In eukaryotic cells, the genetic system and its functions are separated from the remainder of the cell by a double-membraned barrier, the nuclear envelope (NE). The necessary molecular exchange between nucleus and cytoplasm is accomplished through nuclear pores, where the two membranes have fused, forming an open passage. In these pores we find an elaborate macromolecular assembly, the nuclear pore complex (NPC), consisting of about 100 proteins and glycoproteins. These are organized into specialized structures, forming aqueous channels for free diffusion of ions and proteins smaller that 9nm. and structures for directed, energy requiring transport of selected molecules. Using low voltage SEM at the IMR I have been able to visualize the 3D structure of the """"""""fishtrap"""""""" shaped NPC. This research continues the development of methods for simultaneous preservation of the lamin network, nuclear pore complexes, and the intranuclear pore connecting cable system. In recent studies, I have developed a nuclear isolation buffer that prevents the swelling of the nucleus which leads to stretching and disruption of the lamin net and the pre connecting cables. This buffer also disperses the nuclear gel to reveal the cable structures. To avoid the disruptive flattening of the nuclear envelope in preparing whole mounts, isolated nuclei will be mounted in a spherical depression. Another approach to preserve the spherical shape will be embedding of nuclear envelopes in epon and imaging of thick sections by FESEM after epon extraction and by energy filtering TEM operated at zero loss. This approach will provide information on how far the cables penetrate into the nuclear interior and whether there are any open ends which might suggest their involvement in nuclear transport
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