Microvasculatures-on-a-chip, i.e. in vitro models that mimic important features of microvessel networks, have gained increasing interest in recent years. Such devices have allowed investigating ...pathophysiological situations involving abnormal biophysical interactions between blood cells and vessel walls. Still, a central question remains regarding the presence, in such biomimetic systems, of the endothelial glycocalyx. The latter is a glycosaminoglycans-rich surface layer exposed to blood flow, which plays a crucial role in regulating the interactions between circulating cells and the endothelium. Here, we use confocal microscopy to characterize the layer expressed by endothelial cells cultured in microfluidic channels. We show that, under our culture conditions, endothelial cells form a confluent layer on all the walls of the circuit and display a glycocalyx that fully lines the lumen of the microchannels. Moreover, the thickness of this surface layer is found to be on the order of 600 nm, which compares well with measurements performed ex or in vivo on microcapillaries. Furthermore, we investigate how the presence of endothelial cells in the microchannels affects their hydrodynamic resistance and the near-wall motion of red blood cells. Our study thus provides an important insight into the physiological relevance of in vitro microvasculatures.
The design of original twisted charge transfer antennae in which a non‐planar geometry is enforced thanks to one or two bulky ortho‐Me substituents allows us to prepare the corresponding ultra‐bright ...TbIII and DyIII bioprobes. The brightness of the TbIII derivative compares well with that of the benchmark Tb‐Lumi4 complex. The first bio‐imaging experiments with a DyIII luminescent bioprobe are also reported.
Translation
La conception de nouvelles antennes biphényle présentant une rotation bloquée due à l′introduction de groupements méthyle encombrant en position ortho nous a permis de préparer des complexes de TbIII et de DyIII extrêmement brillants. La brillance du complexe de TbIII est du même ordre de grandeur que le celle du Tb‐Lumi4, composé de référence dans le domaine. Les premières expériences d'imagerie biologique avec un complexe de DyIII sont également décrites.
Twist and bright: The design of original twisted charge‐transfer antennae in which a non‐planar geometry is enforced due to one or two bulky ortho‐Me substituents allowed the preparation of the corresponding ultra‐bright TbIII and DyIII bioprobes. The brightness of the TbIII derivative compares well with that of the benchmark Tb‐Lumi4 complex. The first bio‐imaging experiments with a DyIII luminescent bioprobe are also reported.
Platelets float in the blood as discoid particles. Their shape is maintained by microtubules organized in a ring structure, the so-called marginal band (MB), in the periphery of resting platelets. ...Platelets are activated after vessel injury and undergo a major shape change known as disc to sphere transition. It has been suggested that actomyosin tension induces the contraction of the MB to a smaller ring. In this paper, we show that antagonistic microtubule motors keep the MB in its resting state. During platelet activation, dynein slides microtubules apart, leading to MB extension rather than contraction. The MB then starts to coil, thereby inducing the spherical shape of activating platelets. Newly polymerizing microtubules within the coiled MB will then take a new path to form the smaller microtubule ring, in concerted action with actomyosin tension. These results present a new view of the platelet activation mechanism and reveal principal mechanistic features underlying cellular shape changes.
Although live imaging of dynamic processes in platelets is a challenging task, several important observations have been published during the last 20 years. We will discuss the amazing insights that ...have been achieved, the difficulties that can be encountered as well as some questions still open and the future technical perspectives.
ABSTRACT
The phagocyte NADPH oxidase 2 (Nox2) is an enzymatic complex that is involved in innate immunity, notably via its capacity to produce toxic reactive oxygen species. Recently, a proteomic ...analysis of the constitutively active Nox2 complex, isolated from neutrophil fractions, highlighted the presence of 6‐phosphofructo‐2‐kinase (PFK‐2). The purpose of this workwas to study the relationship between PFK‐2 and NADPHoxidase in neutrophils. Data have underlined a specific association of the active phosphorylated form of PFK‐2 with Nox2 complex in stimulated neutrophils. In its active form, PFK‐2 catalyzes the production of fructose‐2, 6‐bisphosphate, which is the main allosteric activator of phosphofructo‐1‐kinase, the limiting enzyme in glycolysis. Pharmacologic inhibition of PFK‐2 phosphorylation and cell depletion in PFK‐2 by a small interfering RNA strategy led to a decrease in the glycolysis rate and a reduction in NADPH oxidase activity in stimulated cells. Surprisingly, alteration of Nox2 activity impacted the glycolysis rate, which indicated that Nox2 in neutrophils was not only required for reactive oxygen species production but was also involved in supporting the energeticmetabolismincrease thatwas induced by inflammatory conditions. PFK‐2 seems to be a strategic element that links NADPH oxidase activation and glycolysismodulation, and, as such, is proposedas a potential therapeutic target in inflammatory diseases.—Baillet, A., Hograindleur, M.‐A., El Benna, J., Grichine, A., Berthier, S., Morel, F., Paclet, M.‐H. Unexpected function of the phagocyteNADPHoxidase in supporting hyperglycolysis in stimulated neutrophils:key role of 6‐phosphofructo‐2‐ kinase. FASEB J. 31, 663–673 (2017). www.fasebj.org
During the course of our research studies aiming to combine the unique photophysical properties of lanthanide complexes and the intrinsic advantage of a nonlinear two-photon excitation, we described ...the proof-of-concept of a biphotonic multiplexing bio-imaging experiment. To that end, a new Tb-luminescent bioprobe TbL(2) was designed using the TACN-trispicolinate platform. The biphotonic imaging of T24 fixed cells stained either with TbL(2) or with the already described EuL(1) complexes is reported. Finally the biphotonic multiplexing experiment was performed using a combination of both probes using the spectral mode of the two-photon microscope.
Organisms respond to circumstances threatening the cellular protein homeostasis by activation of heat-shock transcription factors (HSFs), which play important roles in stress resistance, development, ...and longevity. Of the four HSFs in vertebrates (HSF1-4), HSF1 is activated by stress, whereas HSF2 lacks intrinsic stress responsiveness. The mechanism by which HSF2 is recruited to stress-inducible promoters and how HSF2 is activated is not known. However, changes in the HSF2 expression occur, coinciding with the functions of HSF2 in development. Here, we demonstrate that HSF1 and HSF2 form heterotrimers when bound to satellite III DNA in nuclear stress bodies, subnuclear structures in which HSF1 induces transcription. By depleting HSF2, we show that HSF1-HSF2 heterotrimerization is a mechanism regulating transcription. Upon stress, HSF2 DNA binding is HSF1 dependent. Intriguingly, when the elevated expression of HSF2 during development is mimicked, HSF2 binds to DNA and becomes transcriptionally competent. HSF2 activation leads to activation of also HSF1, revealing a functional interdependency that is mediated through the conserved trimerization domains of these factors. We propose that heterotrimerization of HSF1 and HSF2 integrates transcriptional activation in response to distinct stress and developmental stimuli.
The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers, focusing also on the role of cytoskeletal protein ...tubulin βII isotype in mitochondrial metabolism and organization. By analyzing accessibility of mitochondrial ADP, using respirometry and pyruvate kinase–phosphoenolpyruvate trapping system for ADP, we show that the apparent affinity of respiration for ADP can be directly linked to the permeability of the mitochondrial outer membrane (MOM). Previous studies have shown that MOM permeability in cardiomyocytes can be regulated by VDAC interaction with cytoskeletal protein, βII tubulin. We found that in oxidative soleus skeletal muscle the high apparent Km for ADP is associated with low MOM permeability and high expression of non-polymerized βII tubulin. Very low expression of non-polymerized form of βII tubulin in glycolytic muscles is associated with high MOM permeability for adenine nucleotides (low apparent Km for ADP).
•Mitochondrial sensitivity to ADP is dependent on the outer membrane permeability.•Free βII tubulin may regulate mitochondrial ADP sensitivity in oxidative muscles.•Polymerized βII tubulin contributes to mitochondria location in striated muscles.
In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The ...composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer.
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