We have applied a new generation of short cantilevers with high resonant frequencies to tapping mode atomic force microscopy of a process in situ. Crystal growth in the presence of protein has been imaged stably at 79 lines/s (1.6 s/image), using a 26 jim long cantilever with a spring constant of 0.66 N/rn at a tapping frequency of 90.9 kHz. This high scan speed nearly eliminated distortion in the step edge motion and allowed imaging of finer features along the step edges. Atomic force microscopy with short cantilevers therefore allows higher resolution imaging of crystal growth in space as well as time.
Understanding intermolecular forces in liquids at distances below a few nanometers is extremely important in many fields because forces on this length scale determine properties such as chemical bonding, wetting, and specific molecular recognition. However, it is difficult to measure forces over these length scales and perhaps even more difficult to understand them theoretically. The problem stems from the fact that it is hard to make simplifying approximations on these length scales. For example, continuum models usually break down because the finite size of the solvent molecules (typically several angstroms) can not be ignored. Molecular dynamics must often be used to study these systems, limiting the accessible time scales to, at most, many nanoseconds with current computing power.Atomic force microscopy (AFM) is a relatively new technique that is proving useful in the measurement of forces at these small length scales. Probably the oldest and most direct way to measure a force is through the deflection of a spring, and in many ways, the AFM is simply a much smaller version of more familiar spring based instruments that measure force such as a weight scale.
During vascular development endothelial junctions mature and vessel integrity is established to form the endothelial barrier. The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we uncover the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic, but not blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, while large blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, while cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to downregulation of NOTCH signaling. Vice versa, DLL4-induced NOTCH activation restores junctional maturation in PDE2A-deficient lymphatic endothelial cells. Our data demonstrate that PDE2A selectively modulates a crosstalk between cGMP and NOTCH signaling to finetune lymphatic development and suggest that PDE2A may be a druggable target to control lymphatic leakage and regeneration.
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