Ran Localizes around the Microtubule Spindle In Vivo during Mitosis in Drosophila Embryos

Trieselmann, Nadia; Wilde, Andrew

doi:10.1016/s0960-9822(02)00934-x

Cited by 49 publications

(53 citation statements)

References 29 publications

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“…3Ba,b) (see also Kéryer et al, 2003;Ciciarello et al, 2004). This distribution is consistent with that reported in Drosophila embryos for RanL43E (a mutant mimicking GTP-bound Ran), which co-localizes with spindle MTs, whereas RanT24N (mimicking GDP-bound Ran) co-localizes with chromatin (Trieselmann and Wilde, 2002). When RANBP1-interfered mitoses were incubated with AR12, localized signals only remained concentrated at asters and spindle poles; AR12 had an otherwise diffuse distribution and failed to colocalize with the spindle MTs ( Fig.…”

Section: Journal Of Cell Science 120 (21)supporting

confidence: 86%

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

Tedeschi

Ciciarello

Mangiacasale

et al. 2007

Journal of Cell Science

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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.

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Section: Journal Of Cell Science 120 (21)supporting

confidence: 86%

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

Tedeschi

Ciciarello

Mangiacasale

et al. 2007

Journal of Cell Science

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“…ketel GFP differed from the dynamics described previously for wild-type Ketel (Trieselmann and Wilde, 2002) and rather reproduced the behavior of Nup153 (compare Supplemental Figure 3 and Figure 3C). Because importin ␤ has been reported to have higher affinity for Nup153 compared with other vertebrate FG-repeat nucleoporins (Ben-Efraim and Gerace, 2001), the increased residency of the ketel GFP fusion at NPCs, and potentially its dynamics in mitosis, may thus reflect its binding to Nup153.…”

Section: K R Katsani Et Alcontrasting

confidence: 37%

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

Katsani

Karess

Dostatni

et al. 2008

MBoC

115

128

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Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells. INTRODUCTIONIn eukaryotes, the nuclear envelope (NE) defines the limits between the nucleus and the cytoplasm. The outer NE membrane is considered to be structurally and functionally part of the endoplasmic reticulum network, whereas the inner membrane, with its distinct protein composition, provides anchoring points for the chromatin and nuclear lamina. Nuclear pore complexes (NPCs) are embedded at the points of fusion between the inner and outer NE membrane and represent the sole channels of transport across the NE. NPCs are composed of multiple copies of ϳ30 different proteins termed nucleoporins (Nups), most of which are organized into subcomplexes that associate with each other to build up the mature NPCs (for reviews, see Hetzer et al., 2005;Schwartz, 2005;Lim and Fahrenkrog, 2006;Tran and Wente, 2006). During cell division, the NE and NPCs are subjected to major rearrangements. However, the extent to which the NE and NPCs disassemble at mitotic entry varies among organisms (for reviews, see Margalit et al., 2005;Prunuske and Ullman, 2006). Unlike in most yeast and fungi, characterized by a "closed mitosis," NE disassembly is required in animal cells to allow spindle microtubule access to chromosomes. In vertebrates, cell division leads to complete NE breakdown at the prophase-prometaphase transition. During this "open mitosis," integral membrane proteins of the NE and the soluble subcomplexes of the NPCs redistribute throughout the endoplasmic reticulum and the mitotic cytoplasm (for reviews, see Hetzer et al., 2005;Margalit et al., 2005;Prunuske and Ullman, 2006). In Drosophila and Caenorhabditis elegans embryos, however, the NE only partially disassembles near spindle poles in early mitosis. NPCs disassemble during prometapha...

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“…Most cellular RCC1 in vertebrate cells remains associated with chromatin throughout the cell cycle as indicated by biochemical fractionation (Ohtsubo et al, 1989), indirect immunofluorescent localization of RCC1 (Guarguaglini et al, 2000;Moore et al, 2002), and by the localization of GFP-RCC1 in living cells Trieselmann and Wilde, 2002;Li et al, 2003). To analyze the association of RCC1 with mitotic and interphase chromatin in living cells under as physiological conditions as possible, we created a stable GFP-RCC1-expressing cell line from the tsBN2 hamster cell line that contains a point mutation in RCC1 (Ohtsubo et al, 1989).…”

Section: Resultsmentioning

confidence: 99%

The Dynamic Association of RCC1 with Chromatin Is Modulated by Ran-dependent Nuclear Transport

Cushman

Stenoien

Moore

2004

MBoC

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Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crm1/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or ␣-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo. INTRODUCTIONRegulator of chromosome condensation (RCC1) stimulates guanine nucleotide exchange by the small GTPase Ran (Bischoff and Ponstingl, 1991a). Inside the cell, RCC1 is essential for the efficient conversion of RanGDP to RanGTP. RanGTP in turn is essential for several key cellular processes, including nucleocytoplasmic transport, regulation of spindle formation and nuclear envelope reassembly at mitosis, and prevention of rereplication of DNA during S phase (Dasso, 2002;Yamaguchi and Newport, 2003).The local concentration of RanGTP is a positional cue used by nuclear import and export complexes to distinguish between the cytoplasm and nuclear interior, and this regulates the assembly and disassembly of these transport complexes in the correct compartment. RanGTP is kept high inside the nucleus by localization of RCC1 to chromatin in the nuclear interior, and low inside the cytoplasm by the RanGAP (GTPase activating protein) that is restricted to that compartment. The differential placement of these two Ran accessory factors forms a gradient of RanGTP across the nuclear pore complex essential for most nuclear transport during interphase . All nuclear carriers of the karyopherin-␤ (Kap-␤) (importin ␤) family bind RanGTP Gorlich and Kutay, 1999). Binding of RanGTP is required for Kap-␤ export carriers to simultaneously bind their cargo inside the nucleus. These export complexes disassemble after encounter with the RanGAP (and a cofactor RanBP1) in the cytoplasm and the subsequent conversion of complexed RanGTP to RanGDP. Conversely, import complexes consisting of the carrier with bound cargo assemble only in the absence of RanGTP (the cytoplasm) and disassemble upon encoun...

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Ran Localizes around the Microtubule Spindle In Vivo during Mitosis in Drosophila Embryos

Cited by 49 publications

References 29 publications

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

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

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

The Dynamic Association of RCC1 with Chromatin Is Modulated by Ran-dependent Nuclear Transport

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