Cell Biology and BiophysicsEllenberg Group
Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.
Szymborska, A., de Marco, A., Daigle, N., Cordes, V.C., Briggs, J.A. & Ellenberg, J.
Science. 2013 Aug 9;341(6146):655-8. doi: 10.1126/science.1240672. Epub 2013 Jul11.
Much of life's essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the NPC, with single particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.
Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes.
Kitajima, T.S., Ohsugi, M. & Ellenberg, J.
Cell. 2011 Aug 19;146(4):568-81. doi: 10.1016/j.cell.2011.07.031.
Chromosomes must establish stable biorientation prior to anaphase to achieve faithful segregation during cell division. The detailed process by which chromosomes are bioriented and how biorientation is coordinated with spindle assembly and chromosome congression remain unclear. Here, we provide complete 3D kinetochore-tracking datasets throughout cell division by high-resolution imaging of meiosis I in live mouse oocytes. We show that in acentrosomal oocytes, chromosome congression forms an intermediate chromosome configuration, the prometaphase belt, which precedes biorientation. Chromosomes then invade the elongating spindle center to form the metaphase plate and start biorienting. Close to 90% of all chromosomes undergo one or more rounds of error correction of their kinetochore-microtubule attachments before achieving correct biorientation. This process depends on Aurora kinase activity. Our analysis reveals the error-prone nature of homologous chromosome biorientation, providing a possible explanation for the high incidence of aneuploid eggs observed in mammals, including humans.
Live imaging of single nuclear pores reveals unique assembly kinetics and mechanism in interphase.
Dultz, E. & Ellenberg, J.
J Cell Biol. 2010 Oct 4;191(1):15-22. Epub 2010 Sep 27.
In metazoa, new nuclear pore complexes (NPCs) form at two different cell cycle stages: at the end of mitosis concomitant with the reformation of the nuclear envelope and during interphase. However, the mechanisms of these assembly processes may differ. In this study, we apply high resolution live cell microscopy to analyze the dynamics of single NPCs in living mammalian cells during interphase. We show that nuclear growth and NPC assembly are correlated and occur at a constant rate throughout interphase. By analyzing the kinetics of individual NPC assembly events, we demonstrate that they are initiated by slow accumulation of the membrane nucleoporin Pom121 followed by the more rapid association of the soluble NPC subcomplex Nup107-160. This inverse order of recruitment and the overall much slower kinetics compared with postmitotic NPC assembly support the conclusion that the two processes occur by distinct molecular mechanisms.
Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.
Neumann, B., Walter, T., Heriche, J.K., Bulkescher, J., Erfle, H., Conrad, C., Rogers, P., Poser, I., Held, M., Liebel, U., Cetin, C., Sieckmann, F., Pau, G., Kabbe, R., Wünsche, A., Satagopam, V., Schmitz, M.H., Chapuis, C., Gerlich, D.W., Schneider, R., Eils, R., Huber, W., Peters, J.M., Hyman, A.A., Durbin, R., Pepperkok, R. & Ellenberg, J.
Nature. 2010 Apr 1;464(7289):721-7.
Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.