Myosin II Activity Is Selectively Needed for Migration in Highly Confined Microenvironments in Mature Dendritic Cells

Upon infection, mature dendritic cells (mDCs) migrate from peripheral tissue to lymph nodes (LNs) to activate T lymphocytes and initiate the adaptive immune response. This fast and tightly regulated process is tuned by different microenvironmental factors, such as the physical properties of the tissue. Mechanistically, mDCs migration mostly relies on acto-myosin flow and contractility that depend on non-muscular Myosin IIA (MyoII) activity. However, the specific contribution of this molecular motor for mDCs navigation in complex microenvironments has yet to be fully established. Here, we identified a specific role of MyoII activity in the regulation of mDCs migration in highly confined microenvironments. Using microfluidic systems, we observed that during mDCs chemotaxis in 3D collagen gels under defined CCL21 gradients, MyoII activity was required to sustain their fast speed but not to orientate them toward the chemokine. Indeed, despite the fact that mDCs speed declined, these cells still migrated through the 3D gels, indicating that this molecular motor has a discrete function during their motility in this irregular microenvironment. Consistently, using microchannels of different sizes, we found that MyoII activity was essential to maintain fast cell speed specifically under strong confinement. Analysis of cell motility through micrometric holes further demonstrated that cell contractility facilitated mDCs passage only over very small gaps. Altogether, this work highlights that high contractility acts as an adaptation mechanism exhibited by mDCs to optimize their motility in restricted landscapes. Hence, MyoII activity ultimately facilitates their navigation in highly confined areas of structurally irregular tissues, contributing to the fine-tuning of their homing to LNs to initiate adaptive immune responses

Size control in mammalian cells involves modulation of both growth rate and cell cycle duration

Despite decades of research, how mammalian cell size is controlled remains unclear because of the difficulty of directly measuring growth at the single-cell level. Here we report direct measurements of single-cell volumes over entire cell cycles on various mammalian cell lines and primary human cells. We find that, in a majority of cell types, the volume added across the cell cycle shows little or no correlation to cell birth size, a homeostatic behavior called “adder”. This behavior involves modulation of G1 or S-G2 duration and modulation of growth rate. The precise combination of these mechanisms depends on the cell type and the growth condition. We have developed a mathematical framework to compare size homeostasis in datasets ranging from bacteria to mammalian cells. This reveals that a near-adder behavior is the most common type of size control and highlights the importance of growth rate modulation to size control in mammalian cells

Modifications de surfaces et intégration de MEMS pour les laboratoires sur puce

This thesis presents various applications of radical photopolymerization in microfluidic chips. In a first part, we describe the interest and importance of surface modification rendering the surface of microfluidic channels hydrophilic and neutral. We present surfaces modifications based on radical photopolymerization of polyacrylamide, first in PDMS chips, in which it is a challenge to obtain stable coatings and then on COC surface which is chemically inert. In an other application, photopolymerization is developed for MEMS in-situ implementation in microchannels. We present the integration of arrays of columns functionalized with proteins, as well as flow sensors. A flow sensor based on the elongation of a deformable structure showed a large range of measurable flow range, good sensibility and reproducibility. The second flow sensor measurement principle is based on the rotation of a structure around an axel. Although it has lower performances, its measurement does not depend on fluid's viscosity.Cette thèse présente diverses applications de la photopolymérisation radicalaire dans les puces microfluidiques. Dans un premier temps, nous décrirons l'importance des modifications de surfaces des puces microfluidiques afin de conférer à la surface un caractère hydrophile et neutre. Nous présenterons une modification de surface par photopolymérisation radicalaire in-situ de polyacrylamide pour des puces d'une part en PDMS, sur lequel la longévité des modifications de surface est difficile à obtenir, et d'autre part sur le COC qui étant inerte chimiquement, est difficilement modifiable. Dans une autre application la photopolymérisation sera effectuée en volume et nous permettra d'intégrer très simplement des MEMS, in-situ dans le microcanal. L'intégration de réseaux de colonnes fonctionnalisées avec des protéines sera présentée, ainsi que l'implémentation de deux capteurs de flux. Un capteur de flux basé sur l'élongation d'une structure déformable s'est montré très performant en terme de large gamme de mesures, de sensibilité et de reproductibilité. Le deuxième capteur de flux est basé sur la rotation d'un objet autour d'un axe. Sa mesure est indépendante de la viscosité du fluide malgré ses moindres performances

Leukocyte Migration and Deformation in Collagen Gels and Microfabricated Constrictions

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Cultivation and Imaging of S. latissima Embryo Monolayered Cell Sheets Inside Microfluidic Devices.

The culturing and investigation of individual marine specimens in lab environments is crucial to further our understanding of this highly complex ecosystem. However, the obtained results and their relevance are often limited by a lack of suitable experimental setups enabling controlled specimen growth in a natural environment while allowing for precise monitoring and in-depth observations. In this work, we explore the viability of a microfluidic device for the investigation of the growth of the alga Saccharina latissima to enable high-resolution imaging by confining the samples, which usually grow in 3D, to a single 2D plane. We evaluate the specimen's health based on various factors such as its growth rate, cell shape, and major developmental steps with regard to the device's operating parameters and flow conditions before demonstrating its compatibility with state-of-the-art microscopy imaging technologies such as the skeletonisation of the specimen through calcofluor white-based vital staining of its cell contours as well as the immunolocalisation of the specimen's cell wall. Furthermore, by making use of the on-chip characterisation capabilities, we investigate the influence of altered environmental illuminations on the embryonic development using blue and red light. Finally, live tracking of fluorescent microspheres deposited on the surface of the embryo permits the quantitative characterisation of growth at various locations of the organism.This work was supported by the CNRS, Sorbonne Université, the University of Cambridge, and, in part, by a grant from the Infinitus (China) Company Ltd (Contract Number RG82367) to G.S.K.S., by a career grant from the Swiss National Science Foundation (Grant Number P2EZP2_199843) to N.F.L., a project grant from the ARED Région Bretagne (Grant Number COH20020) to S.B., a CNRS grant (MITI “Lame Brune”) to B.C., and a project grant from the Gordon and Betty Moore Foun-dation (Symbiochip, Grant Number GBMF9333) to R.A.. The authors acknowledge open access funding by the Swiss National Science Foundation

Myosin II Activity Is Selectively Needed for Migration in Highly Confined Microenvironments in Mature Dendritic Cells

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Macropinocytosis Overcomes Directional Bias in Dendritic Cells Due to Hydraulic Resistance and Facilitates Space Exploration

International audienceThe migration of immune cells can be guided by physical cues imposed by the environment, such as geometry, rigidity, or hydraulic resistance (HR). Neutrophils preferentially follow paths of least HR in vitro, a phenomenon known as barotaxis. The mechanisms and physiological relevance of barotaxis remain unclear. We show that barotaxis results from the amplification of a small force imbalance by the actomyosin cytoskeleton, resulting in biased directional choices. In immature dendritic cells (DCs), actomyosin is recruited to the cell front to build macropinosomes. These cells are therefore insensitive to HR, as macropinocytosis allows fluid transport across these cells. This may enhance their space exploration capacity in vivo. Conversely, mature DCs down-regulate macropinocytosis and are thus barotactic. Modeling suggests that HR may help guide these cells to lymph nodes where they initiate immune responses. Hence, DCs can either overcome or capitalize on the physical obstacles they encounter, helping their immune-surveillance function