Exportins can inhibit major mitotic assembly events in vitro: membrane fusion, nuclear pore formation, and spindle assembly

Abstract

Xenopus egg extracts are a powerful in vitro tool for studying complex biological processes, including nuclear reconstitution, nuclear membrane and pore assembly, and spindle assembly. Extracts have been further used to demonstrate a moonlighting regulatory role for nuclear import receptors or importins on these cell cycle assembly events. Here we show that exportins can also play a role in these events. Addition of Crm1, Exportin-t, or Exportin-5 decreased nuclear pore assembly in vitro. RanQ69L-GTP, a constitutively active form of RanGTP, ameliorated inhibition. Both Crm1 and Exportin-t inhibited fusion of nuclear membranes, again counteracted by RanQ69L-GTP. In mitotic extracts, Crm1 and Exportin-t negatively impacted spindle assembly. Pulldowns from the extracts using Crm1-or Exportin-t-beads revealed nucleoporins known to be essential for both nuclear pore and spindle assembly, with RanQ69L-GTP decreasing a subset of these target interactions. This study suggests a model where exportins, like importins, can regulate major mitotic assembly events.

Nuclear assembly in giant unilamellar vesicles encapsulatingXenopusegg extract

“…3a and Supplementary Fig. 7), we conducted immunostaining of nuclear pore complexes (NPCs) using NPC antibodies (mAb414) [27,28]. GUVs containing a nucleus-like structure were fixed with 2.0% glutaraldehyde and stained with mAb414-Alexa Fluor 594 (mAb414-AF594; see 'Methods' for the details on immunostaining).…”

Therapeutic targeting of nuclear export and import receptors in cancer and their potential in combination chemotherapy

“…21,22 Although the mitotic roles of XPO1 are poorly characterized relative to those of KPNB1, recent literature by Nord et al illustrates that XPO1, in concert with Exportin 5 and Exportin T, has similar inhibitory effects on major mitotic events, such as membrane fusion and nuclear pore formation via interactions with various nucleoporins and RanGTP-dependent mechanism. 23 Nuclear receptors globally control the distribution of proteins across the nuclear membrane. Given the extent to which spatiotemporal localization dictates the activity of both receptor and cargo, it is not surprising that aberrant functioning of the nuclear transport machinery is associated with pathogenic disease states, such as cancer.…”

“…Phosphorylation of XPO1 serine 391 by CDK1‐cyclin B promotes the transfer of sequestered RanBP1 from KPNB1 to XPO1, thereby facilitating RanBP1 localization to kinetochores, an event which is crucial for kinetochore function after chromosomal attachment 21,22 . Although the mitotic roles of XPO1 are poorly characterized relative to those of KPNB1, recent literature by Nord et al illustrates that XPO1, in concert with Exportin 5 and Exportin T, has similar inhibitory effects on major mitotic events, such as membrane fusion and nuclear pore formation via interactions with various nucleoporins and RanGTP‐dependent mechanism 23 …”

“… 21 , 22 Although the mitotic roles of XPO1 are poorly characterized relative to those of KPNB1, recent literature by Nord et al illustrates that XPO1, in concert with Exportin 5 and Exportin T, has similar inhibitory effects on major mitotic events, such as membrane fusion and nuclear pore formation via interactions with various nucleoporins and RanGTP‐dependent mechanism. 23 …”

NUP85 alleviates lipid metabolism and inflammation by regulating PI3K/AKT signaling pathway in nonalcoholic fatty liver disease

“…Nuclear pore complexes (NPCs) are large macromolecular complexes embedded in the nuclear membrane that control the bidirectional molecular transport channels between cytoplasm and nucleus 9 , 10 . NPCs are composed of a variety of nucleoporins (NUPs), including NUP160, NUP155, and NUP85 11 , 12 . More and more evidences have confirmed that NUPs are related to lipid metabolism and inflammation of liver diseases 13 .…”