Power laws in microrheology experiments on living cells: comparative analysis and modelling

We compare and synthesize the results of two microrheological experiments on the cytoskeleton of single cells. In the first one, the creep function J(t) of a cell stretched between two glass plates is measured after applying a constant force step. In the second one, a micrometric bead specifically bound to transmembrane receptors is driven by an oscillating optical trap, and the viscoelastic coefficient $G_e(\omega)$ is retrieved. Both $J(t)$ and $G_e(\omega)$ exhibit power law behavior: $J(t)= A(t/t_0)^\alpha$ and $\bar G_e(\omega)\bar = G_0 (\omega/\omega_0)^\alpha$, with the same exponent $\alpha\approx 0.2$. This power law behavior is very robust ; $\alpha$ is distributed over a narrow range, and shows almost no dependance on the cell type, on the nature of the protein complex which transmits the mechanical stress, nor on the typical length scale of the experiment. On the contrary, the prefactors $A_0$ and $G_0$appear very sensitive to these parameters. Whereas the exponents $\alpha$ are normally distributed over the cell population, the prefactors $A_0$ and $G_0$ follow a log-normal repartition. These results are compared with other data published in the litterature. We propose a global interpretation, based on a semi-phenomenological model, which involves a broad distribution of relaxation times in the system. The model predicts the power law behavior and the statistical repartition of the mechanical parameters, as experimentally observed for the cells. Moreover, it leads to an estimate of the largest response time in the cytoskeletal network: $\tau_m \approx 1000$ s.Comment: 47 pages, 14 figures // v2: PDF file is now Acrobat Reader 4 (and up) compatible // v3: Minor typos corrected - The presentation of the model have been substantially rewritten (p. 17-18), in order to give more details - Enhanced description of protocols // v4: Minor corrections in the text : the immersion angles are estimated and not measured // v5: Minor typos corrected. Two references were clarifie

Modifications mécaniques et biologiques induites dans des cellules en culture par application locale d'une force contrôlée

Adherent cells can control their mechanical properties in order to perform crucial biological functions, like division, migration or differenciation. It has now been proved that cells are very sensitive to the mechanical properties of their substrate, which they sense through integrins. Integrins are transmembrane proteins that link the actin cytoskeleton to the extracellular matrix through scaffolding proteins. We designed an optical tweezers setup controlled by a feedback loop, which allows the application of a constant local force via microbeads bound to the cell integrins. We can thus measure the creep function of a single cell and retrieve an estimate of its rigidity. Simultaneous fluorescence observations allow us to evaluate the impact of force application on the actin repartition within the cell. We observed that cells stiffen under force application but keep the same rheological response - the creep function still exhibits a power law behavior : J(t) = At^(alpha), in which A decreases on a long time range. Stiffening is coupled to actin recruitment both in the contacts and in the cytoskeleton networtk - up to several µm from the force application point. Stiffening and recruitment dynamics seem strongly correlated. This work presents an evaluation of the dynamics of cell stiffening under stress, which is a novel insight into the elucidation of the more general phenomenon of mechanotransduction.Les propriétés mécaniques des cellules adhérentes ont une importance capitale pour l'ensemble de leurs fonctions : division, migration, différenciation, etc. De plus, on sait désormais qu'elles sont très sensibles aux caractéristiques mécaniques de leur substrat, auquel elles sont ancrées par l'intermédiaire des intégrines. Ces récepteurs transmembranaires lient indirectement le cytosquelette d'actine intracellulaire aux protéines de la matrice extracellulaire.Nous avons conçu un dispositif de pinces optiques contrôlées par une boucle de rétroaction, qui permet d'appliquer aux cellules une force locale constante, via des microbilles liées aux intégrines.Nous pouvons ainsi mesurer la fonction de fluage de chaque cellule et en tirer une estimation de sa rigidité. Des observations simultanées en épifluorescence permettent par ailleurs d'évaluer les effets de l'application de la force sur la répartition d'actine locale.Nous avons constaté que les cellules se rigidifient sous l'application prolongée d'une force, tout en gardant le même comportement rhéologique : une fonction de fluage en loi de puissance du temps, J(t) = At^(alpha), où A décroît aux temps longs. Cette rigidification est couplée à un recrutement d'actine au niveau des contacts et au sein du réseau cytsoquelettique (jusqu'à plusieurs µm du point d'application de la force). De plus, les dynamiques de ces deux phénomènes semblent fortement corrélées. Ce travail présente une évaluation de la dynamique de renforcement cellulaire sous contrainte, et ouvre des perspectives prometteuses vers l'élucidation des phénomènes intervenant dans la mécanotransduction

Modifications mécaniques et biologiques induites dans des cellules en culture par application locale d'une force contrôlée

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Relationship between discordant response to HAART, Tregs, immune activation and low-level viraemia

Introduction: The incomplete immune recovery upon effective long-term highly active antiretroviral therapy (HAART) has been associated with increased morbidity and mortality in HIV infected patients [1]. Immune cellular activation, Tregs or very low-level viraemia has been alternatively suspected, but never investigated simultaneously [2]. Materials and Methods: We performed a cross-sectional study in 87 aviraemic patients (men=62, mean CD4+T cells=570/mm3, mean duration of HAART=12 years). Patients with at least 500 CD4+ T cells /mm3 were classified as complete immunological responders (cIR), whereas remaining patients were classified as inadequate immunological responder (iIR). Tregs were characterized based on CD4+CD25highFoxP3+phenotype using a one-step intracellular staining. Effector Tregs and terminal effectors Tregs were respectively defined as CD4+CD25+FoxP3+CD45RA-, and CD4+CD25+FoxP3+CD45RA-HLADR+phenotypes as recently described [3]. Activated T cells were identified using (i) elevated HLA-DR expression for CD4+T cells, and (ii) increased expressions of HLA-DR, or CD38, or both (HLADR+CD38+cells) for CD8+T cells. Very low-level viraemia was defined as detectable viraemia between 1 and 39 cp/mL. Univariate and multivariate analyses were performed to identify determinants of iIR. Results: Thirty-nine patients were classified as iIR, and 48 as cIR. Patients from the iIR group were significantly older (55 vs 50 years, p=0.027), and had percentages of activated CD4+ T cells, Tregs, effector Tregs and terminal effector Tregs significantly higher (5.3 vs 4%, p=0.014; 9 vs 7.5%, p=0,022; 8 vs 6.3%, p=0.01 and 1.8 vs 1.3%, p=0,033 among CD4+T cells, respectively). Neither the percentage of activated CD8+T cell nor very low-level viraemia were found to be associated with iIR. In the multivariate analysis, nadir of CD4+T cell count and percentage of Tregs were the only two parameters independently associated with iIR (OR=2.339, p=0.001, and OR=0.803, p=0.041, respectively). Conclusions: We present here the largest study investigating simultaneously immune response to long-term HAART, immune activation of CD4+ and CD8+ T cells, Tregs percentages and very low-level viraemia. Our results highlight the importance of Tregs in CD4 homeostasis. This aspect should now be prospectively explored in a large cohort of patients

Relationship between discordant response to HAART, Tregs, immune activation and low-level viraemia

Introduction: The incomplete immune recovery upon effective long-term highly active antiretroviral therapy (HAART) has been associated with increased morbidity and mortality in HIV infected patients [1]. Immune cellular activation, Tregs or very low-level viraemia has been alternatively suspected, but never investigated simultaneously [2]. Materials and Methods: We performed a cross-sectional study in 87 aviraemic patients (men=62, mean CD4+T cells=570/mm3, mean duration of HAART=12 years). Patients with at least 500 CD4+ T cells /mm3 were classified as complete immunological responders (cIR), whereas remaining patients were classified as inadequate immunological responder (iIR). Tregs were characterized based on CD4+CD25highFoxP3+phenotype using a one-step intracellular staining. Effector Tregs and terminal effectors Tregs were respectively defined as CD4+CD25+FoxP3+CD45RA-, and CD4+CD25+FoxP3+CD45RA-HLADR+phenotypes as recently described [3]. Activated T cells were identified using (i) elevated HLA-DR expression for CD4+T cells, and (ii) increased expressions of HLA-DR, or CD38, or both (HLADR+CD38+cells) for CD8+T cells. Very low-level viraemia was defined as detectable viraemia between 1 and 39 cp/mL. Univariate and multivariate analyses were performed to identify determinants of iIR. Results: Thirty-nine patients were classified as iIR, and 48 as cIR. Patients from the iIR group were significantly older (55 vs 50 years, p=0.027), and had percentages of activated CD4+ T cells, Tregs, effector Tregs and terminal effector Tregs significantly higher (5.3 vs 4%, p=0.014; 9 vs 7.5%, p=0,022; 8 vs 6.3%, p=0.01 and 1.8 vs 1.3%, p=0,033 among CD4+T cells, respectively). Neither the percentage of activated CD8+T cell nor very low-level viraemia were found to be associated with iIR. In the multivariate analysis, nadir of CD4+T cell count and percentage of Tregs were the only two parameters independently associated with iIR (OR=2.339, p=0.001, and OR=0.803, p=0.041, respectively). Conclusions: We present here the largest study investigating simultaneously immune response to long-term HAART, immune activation of CD4+ and CD8+ T cells, Tregs percentages and very low-level viraemia. Our results highlight the importance of Tregs in CD4 homeostasis. This aspect should now be prospectively explored in a large cohort of patients

Association between discordant immunological response to highly active anti-retroviral therapy, regulatory T cell percentage, immune cell activation and very low-level viraemia in HIV-infected patients

International audienceThe mechanisms sustaining the absence of complete immune recovery in HIV-infected patients upon long-term effective highly active anti-retroviral therapy (HAART) remain elusive. Immune activation, regulatory T cells (T(regs)) or very low-level viraemia (VLLV) have been alternatively suspected, but rarely investigated simultaneously. We performed a cross-sectional study in HIV-infected aviraemic subjects (mean duration of HAART: 12 years) to concomitantly assess parameters associated independently with inadequate immunological response. Patients were classified as complete immunological responders (cIR, n = 48) and inadequate immunological responders (iIR, n = 39), depending on the CD4(+) T cell count (\textgreater or \textless 500/mm(3)). Clinical and virological data (including very low-level viraemia) were collected. In parallel, immunophenotyping of CD4(+) lymphocytes, including T(reg) subsets, and CD8(+) T cells was performed. Percentages of activated CD4(+) T cells, T(regs), effector T(regs) and terminal effector T(regs) were found to be significantly elevated in iIR. Neither the percentage of activated CD8(+) T cells nor VLLV were found to be associated with iIR. In the multivariate analysis, nadir of CD4(+) T cell count and percentage of T(regs) were the only two parameters associated independently with iIR [odds ratio (OR) = 2\textperiodcentered339, P = 0\textperiodcentered001, and OR = 0\textperiodcentered803, P = 0\textperiodcentered041]. We present here the largest study investigating simultaneously the immune response to long-term HAART, activation of CD4(+) and CD8(+) T cells, T(reg) percentages and very low-level viraemia. Causative interactions between T(regs) and CD4(+) T cells should now be explored prospectively in a large patients cohort

Online self-sampling kits to screen multipartner MSM for HIV and other STIs: Participant characteristics and factors associated with kit use in the first three months of the MemoDepistages program, France, 2018

International audienc

PROFIL DES UTILISATEURS DU PREMIER KIT DE DÉPISTAGE PAR AUTOPRÉLÈVEMENT DU PROGRAMME MÉMODÉPISTAGES PROPOSÉ AUX HSH MULTIPARTENAIRES EN FRANCE EN 2018

International audienceIn 2017, the French National Authority for Health’s guidelines for HIV screening stated that men who havesex with men (MSM) should be screened every three months. This pace was defined to reduce the intervalbetween infection and diagnosis and the proportion of undiagnosed people for HIV. To support the implementationof these guidelines, a wide range of screening options were available – laboratory, anonymousscreening centers, self-administered tests, community screening– but there were fewer options for othersexually transmitted infections (STIs). To help MSM repeat the HIV screening while taking into considerationthe STIs issue, Santé publique France developed the MemoDepistages program designed for multi-partnerMSM. Advertised online in spring 2018, the program offered MSM with multiple partners a self-samplingkit (SSK) for HIV, HBV, HCV, syphilis, and gonococcal and chlamydia infections. Among the 7,158 men whowere offered the kit, 27.2% used it to make at least one of the four tests proposed. Users lived mainly in bigcities, were highly educated and frequent users of gay meeting venues. Sociodemographic factors (age,diploma) were strongly associated with using the kit. SSK was used by 30% of our sample with small variationbetween the different groups, and addressed a diverse population regarding socio-demographic and HIVscreening habits at baseline.En 2017, la Haute Autorité de Santé (HAS) a fixé à trois mois la fréquence optimale de dépistage pour le VIHdes hommes ayant des relations sexuelles avec d’autres hommes (HSH) afin de réduire le délai entre la datede l’infection et le diagnostic et la part des personnes vivant avec le VIH non diagnostiquées. Ces objectifss’appuientsur une offre variée de dépistage du VIH – laboratoires, centres de dépistages anonymes, autotests,dépistages communautaires – mais moins diversifiée pour les autres infections sexuellement transmissibles (IST).Afin d’encouragerle dépistage répété du VIH tout en tenant compte des enjeux relatifs au dépistage des autresIST, Santé publique France a construit le programme MémoDépistages à destination de HSH multipartenaires.Promu en ligne au printemps 2018, ce programme proposait un kit d’autoprélèvement pour réaliser le dépistagedu VIH, du VHB, du VHC, de la syphilis, des infections à chlamydia et à gonocoques. Parmi les 7 158 hommeséligibles auxquels un kit d’autoprélèvement a été proposé, 27,2% l’ont utilisé pour réaliser au moins l’un desquatre prélèvements proposés. Il s’agissait majoritairement d’hommes citadins, avec un haut niveau d’étude,familiers des lieux de convivialité gay. Ce sont principalement les facteurs sociodémographiques (âge, niveaud’étude) qui étaient associés à un taux élevé d’utilisation de l’autoprélèvement dans l’étude. Utilisé par près de30% des hommes auxquels il est proposé, le kit d’autoprélèvement permet d’amener au dépistage une populationdiversifiée tant en termes sociodémographiques qu’en termes de comportements face au dépistage

Spatiotemporal Analysis of Cell Response to a Rigidity Gradient: A Quantitative Study Using Multiple Optical Tweezers

We investigate the dynamic response of single cells to weak and local rigidities, applied at controlled adhesion sites. Using multiple latex beads functionalized with fibronectin, and each trapped in its own optical trap, we study the reaction in real time of single 3T3 fibroblast cells to asymmetrical tensions in the tens of pN · μm−1 range. We show that the cell feels a rigidity gradient even at this low range of tension, and over time develops an adapted change in the force exerted on each adhesion site. The rate at which force increases is proportional to trap stiffness. Actomyosin recruitment is regulated in space and time along the rigidity gradient, resulting in a linear relationship between the amount of recruited actin and the force developed independently in trap stiffness. This time-regulated actomyosin behavior sustains a constant and rigidity-independent velocity of beads inside the traps. Our results show that the strengthening of extracellular matrix-cytoskeleton linkages along a rigidity gradient is regulated by controlling adhesion area and actomyosin recruitment, to maintain a constant deformation of the extracellular matrix