Nuclear transport factors: global regulation of mitosis

Abstract

The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator - the γ-TuRC complex - and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.

Figure 1. Karyopherins and RanGTP in interphase and mitosis

(A) In interphase, RanGTP (yellow) is primarily found only in the nucleus, due to the localization of the RanGEF, RCC1 (purple), to chromatin. RanGDP (light grey) is found in the cytoplasm where the RanGAP and its accessory protein RanBP1 (both not shown) are also localized and induce RanGTP hydrolysis. (B) In mitosis, RCC1 continues to be bound to the chromatin of the mitotic chromosomes and produces a “cloud” of RanGTP, which dissociates any adjacent importin beta/SAF or transportin/SAF pairs. The freed spindle assembly factors or SAFs thus promote spindle assembly only around the mitotic chromosomes. At a distance from the chromosomes, the SAFs are held inactive by the binding of the transport receptors importin beta and transportin [9,49,52]. Thus, overall RanGTP appears to act as a spatial cue for assembly of the mitotic spindle and, later in mitosis, for assembly of the nuclear membranes and nuclear pores around chromatin. (C) For reference, the schematic shows the details of nuclear import of an NLS cargo protein by a generic importin receptor. The importin/NLS cargo complex is dissociated by nuclear RanGTP. Also shown is the export of an NES cargo protein by a generic exportin receptor. In this case, the export complex requires RanGTP as a co-factor in its formation. After export, the exportin/NES cargo/RanGTP complex is dissociated upon RanGTP hydrolysis by cytoplasmic RanGAP/RanBP1 (not shown).

Figure 2. Microtubule nucleation occurs at multiple locales within the spindle and is regulated by RanGTP in each

(A) In mitosis, microtubules have been shown to grow from γ-TuRC complexes recruited to the kinetochore by the Nup107–160 nucleoporin complex [42]. In humans, the export receptor Crm1 is also present at the kinetochore with its binding partners RanGap1, RanBP1, and RanBP2. The RanGEF RCC1 is present on mitotic chromosomes and, as described in Legend 1B, produces a gradient (“cloud”) of RanGTP around the chromosomes. RanGTP frees local SAFs from inhibition by importin beta (multiple SAFs) and transportin (the Nup107/160 complex and Elys SAFs) [9,10,49,52], such that k-fiber microtubules can grow from the kinetochores. (B) A newly discovered branching mechanism acting in spindle microtubule assembly is depicted. Here assembly can occur not only from centrosomes and kinetochores, as has long been known, but also from the sides of existing spindle microtubules. Augmin and γ-TuRC complexes are involved in the initiation of branching; RanGTP and TPX2 are also involved, but their exact mechanistic roles remain unknown [–55]. (C) Spindle microtubules also initiate strongly from the centrosomes in many instances [35], nucleated by γ-TuRC complexes. Exportin Crm1/RanGTP aids in the recruitment of pericentrin to the centrosome region, which then recruits the γ-TuRC complexes [91].

Figure 3. Multiple different arenas are regulated by karyopherins and RanGTP

(A) Spindle assembly. During normal mitotic conditions (left panel), as also described in Legend 1B, importin beta (Imp β, red) and transportin (Trn, green) bind to and inhibit Spindle Assembly Factors (inactive SAFs; grey) in areas far from chromatin. This inhibition prevents mitotic microtubule assembly at a distance from the chromosomes. A RanGTP cloud (yellow area), produced by the RanGEF RCC1 (not shown), around the mitotic chromosomes causes localized release of the SAFs from adjacent importins; the released SAFs are now active and promote spindle assembly (active SAF; blue) in the correct location, i.e.,, around the chromosomes. In extracts, as in cells, the initial balance of RanGTP, karyopherins AND SAFs determines the influence of karyopherins and Ran on spindle assembly. If excess importin beta is added (middle panel), the importin beta (red) overwhelms the amount of RanGTP produced (not shown for clarity) and sequesters even the SAFs close to the chromosomes preventing formation of a mitotic spindle. In contrast, if excess RanGTP is added (right panel, yellow), the release of SAFs from importin beta and transportin (not shown) occurs throughout the extract, independently of their position with respect to chromatin. Thus, microtubules nucleate throughout the reaction, generating not only a mitotic spindle, but abundant microtubule asters (stars). (B) Nuclear membrane assembly. Normally the coordinated action of RanGTP and importin beta promote the formation of a double nuclear membrane surrounding the chromatin during telophase (green lines; left panel). If excess importin beta is added (middle panel), this is found to prevent the fusion of the ER membrane vesicles and tubules that normally form the double nuclear membrane, resulting in unfused vesicles (green circles). This occurs presumably by inhibiting one or more “membrane assembly factors” (M-AFs), whose nature is still unknown and could be either soluble or membrane-bound. If instead too much RanGTP is added (right panel), this causes excess nuclear membrane production, which appears as invaginated nuclear membranes replete with nuclear pores (light grey) around the chromatin. (C) Nuclear pore assembly. In normal conditions, cells possess a double nuclear membrane studded with nuclear pore complexes (purple cylinders; right panel). When excess importin beta is added to a pore-free nuclear assembly intermediate (containing fused nuclear membranes but no nuclear pores that has been induced by BAPTA [52]; middle panel), the excess importin beta binds to and inhibits nuclear pore complex assembly factors (NPC-AFs: purple color), and thus the nucleus remains devoid of nuclear pores. These NPC-AFs are disassembled nuclear pore proteins that act as NPC assembly factors at the end of mitosis. If, instead, excess RanGTP is included with the excess importin beta (left panel), the balance between the two is restored and nuclear pore assembly occurs as normal. Note: In (A), (B) and (C), an excess of transportin would cause the same effect that an excess of importin beta does (middle panels). (D) Spindle scaling. In nature, mitotic spindle size mirrors organismal size. For example, the spindle of the large frog, X laevis (left panel), is larger than the spindle of a smaller frog, X tropicalis (middle panel). This spindle scaling has been shown to be dependent on TPX2 levels. Indeed, adding an excess of TPX2 to X laevis spindle assembly reactions reduces the size of the X laevis spindle to that of X. tropicalis (right panel). Note: Karyopherins and RanGTP are present, but are dominated by higher TPX2 concentrations [65]. (E) Spindle positioning in the cell. The mitotic spindle orients lengthwise in a normal cell (left panel) due to the pulling forces that cortically-bound LGN and NuMA (green) generate on the astral microtubules (black lines). Inhibition of importin β, using the inhibitor importazole, causes misoriented spindles due to a loss of LGN and NuMA from the cortex (right panel).