XB-ART-57250Nucleus January 1, 2020; 11 (1): 178-193.
Exportins can inhibit major mitotic assembly events in vitro: membrane fusion, nuclear pore formation, and spindle assembly.
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.
PubMed ID: 32762441
PMC ID: PMC7540616
Article link: Nucleus
Species referenced: Xenopus
Genes referenced: nup133 nup98 post xpo5 xpot
GO keywords: nuclear pore
Article Images: [+] show captions
|Figure 1. Crm1, Exportin-t, and Exportin-5 inhibit nuclear pore formation in pore-free BAPTA nuclei.|
|Figure 2. The Exportins Crm1 and Exportin-t inhibit nuclear membrane fusion and can be counteracted by RanQ69L-GTP. (a) Crm1 and Exportin-t were added to extracts together with sperm chromatin interphase egg extracts and nuclear membrane fusion was assayed after 1 hour at room temperature. The primary measure of fully fused nuclear membrane was the ability to exclude TRITC-70 kDa Dextran (red); fusion is also supported by the appearance of a smooth membrane around the DNA (green, DHCC). The conditions assayed included the following addition of recombinant protein. a: 25 μM GST, b: 37.5 μM RanQ69L-GTP, c: 25 μM Transportin, d: 25 μM Transportin + 37.5 μM RanQ69L-GTP, e: 25 μM Crm1, f: 25 μM Crm1 + 37.5 μM RanQ69L-GTP, g: 25 μM Exportin-t, and h: 25 μM Exportin-t + 37.5 μM RanQ69L-GTP. Note: RanGTP alone causes abundant membrane fusion, producing a nuclear membrane with many outfoldings, which is less smooth than seen in GST alone nuclei. (b) Quantification of membrane fusion assays. The percentages shown were determined by counting at least 50 nuclei from each condition. Each experiment was repeated three times. Error bars represent the Standard Error from the Mean. Trn = Transportin, Ran = RanQ69L-GTP, Xpo-t = Exportin-t.|
|Figure 3. Crm1 inhibits spindle assembly. Recombinant Crm1 (c-g) was added to mitotic extract supplemented with rhodamine-labeled tubulin and compared to GST (a) and Transportin controls (b). Typical images for each reaction are shown. GST (A) addition did not interfere with the production of strong bipolar spindles, while 15 μM Transportin (B) strongly inhibited bipolar spindle formation. Low concentrations (1–2 μM) of Crm1 (c and d) had little effect on bipolar spindle formation. However, increasing concentrations (8–10 μM) Crm1 had an increasingly deleterious effect on bipolar spindle formation (e and f). Bipolar spindle assembly was completely inhibited by 15 μM Crm1 (g).|
|Figure 4. Spindle assembly with Exportin-t and RanGTP. Exportin-t was analyzed in mitotic extract to assess its effect on spindle assembly. (a): In control 15 μM GST conditions, 75% of the structures observed were bipolar spindles (* the remaining 25% of the structures were half spindles). (b): RanQ69L-GTP addition created an abundance of microtubule nucleations called asters (^ the remaining 55% of structures were bipolar spindles and multi-polar spindles). When 15 μM Exportin-t was added, two major phenotypes were observed, as shown in (c): 48% of the structures were very small microtubule nucleations on opposite sides of the mitotic chromatin, and (d): 52% were mitotic chromatin completely inhibited for spindle assembly. When 15 μM Exportin-t and 15 μM RanQ69L-GTP were added simultaneously, several phenotypes were observed. These percentage of structures observed were as follows: (e): a small spindle directly adjacent to mitotic chromatin (35%), (f): multipolar spindles around mitotic chromatin (16%), (g): groups of microtubule nucleations called asters, commonly seen in extracts with high RanGTP (45%), and (h): mitotic chromatin with no spindle associated (5%).|
|Figure 5. Crm1 and Exportin-t nucleoporin interactions in Xenopus egg cytosol. GST, GST-Crm1 and GST-Exportin-t were bound to glutathione beads and incubated in cytosol with or without RanQ69L-GTP. The beads were washed in PBS+0.2% NP-40. The bound proteins were eluted from the beads, resolved on a gel (lanes 2–7), then transferred and probed with the following antibodies: 414 anti-FG Nups, anti-Nup133, anti-Nup98, and anti-actin. Actin showed a clean signal for the input extract cytosol, but was not present in the GST-, GST-Crm1, or exportin-t bead pulldown lanes (data not shown). Lane 1: cytosol input (0.5 μL of interphase cytosol). Lane 2: GST. Lane 3: GST + RanQ69L-GTP. Lane 4: GST-Crm1. Lane 5: GST-Crm1 + RanQ69L-GTP. Lane 6: GST-Exportin-t. Lane 7: GST-Exportin-t + RanQ69L-GTP.|
|Figure 6. A model for Exportin action in nuclear pore assembly.|
References [+] :
Arnaoutov, Crm1 is a mitotic effector of Ran-GTP in somatic cells. 2005, Pubmed