pombe ars1 origin
Marilley M, Milani P, Thimonier J, Rocca-Serra J. Nucleic Acid Res. 2007;35(20):6832-6845.
The replication origins (ORIs) of Schizosaccharomyces pombe, like those in most eukaryotes, are long chromosomal regions localized within A+T-rich domains. Although there is no consensus sequence, the interacting proteins are strongly conserved, suggesting that DNA structure is important for ORI function. We used atomic force microscopy in solution and DNA modelling to study the structural properties of the Spars1 origin. We show that this segment is the least stable of the surrounding DNA (9 kb), and contains regions of intrinsically bent elements (strongly curved and inherently supercoiled DNAs). The pORC-binding site co-maps with a superhelical DNA region, where the spatial arrangement of adenine/thymine stretches may provide the binding substrate. The replication initiation site (RIP) is located within a strongly curved DNA region. On pORC unwinding, this site shifts towards the apex of the curvature, thus potentiating DNA melting there. Our model is entirely consistent with the sequence variability, large size and A+T-richness of ORIs, and also accounts for the multistep nature of the initiation process, the specificity of pORC-binding site(s), and the specific location of RIP. We show that the particular DNA features and dynamic properties identified in Spars1 are present in other eukaryotic origins.
17. Stromal protein βig-h3 reprogrammes tumour microenvironment in pancreatic cancer
Goehrig D, Nigri J, Samain R, Wu Z, Cappello P, Gabiane G, Zhang X, Zhao Y, Kim IS, Chanal M, Curto R, Hervieu V, de la Fouchardière C, Novelli F, Milani P, Tomasini R, Bousquet C, Bertolino P, Hennino A.
OBJECTIVE : Pancreatic cancer is associated with an abundant stromal reaction leading to immune escape and tumour growth. This massive stroma drives the immune escape in the tumour. We aimed to study the impact of βig-h3 stromal protein in the modulation of the antitumoural immune response in pancreatic cancer.
DESIGN : We performed studies with p48-Cre;Kras, pdx1-Cre;Kras;Ink4a/Arf , pdx1-Cre;Kras; p53 mice and tumour tissues from patients with pancreatic ductal adenocarcinoma (PDA). Some transgenic mice were given injections of anti-βig-h3, anti-CD8, anti-PD1 depleting antibodies. Tumour growth as well as modifications in the activation of local immune cells were analysed by flow cytometry, immunohistochemistry and immunofluorescence. Tissue stiffness was measured by atomic force microscopy.
RESULTS : We identified βig-h3 stromal-derived protein as a key actor of the immune paracrine interaction mechanism that drives pancreatic cancer. We found that βig-h3 is highly produced by cancer-associated fibroblasts in the stroma of human and mouse. This protein acts directly on tumour-specific CD8+ T cells and F4/80 macrophages. Depleting βig-h3 in vivo reduced tumour growth by enhancing the number of activated CD8+ T cell within the tumour and subsequent apoptotic tumour cells. Furthermore, we found that targeting βig-h3 in established lesions released the tissue tension and functionally reprogrammed F4/80 macrophages in the tumour microenvironment.
CONCLUSIONS : Our data indicate that targeting stromal extracellular matrix protein βig-h3 improves the antitumoural response and consequently reduces tumour weight. Our findings present βig-h3 as a novel immunological target in pancreatic cancer.