Key Points• PI3K-C2a controls platelet membrane structure and remodeling.• PI3K-C2a is a key regulator of a basal housekeeping PI3P pool in platelets.The physiologic roles of the class II phosphoinositide 3-kinases (PI3Ks) and their contributions to phosphatidylinositol 3-monophosphate (PI3P) and PI(3,4)P 2 production remain elusive. Here we report that mice heterozygous for a constitutively kinase-dead PI3K-C2a display aberrant platelet morphology with an elevated number of barbell-shaped proplatelets, a recently discovered intermediate stage in the final process of platelet production. Platelets with heterozygous PI3K-C2a inactivation have critical defects in a-granules and membrane structure that are associated with modifications in megakaryocytes. These platelets are more rigid and unable to form filopodia after stimulation. Heterozygous PI3K-C2a inactivation in platelets led to a significant reduction in the basal pool of PI3P and a mislocalization of several membrane skeleton proteins known to control the interactions between the plasma membrane and cytoskeleton. These alterations had repercussions on the performance of platelet responses with delay in the time of arterial occlusion in an in vivo model of thrombosis and defect in thrombus formation in an ex vivo blood flow system. These data uncover a key role for PI3K-C2a activity in the generation of a basal housekeeping PI3P pool and in the control of membrane remodeling, critical for megakaryocytopoiesis and normal platelet production and function. (Blood. 2015;126(9):1128-1137

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It is well-established that, during microcontact printing (muCP) using poly(dimethylsiloxane) (PDMS)-based stamps, some unexpected siloxane fragments can be transferred from the stamp to the surface of the sample. This so-called contamination effect coexists with the delivery of the molecules constituting the ink and by this way influences the printing process. The real impact of this contamination for the muCP technique is still partially unknown. In this work, we investigate the kinetics of this contamination process through the surface characterization of both the sample and the stamp after imprinting. The way both the curing conditions of the PDMS material and the contact time influence the degree of contamination of the surface is investigated on silicon and glass substrates. We propose a cleaning process of the stamp during several hours which eliminates any trace of contamination during printing. We show that hydrophobicity recovery of PDMS surfaces after hydrophilic treatment using oxygen plasma is considerably slowed down when the PDMS material is cleaned using our procedure. Finally, by comparing cleaned and uncleaned PDMS stamps, we show the influence of contamination on the quality of muCP using fluorescent DNA molecules as an ink. Surprisingly, we observe that the amount of DNA molecules transferred during muCP is higher for the uncleaned stamp, highlighting the positive impact of the presence of low molecular weight siloxane fragments on the muCP process. This result is attributed to the better adsorption of oligonucleotides on the stamp surface in presence of these contaminating molecules.

We use single cell force spectroscopy to compare elasticity, adhesion and tether extrusion on four breast cancer cell lines with an increasing invasive potential. We perform cell attachment/detachment experiments either on fibronectin or on another cell using an Atomic Force Microscope. Our study on the membrane tether formation from cancer cells show that they are easier to extrude from aggressive invasive cells. Measured elastic modulus values confirm that more invasive cells are softer. Moreover, the adhesion force increases with the invasive potential. Our results provide a mechanical signature of breast cancer cells that correlates with their invasivity.

Micropatterning and manipulation of mammalian and bacterial cells are important in biomedical studies to perform in vitro assays and to evaluate biochemical processes accurately, establishing the basis for implementing biomedical microelectromechanical systems (bioMEMS), point-of-care (POC) devices, or organs-on-chips (OOC), which impact on neurological, oncological, dermatologic, or tissue engineering issues as part of personalized medicine. Cell patterning represents a crucial step in fundamental and applied biological studies in vitro, hence today there are a myriad of materials and techniques that allow one to immobilize and manipulate cells, imitating the 3D in vivo milieu. This review focuses on current physical cell patterning, plus chemical and a combination of them both that utilizes different materials and cutting-edge micro-nanofabrication methodologies.

Structural and mechanical mapping at the nanoscale by novel high-speed multiparametric Quantitative Imaging (QI) and PeakForce Quantitative Nanomechanical Mapping (PF-QNM) AFM modes was compared to the classical Force Volume (FV) mapping for the case of living Pseudomonas aeruginosa bacterial cells. QI and PF-QNM modes give results consistent with FV for the whole cells in terms of morphology and elastic modulus, while providing higher resolution and shorter acquisition time. As an important complement, the influence of scanning parameters on elastic modulus values was explored for small 0.2(2)μm(2) central area on top of cells. The modulus decreases with the indentation depth due to the effect of the hard cell wall, while it increases vs. tip oscillation frequency, displaying viscoelastic behaviour of the living bacterial cells. The ability of different AFM modes to follow correctly the bacteria viscoelastic behaviour at high oscillation frequency was tested.

Three-dimensional spheroids are widely used as cancer models to study tumor cell proliferation and to evaluate new anticancer drugs. Growth-induced stress (i.e., stress that persists in tumors after external loads removal) influences tumor growth and resistance to treatment. However, it is not clear whether spheroids recapitulate the tumor physical properties. Here, we demonstrated experimentally and with the support of mathematical models that, like tumors, spheroids accumulate growth-induced stress. Moreover, we found that this stress is lower in spheroids made of 5,000 cancer cells and grown for 2 days than in spheroids made of 500 cancer cells and grown for 6 days. These two culture conditions associated with different growth-induced stress levels also had different effects on the spheroid shape (using light sheet microscopy) and surface topography and stiffness (using scanning electron microscopy and atomic force microscopy). Finally, the response to irinotecan was different in the two spheroid types. Taken together, our findings bring new insights into the relationship between the spheroid physical properties and their resistance to antitumor treatment that should be taken into account by the experimenters when assessing new therapeutic agents using in vitro 3D models or when comparing studies from different laboratories.

This paper reports a methodology which includes an algorithm able to move an AFM tip onto a single cell and through several cells combined with a smart strategy of cell immobilization.