Visualizing Nuclear Pore Complex Assembly In Situ in Human Cells at Nanometer Resolution by Correlating Live Imaging with Electron Microscopy.

In eukaryotic cells that undergo open mitosis, nuclear pore complex assembly proceeds via two distinct pathways: postmitotic and interphase assembly. Studying both assembly processes is challenging because postmitotic assembly is fast, interphase assembly is rare and sporadic, and assembly intermediates in both pathways are very small with a diameter below 100 nm. Here, we present a protocol for studying nuclear pore complex biogenesis in situ in cultured human cells in a spatiotemporally resolved and quantitative manner by combining live imaging with three-dimensional electron microscopy. The method described here can also be applied for studying other cell cycle-associated events with high spatiotemporal resolution.