Macromolecules and Microsystems in Biology and Medicine (MMBM)

Keywords: lab on chips, magnetic tweezers, microfluidics, homologous recombination, chromatin, protein assays, mutation screening

Read the scientific activity report. (pdf 1,7Mo, last update 19th, april 2010)

MMBM is an interdisciplinary group of about 30 persons, dedicated to the application of physics and chemistry to biology and medicine. We develop both original methodologies for the study of DNA-protein transactions.
involved in cancer, and diagnosis methods directly relevant to cancer, such as mutation analysis, or the search and sorting of cancer cells. The group is also involved in the development of diagnosis tools for other types of pathologies, such as Alzheimer's disease, or infectious diseases.

Our group has three main lines of research :
The first is the development of bioanalytical tools and methods. Our group is a pioneer in microfluidics and lab on chips, developing in the area innovative technologies: magnetic and convective self-assembly, flow control, non-conventional microfabrication strategies and surface treatments, high throughput droplet microfluidics. Using these technologies, our group is developing several diagnosis-oriented projects in collaboration with clinicians, e.g.:

• Development of new media and strategies for mutation analysis (now in use in routine in several hospitals in France).
• Capture and molecular typing of tumour cells from patients, for the evaluation of metastatic relapse and treatment orientation. We are the coordinator of the European Project Caminems (cf charte d'aide à la lecture : un acronyme qui se lit comme un mot, sans l'épeler, s'écrit en minuscules) on this topic, and collaborating with several hospitals and research groups in France and abroad.
• Early diagnosis of neurodegenerative diseases (prion diseases, Alzheimer) by microfluidic methods, within the European consortium NeuroTAS
• Original systems for the oriented culture of neurones, and the study of neurons degenerescence
• Portable “point of care” microfluidic device for fast genetic analysis of pathogens, and the diagnosis of nosocomial infections (ANR project “Redloc”)

Fig. 1 

Our group is also involved in fundamental studies of molecular motors DNA-protein interactions and protein-protein interactions, at the single molecule and single cell level, developing for that novel nanomanipulation instruments. Currently we focus on the mechanisms of homologous recombination at the single molecule level, the organization and structural plasticity of chromatin, also studied by single molecule nanomanipulation, and the study of single molecular motors and trafficking at the single organelle level in vivo.

Fig. 2 

We also pursue projects using micrometric colloids to explore and engineer various biological cell or tissue functions and properties as T-cell activation. This cell plays a central role in mammalian immune response or single cell and collective bacterial adhesion which raises important public health problems.

In the next years, the group plans to intensify these research lines, with a particular focus on cell biology and cellular diagnosis.

Last update: June 2010

Key publications

2010

• Paglia LL, Laugé A, Weber J, Champ J, Cavaciuti E, Russo A, Viovy JL, Stoppa-Lyonnet D.
  ATM germline mutations in women with familial breast cancer and a relative with haematological malignancy
  Breast cancer research and treatment, 119(2):443-52 - Abstract
  Biallelic inactivation of the ATM gene causes ataxia-telangiectasia (A-T), a complex neurological disease associated with a high risk of leukaemias and lymphomas. Mothers of A-T children, obligate ATM heterozygote mutation carriers, have a breast cancer (BC) relative risk of about 3. The frequency of ATM carriers in BC women with a BC family history has been estimated to be 2.70%. To further our clinical understanding of familial BC and examine whether haematological malignancies are predictive of ATM germline mutation, we estimated the frequency of heterozygote mutation carriers in a series of 122 BC women with a family history of both BC and haematological malignancy and without BRCA1/2 mutation. The gene screening was performed with a new high throughput method, EMMA (enhanced mismatch mutation analysis). Amongst 28 different ATM variants, eight mutations have been identified in eight patients: two mutations leading to a putative truncated protein and six being likely deleterious mutations. One of the truncating mutations was initially interpreted as a missense mutation, p.Asp2597Tyr, but is actually a splice mutation (c.7789G>T/p.Asp2597_Lys2643>LysfsX3). The estimated frequency of ATM heterozygote mutation carriers in our series is 6.56% (95% CI: 2.16-10.95), a significantly higher figure than that observed in the general population, estimated to be between 0.3 and 0.6%. Although a trend towards an increased frequency of ATM carriers was observed, it was not different from that observed in a population of familial BC women not selected for haematological malignancy as the frequency of ATM carriers was 2.70%, a value situated in the confidence interval of our study.

2009

• Carpentier B, Pierobon P, Hivroz C, Henry N.
  T-cell artificial focal triggering tools: linking surface interactions with cell response
  PLoS One. 2009, 4(3): e4784 - Abstract
  -cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy.
• Attia R, Pregibon DC, Doyle PS, Viovy JL, Bartolo D
  Soft microflow sensors
  Lab on a chip, 9(9):1213-8 - Abstract
  We present a rapid prototyping method for integrating functional components in conventional PDMS microfluidic devices. We take advantage of stop-flow lithography (D. Dendukuri, S. S. Gu, D. C. Pregibon, T. A. Hatton and P. S. Doyle, Lab Chip, 2007, 7, 818)1 to achieve the in situ fabrication of mobile and deformable elements with a controlled mechanical response. This strategy is applied to the fabrication of microflow sensors based on a deformable spring-like structure. We show that these sensors have a large dynamic range, typically 3 to 4 orders of magnitude, and that they can be scaled down to measure flows in the nl per min range. We prepared sensors with different geometries, and their flow-elongation characteristics were modeled with a simple hydrodynamic model, with good agreement between model and experiments.

2008

• Roos WH, Campàs O, Montel F, Woehlke G, Spatz JP, Bassereau P, Cappello G
  Dynamic kinesin-1 clustering on microtubules due to mutual attractive interactions
  Phys. Biol., (5) :046004 - Abstract
  Molecular motors often work collectively inside the cell. While the properties of individual motors have been extensively studied over the last decade, much less is known on how motors coordinate their action when working in ensembles. The motor collective behaviour in conditions where they contact each other, as in intracellular transport, may strongly depend on their mutual interactions. In particular, mutual interactions may result in motor clustering without the need of additional proteins. Here we study the interactions between kinesin-1 molecules by analysing their attachment/detachment kinetics on microtubules in the absence of motor motion. Our in vitro experiments show that kinesins-1 remain longer attached to the microtubule in the presence of neighbouring motors, resulting in the formation of motor clusters. Numerical simulations of the motor attachment/detachment dynamics show that the presence of attractive interactions between motors quantitatively accounts for the experimental observations. From the comparison of the numerical results and the experimental data we estimate the interaction energy between kinesin-1 molecules to be 1.6 +/- 0.5K(B)T. The existence of attractive interactions between kinesins-1 provides a new insight into the coordination mechanism between motor proteins and may be crucial to understand the large scale traffic in cells.
• Beloin C, Houry A, Froment M, Ghigo JM, Henry N
  A Short-Time Scale Colloidal System Reveals Early Bacterial Adhesion Dynamic
  PLoS Biology, 6(7), e167 - Abstract
  The development of bacteria on abiotic surfaces has important public health and sanitary consequences. However, despite several decades of study of bacterial adhesion to inert surfaces, the biophysical mechanisms governing this process remain poorly understood, due, in particular, to the lack of methodologies covering the appropriate time scale. Using micrometric colloidal surface particles and flow cytometry analysis, we developed a rapid multiparametric approach to studying early events in adhesion of the bacterium Escherichia coli. This approach simultaneously describes the kinetics and amplitude of early steps in adhesion, changes in physicochemical surface properties within the first few seconds of adhesion, and the self-association state of attached and free-floating cells. Examination of the role of three well-characterized E. coli surface adhesion factors upon attachment to colloidal surfaces-curli fimbriae, F-conjugative pilus, and Ag43 adhesin-showed clear-cut differences in the very initial phases of surface colonization for cell-bearing surface structures, all known to promote biofilm development. Our multiparametric analysis revealed a correlation in the adhesion phase with cell-to-cell aggregation properties and demonstrated that this phenomenon amplified surface colonization once initial cell-surface attachment was achieved. Monitoring of real-time physico-chemical particle surface properties showed that surface-active molecules of bacterial origin quickly modified surface properties, providing new insight into the intricate relations connecting abiotic surface physicochemical properties and bacterial adhesion. Hence, the biophysical analytical method described here provides a new and relevant approach to quantitatively and kinetically investigating bacterial adhesion and biofilm development.
• Le Nel A, Krenkova J, Kleparnik K, Smadja C, Taverna M, Viovy JL, Foret F
  On-chip tryptic digest with direct-coupling to Esi/Ms using magnetic nanoparticles
  Electrophoresis, 29 (24) : 4944-47 Nov 08
• Chabert M and Viovy JL
  Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells
  Proc Natl Acad Sci USA, 105(9):3191-6 - Abstract
  We present a purely hydrodynamic method for the high-throughput encapsulation of single cells into picoliter droplets, and spontaneous self-sorting of these droplets. Encapsulation uses a cell-triggered Rayleigh-Plateau instability in a flow-focusing geometry, and self-sorting puts to work two extra hydrodynamic mechanisms: lateral drift of deformable objects in a shear flow, and sterically driven dispersion in a compressional flow. Encapsulation and sorting are achieved on-flight in continuous flow at a rate up to 160 cells per second. The whole process is robust and cost-effective, involving no optical or electrical discrimination, active sorting, flow switching, or moving parts. Successful encapsulation and sorting of 70-80% of the injected cell population into drops containing one and only one cell, with <1% contamination by empty droplets, is demonstrated. The system is also applied to the direct encapsulation and sorting of cancerous lymphocytes from a whole blood mixture, yielding individually encapsulated cancer cells with a >10,000-fold enrichment as compared with the initial mix. The method can be implemented in simple “soft lithography” chips, allowing for easy downstream coupling with microfluidic cell biology or molecular biology protocols.
• Le Nel A, Minc N, Smadja C, Slovakova M, Bilkova Z, Peyrin JM, Viovy JL, Taverna M
  Controlled proteolysis of normal and pathological prion protein in a microfluidic chip
  Lab on a Chip, 8(2):294-301 - Abstract
  A microreactor for proteinase K (PK)-mediated protein digestion was developed as a step towards the elaboration of a fully integrated microdevice for the detection of pathological prion protein (PrP). PK-grafted magnetic beads were immobilized inside a polydimethylsiloxane (PDMS) microchannel using a longitudinal magnetic field parallel to the flow direction and a magnetic field gradient, thereby forming a matrix for enzymatic digestion. This self-organization provided uniform pore sizes, a low flow resistance and a strong reaction efficiency due to a very thin diffusion layer. The microreactor's performance was first evaluated using a model substrate, succinyl-ala-ala-ala-paranitroanilide (SAAAP). Reaction kinetics were typically accelerated a hundred-fold as compared to conventional batch reactions. Reproducibility was around 98% for on-chip experiments. This microsystem was then applied to the digestion of prion protein from brain tissues. Controlled proteolysis could be obtained by varying the on-chip flow rate, while a complete proteolysis of normal protein was achieved in only three minutes. Extracts from normal and pathological brain homogenates were finally compared and strong discrimination between normal and pathological samples was demonstrated.

2007

• Malaquin L, Kraus T, Schmid H, Wolf H.
  High-precision particle alignment through directed assembly
  Langmuir, 23, 11513-11521
• Kraus T, Malaquin L, Schmid H, Riess W, Spencer ND, Wolf H.
  Nanoparticle printing with single-particle resolution
  Nature Nanotechnology, 2, 570