Nanoparticles delivering drugs, disseminating cancer cells, and red blood cells (RBCs) during splenic filtration must deform and pass through the sub-micrometer and high aspect ratio interstices between the endothelial cells lining blood vessels. The dynamics of passage of particles/cells through these slit-like interstices remain poorly understood because the in vitro reproduction of slits with physiological dimensions in devices compatible with optical microscopy observations requires expensive technologies. Here, novel microfluidic PDMS devices containing high aspect ratio slits with sub-micrometer width are molded on silicon masters using a simple, inexpensive, and highly flexible method combining standard UV lithography and anisotropic wet etching. These devices enabled revealing novel modes of deformations of healthy and diseased RBCs squeezing through splenic-like slits (0.6-2 × 5-10 × 1.6-11 µm ) under physiological interstitial pressures. At the slit exit, the cytoskeleton of spherocytic RBCs seemed to be detached from the lipid membrane whereas RBCs from healthy donors and patients with sickle cell disease exhibited peculiar tips at their front. These tips disappeared much slower in patients' cells, allowing estimating a threefold increase in RBC cytoplasmic viscosity in sickle cell disease. Measurements of time and rate of RBC sequestration in the slits allowed quantifying the massive trapping of spherocytic RBCs.
In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.
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