RANBP1 localizes a subset of mitotic regulatory factors on spindle microtubules and regulates chromosome segregation in human cells

Abstract

The GTPase RAN has an established role in spindle assembly and in mitotic progression, although not all mechanisms are fully understood in somatic cells. Here, we have downregulated RAN-binding protein 1 (RANBP1), a RAN partner that has highest abundance in G2 and mitosis, in human cells. RANBP1-depleted cells underwent prolonged prometaphase delay often followed by apoptosis. Cells that remained viable assembled morphologically normal spindles; these spindles, however, were hyperstable and failed to recruit cyclin B1 or to restrict the localization of HURP (DLG7), a microtubule-stabilizing factor, to plus-ends. RANBP1 depletion did not increase the frequency of unattached chromosomes; however, RANBP1-depleted cells frequently showed lagging chromosomes in anaphase, suggesting that merotelic attachments form and are not efficiently resolved. These data indicate that RANBP1 activity is required for the proper localization of specific factors that regulate microtubule function; loss of this activity contributes to the generation of aneuploidy in a microtubule-dependent manner.

RanBP1 downregulation sensitizes cancer cells to taxol in a caspase-3-dependent manner

“…Early apoptotic cells were identified from 4,6-diamidino-2-phenyl indole (DAPI) staining, revealing chromatin condensation, and immunofluorescence (IF) to lamin B1, which is cleaved during early apoptosis. We found a significant increase in apoptotic cells in cultures treated with RanBP1-specific siRNAs compared to GL2-interfered control cultures (Figure 1b), consistent with previous studies (Li et al, 2007;Tedeschi et al, 2007). We next stained the cells with a specific antibody for active caspase-3, an important regulator of apoptosis.…”

“…Having previously found that RanBP1 downregulation yields hyperstable MTs and induction of apoptosis (Tedeschi et al, 2007), we sought to gain insight into the apoptotic machinery involved. To that aim we transfected U2OS osteosarcoma cells for 48 h with two smallinterfering RNA oligonucleotides (siRNAs, indicated as 202 and 459 in the map in Figure 1a) that specifically silence the RanBP1 gene, or with an siRNA targeting the nonhuman luciferase gene (GL2).…”

“…The bulk of RanGTP localizes to chromosomes, but Ran network components also localize at specific structures of the mitotic apparatus in somatic mitotic cells and modulate the activity of MT-regulatory factors therein (reviewed by Di Fiore et al, 2004). In particular, RanBP1 associates with the spindle MTs with an accumulation at spindle poles (Di Fiore et al, 2003), and therein colocalizes with a fraction of Ran and importin-b ; all three components are also present in purified mitotic MT preparations in cosedimentation assays (Tedeschi et al, 2007). Noteworthily, genes encoding Ran and its partners are increasingly found to be aberrantly expressed in transformed and cancer cells (also see Abe et al, 2008;Kurisetty et al, 2008;Rensen et al, 2008;Xia et al, 2008a and references therein).…”

“…Roles of RanBP1 in control of MT function also emerge from RNA interference (RNAi) experiments: RanBP1-downregulated cells assemble mitotic spindles that are apparently normal but have altered properties, including resistance to MT depolymerization induced by chemical or physical agents, failure to associate with cyclin B1 and abnormal recruitment of the MT-stabilizing factor HURP (hepatoma up-regulated protein, itself viewed as a potential oncogene). HURP normally localizes to MT plus-ends and contributes to stabilize MT/kinetochore attachments (Koffa et al, 2006;Sillje et al, 2006;Wong and Fang, 2006), but it acquires a widespread distribution along the entire MT length on RanBP1 inactivation (Tedeschi et al, 2007). The resulting hyperstable MTs prolong or arrest prometaphase in video-recording experiments; many RanBP1-interfered cells actually undergo apoptosis after prolonged prometaphase delay, or progress further into mitosis and undergo inaccurate chromosome segregation (Li et al, 2007;Tedeschi et al, 2007).…”

“…HURP normally localizes to MT plus-ends and contributes to stabilize MT/kinetochore attachments (Koffa et al, 2006;Sillje et al, 2006;Wong and Fang, 2006), but it acquires a widespread distribution along the entire MT length on RanBP1 inactivation (Tedeschi et al, 2007). The resulting hyperstable MTs prolong or arrest prometaphase in video-recording experiments; many RanBP1-interfered cells actually undergo apoptosis after prolonged prometaphase delay, or progress further into mitosis and undergo inaccurate chromosome segregation (Li et al, 2007;Tedeschi et al, 2007). These features are reminiscent of the effects of taxol.…”