Forisome performance in artificial sieve tubes.

In the legume phloem, sieve element occlusion (SEO) proteins assemble into Ca(2+)-dependent contractile bodies. These forisomes presumably control phloem transport by forming reversible sieve tube plugs. This function, however, has never been directly demonstrated, and appears questionable as forisomes were reported to be too small to plug sieve tubes, and failed to block flow efficiently in artificial microchannels. Moreover, plugs of SEO-related proteins in Arabidopsis sieve tubes do not affect phloem translocation. We improved existing procedures for forisome isolation and storage, and found that the degree of Ca(2+)-driven deformation that is possible in forisomes of Vicia faba, the standard object of earlier research, has been underestimated substantially. Forisomes deform particularly strongly under reducing conditions and high sugar concentrations, as typically found in sieve tubes. In contrast to our previous inference, Ca(2+)-inducible forisome swelling certainly seems sufficient to plug sieve tubes. This conclusion was supported by 3D-reconstructions of forisome plugs in Canavalia gladiata. For a direct test, we built microfluidics chips with artificial sieve tubes. Using fluorescent dyes to visualize flow, we demonstrated the complete blockage of these biomimetic microtubes by Ca(2+)-induced forisome plugs, and concluded by analogy that forisomes are capable of regulating phloem flow in vivo.

Towards monitoring transport of single cargos across individual nuclear pore complexes by time-lapse atomic force microscopy.

A new preparation procedure was developed for the stable adsorption of either the cytoplasmic or the nuclear face of native (i.e. in physiological buffer without detergent extraction and in the absence of chemical fixatives) Xenopus oocyte nuclear envelopes (NEs) onto silicon (Si) surfaces. This yields optimal structural preservation of the nuclear pore complexes (NPCs) without compromising their functional properties. The functional viability of thus prepared NPCs was documented by time-lapse atomic force microscopy (AFM) of the reversible calcium-mediated opening (i.e. +Ca(2+)) and closing (i.e. -Ca(2+)) of the iris diaphragm-like distal ring topping the NPCs' nuclear baskets. Moreover, site-specific single colloidal gold particle detection was documented by AFM imaging one and the same NPC before and after immuno-gold labeling the sample with a nucleoporin-specific antibody. With this new preparation protocol at hand, we should eventually be able to follow by time-lapse AFM transport of single gold-conjugated cargos across individual NPCs.