Abstract
BackgroundRan GTPase has multiple functions during the cell division cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. The activity of Ran is determined by both its guanine nucleotide-bound state and its subcellular localization.ResultsHere, we have characterised the localisation and mobility of Ran coupled to green fluorescent protein (GFP) during the cell cycle in live human cells. Ran-GFP is nuclear during interphase and is dispersed throughout the cell during mitosis. GFP-RanQ69L, a mutant locked in the GTP-bound state, is less highly concentrated in the nucleus and associates with nuclear pore complexes within the nuclear envelope. During mitosis, GFP-RanQ69L is excluded from chromosomes and localizes to the spindle. By contrast, GFP-RanT24N, a mutant with low affinity for nucleotides, interacts relatively stably with chromatin throughout the cell cycle and is highly concentrated on mitotic chromosomes.ConclusionThese results show that Ran interacts dynamically with chromatin, nuclear pore complexes and the mitotic spindle during the cell cycle. These interactions are dependent on the nucleotide-bound state of the protein. Our data indicate that Ran-GTP generated at chromatin is highly mobile and interacts dynamically with distal structures that are involved in nuclear transport and mitotic spindle assembly.
Highlights
Ran GTPase has multiple functions during the cell division cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation
A control fusion between green fluorescent protein (GFP) and glutathione-S-transferase (GFP-GST), which was predominantly cytoplasmic in all interphase cells (Figure 1B)
The red fluorescent protein (RFP)-histone signal localised within nuclei with a dappled pattern typical of interphase chromatin, whereas nuclear GFP-RanWT displayed a diffuse localisation, suggesting that it is largely dispersed within the nucleoplasm
Summary
Ran GTPase has multiple functions during the cell division cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. The activity of Ran is determined by both its guanine nucleotide-bound state and its subcellular localization. Ran is a member of the Ras superfamily of small GTPases and has multiple roles in coordinating essential nuclear and mitotic processes in eukaryotes [1]. Like other GTPases, Ran exists in GTP-bound or GDP-bound conformations that differ in their molecular interactions. Conversion between these states requires the interaction of accessory proteins. The intrinsic GTPase activity of Ran is very low but is stimulated greatly by cytoplasmic RanGAP1 in conjunction with Ran-GTP-binding domains of RanBP1 or RanBP2, a large nucleoporin (Nup358) that is located at the cytoplasmic side of the nuclear pore complex (NPC). The spatial separation of these regulators is thought to establish a high concentration of Ran-GTP in the nucleus and a low concentration in the cytoplasm [2]
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