Optical microwell assay of membrane transport kinetics.
In optical single transporter recording, membranes are firmly attached to flat solid substrates containing small wells or test compartments (TC). Transport of fluorescent molecules through TC-spanning membrane patches is induced by solution change and recorded by confocal microscopy. Previously, track-etched membrane filters were used to create solid substrates containing populations of randomly distributed TCs. In this study the possibilities offered by orderly TC arrays as created by laser microdrilling were explored. A theoretical framework was developed taking the convolution of membrane transport, solution change, and diffusion into account. The optical properties of orderly TC arrays were studied and the kinetics of solution change measured. Export and import through the nuclear pore complex (NPC) was analyzed in isolated envelopes of Xenopus oocyte nuclei. In accordance with previous reports nuclear transport receptor NTF2, which binds directly to NPC proteins, was found to be translocated much faster than "inert" molecules of similar size. Unexpectedly, NXT1, a homolog of NTF2 reportedly unable to bind to NPC proteins directly, was translocated as fast as NTF2. Thus, microstructured TC arrays were shown to provide optical single transporter recording with a new basis.
D000465:Algorithms; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000493:pharmacokinetics*; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002462:Cell Membrane / Q000502:physiology*; D002463:Cell Membrane Permeability / Q000502:physiology*; D002478:Cells, Cultured; D004058:Diffusion; D019544:Equipment Failure Analysis / Q000379:methods; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods; D007700:Kinetics; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D008904:Miniaturization; D008954:Models, Biological; D029543:Nucleocytoplasmic Transport Proteins / Q000493:pharmacokinetics*; D055095:Optics and Photonics / Q000295:instrumentation; D014982:Xenopus laevis
D002352:Carrier Proteins; D029543:Nucleocytoplasmic Transport Proteins; C503613:Nutf2 protein, mouse