NUP214/CAN Localizes on Both Sides of the Nuclear Pore Complex in Acute Myeloid Leukaemia, Indicating a Nucleoplasmic Localization of the DEK-CAN Fusion Protein.
Authors: Kraemer Doris , Thiede Christian , Filser Joerg , Fahrenkrog Birthe 
Published: April 2020

Abstract
Background. Acute myeloid leukaemia (AML) is frequently characterized by chromosomal translocations as t (15;17), t (8;21), or inversion 16. The translocation (6;9) is a less frequent translocation in 0,5–1% of all patients suffering from AML. The translocation (6;9) is characterized by fusion of the carboxyterminal part of the nuclear pore protein Nup214/CAN to the aminoterminal part of different nuclear proteins as DEK or SET. As shown by immuno electron microscopy, Nup214/CAN is localized on the cytoplasmic surface of the nuclear pore complex (NPC). The protein serves as binding site for nuclear transport factors through its phenylalanine- and glycine- (FG) rich sequence domain.
Methods. We investigated a commercial cell line (FH1-1) and myeloid blasts of five patients suffering from AML with t (6;9) by immunoelectron microscopy. As controls we used HeLa cells and HL-60 cells. For labelling of Nup214/CAN we expressed a 39-kDa internal segment of the 213,790-Da human Nup214/CAN protein in Escherichia coli and raised monospecific antibodies. The internal segment contained none of the repeat FG-sequences, which are characteristic for nucleoporins. The anti-Nup214/CAN antibody was conjugated to 8-nm colloidal gold particles. For immunoelectron microscopy frozen cells were thawed, washed with PBS, permeabilised with 0.1% Triton X-100 and immunolabelled with the conjugated antibody. After incubation cells were washed with PBS and fixed in 2% glutaraldehyde, postfixed in OsO4, dehydrated and embedded in Epon 812. Thin sections were coated on cupper grids and stained with 6% uranyl acetate and 2% lead citrate. The position of gold particles associated with the NPC was measured from electron micrographs of cross sections and for each gold particle its distance to the central plane of the NPC and from the eightfold symmetry axis of the NPC was determined.
Results. In HeLa cells and in HL-60 cells anti-Nup214/CAN labelled, as expected, the nucleoporin on the cytoplasmic surface of the NPCs. In FH1-1 cells and in myeloid blasts of all our six leukaemia patients, the Nup214/CAN antibody labelled epitopes not only on the cytoplasmic surface of the NPC, but also on the nucleoplasmic side and inside the nucleus.
Conclusions. Nup214/CAN localizes to the cytoplasmic surface of the NPC. In in vitro Nup214/CAN overexpressing cells, the protein is additionally localized at the nucleoplasmic surface of the NPC and in the nucleoplasm (Boer et al., 1997). In this abstract we show for the first time the localization of Nup214/CAN by immunoelectron microscopy in leukemia cells of five patients. In contrast to HeLa cells and HL-60 cells the protein is additionally detectable on the nucleoplasmic surface of the NPC. Since the nucleoporin is involved in nuclear transport, we speculate, that mislocalization of Nup214/CAN in the fusion protein to the nucleoplasmic surface of the pore might disturb transport equilibrium and therefore benefit cell growth in the leukaemia cells.