Elisa Dultz

Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells

During mitosis in higher eukaryotes, nuclear pore complexes (NPCs) disassemble in prophase and are rebuilt in anaphase and telophase. NPC formation is hypothesized to occur by the interaction of mitotically stable subcomplexes that form defined structural intermediates. To determine the sequence of events that lead to breakdown and reformation of functional NPCs during mitosis, we present here our quantitative assay based on confocal time-lapse microscopy of single dividing cells. We use this assay to systematically investigate the kinetics of dis- and reassembly for eight nucleoporin subcomplexes relative to nuclear transport in NRK cells, linking the assembly state of the NPC with its function. Our data establish that NPC assembly is an ordered stepwise process that leads to import function already in a partially assembled state. We furthermore find that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism.

Nuclear pore complex assembly through the cell cycle: Regulation and membrane organization

In eukaryotes, all macromolecules traffic between the nucleus and the cytoplasm through nuclear pore complexes (NPCs), which are among the largest supramolecular assemblies in cells. Although their composition in yeast and metazoa is well characterized, understanding how NPCs are assembled and form the pore through the double membrane of the nuclear envelope and how both processes are controlled still remains a challenge. Here, we summarize what is known about the biogenesis of NPCs throughout the cell cycle with special focus on the membrane reorganization and the regulation that go along with NPC assembly.

A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase

Nup133 links CENP-F, NudE/EL, and the dynein/dynactin complex to anchor centrosomes to the nuclear membrane.

Live imaging of single nuclear pores reveals unique assembly kinetics and mechanism in interphase

Recruitment of nuclear pore complex (NPC) components during interphase occurs in a different order and with slower kinetics than during postmitotic NPC assembly, suggesting the two processes are regulated by distinct mechanisms.

The Signal Peptide of the Mouse Mammary Tumor Virus Rem Protein Is Released from the Endoplasmic Reticulum Membrane and Accumulates in Nucleoli

N-terminal signal sequences mediate endoplasmic reticulum (ER) targeting and insertion of nascent secretory and membrane proteins and are, in most cases, cleaved off by signal peptidase. The mouse mammary tumor virus envelope protein and its alternative splice variant Rem have an unusually long signal sequence, which contains a nuclear localization signal. Although the envelope protein is targeted to the ER, inserted, and glycosylated, Rem has been described as a nuclear protein. Rem as well as a truncated version identical to the cleaved signal sequence have been shown to function as nuclear export factors for intron-containing transcripts. Using transiently transfected cells, we found that Rem is targeted to the ER, where the C-terminal portion is translocated and glycosylated. The signal sequence is cleaved off and accumulates in nucleoli. In a cell-free in vitro system, the generation of the Rem signal peptide depends on the presence of microsomal membranes. In vitro and in cells, the signal peptide initially accumulates in the membrane and is subsequently released into the cytosol. This release does not depend on processing by signal peptide peptidase, an intramembrane cleaving protease that can mediate the liberation of signal peptide fragments from the ER membrane. Our study suggests a novel pathway by which a signal peptide can be released from the ER membrane to fulfill a post-targeting function in a different compartment.

A glucose-starvation response regulates the diffusion of macromolecules

The organization and biophysical properties of the cytosol implicitly govern molecular interactions within cells. However, little is known about mechanisms by which cells regulate cytosolic properties and intracellular diffusion rates. Here, we demonstrate that the intracellular environment of budding yeast undertakes a startling transition upon glucose starvation in which macromolecular mobility is dramatically restricted, reducing the movement of both chromatin in the nucleus and mRNPs in the cytoplasm. This confinement cannot be explained by an ATP decrease or the physiological drop in intracellular pH. Rather, our results suggest that the regulation of diffusional mobility is induced by a reduction in cell volume and subsequent increase in molecular crowding which severely alters the biophysical properties of the intracellular environment. A similar response can be observed in fission yeast and bacteria. This reveals a novel mechanism by which cells globally alter their properties to establish a unique homeostasis during starvation. DOI: http://dx.doi.org/10.7554/eLife.09376.001

Formation of the Nuclear Envelope Permeability Barrier Studied by Sequential Photoswitching and Flux Analysis

In higher eukaryotes, the nuclear envelope breaks down during mitosis. It reforms during telophase, and nuclear import is reestablished within <10 min after anaphase onset. It is widely assumed that import functionality simultaneously leads to the exclusion of bulk cytoplasmic proteins. However, nuclear pore complex assembly is not fully completed when import capacity is regained, which raises the question of whether the transport and permeability barrier functions of the nuclear envelope are indeed coupled. In this study, we therefore analyzed the reestablishment of the permeability barrier of the nuclear envelope after mitosis in living cells by monitoring the flux of the reversibly photoswitchable fluorescent protein Dronpa from the cytoplasm into the nucleus after photoactivation. We performed many consecutive flux measurements in the same cell to directly monitor changes in nuclear envelope permeability. Our measurements at different time points after mitosis in individual cells show that contrary to the general view and despite the rapid reestablishment of facilitated nuclear import, the nuclear envelope remains relatively permeable for passive diffusion for the first 2 h after mitosis. Our data demonstrate that reformation of the permeability barrier of nuclear pore complexes occurs only gradually and is uncoupled from regaining active import functionality.

Global reorganization of budding yeast chromosome conformation in different physiological conditions.

Movement of the GAL locus to the nuclear periphery is part of a large-scale rearrangement of chromosome architecture induced by glucose withdrawal and is regulated by the activities of histone acetyltransferases and histone deacetylases.