Marilena Ciciarello

Spatial control of mitosis by the GTPase Ran

Abstract.Mitosis is the most potentially dangerous event in the life of a cell, during which the cell genetic identity is transmitted to daughters; errors at this stage may yield aneuploid cells that can initiate a genetically unstable clone. The small GTPase Ran is the central element of a conserved signaling network that has a prominent role in mitotic regulation. Pioneering studies with amphibian oocytes indicated that Ran, in the GTP-bound form, activates factors that regulate spindle assembly and dynamics. An increasing body of data indicate higher specificity and complexity in mitotic control operated by Ran in somatic cells. Newly identified target factors of Ran operate with different specificity, and it is emerging that mitotic progression requires the precise positioning of Ran network components and effectors at specific sites of the mitotic apparatus according to a highly regulated schedule in space and time. In this review we summarize our current understanding of Ran control of mitosis and highlight the specificity of mechanisms operating in mammalian somatic cells.

Mitotic Functions of the Ran GTPase Network The Importance of Being in the Right Place at the Right Time

The Ran GTPase has important roles in nucleocytoplasmic transport, cell cycle progression, nuclear organization and nuclear envelope (NE) assembly. In this review, we discuss emerging evidence that implicate the Ran GTPase system in mitotic control in mammalian cells. Recent work indicates that members of the Ran network control two fundamental aspects of the mammalian mitotic apparatus: (i) centrosome and spindle pole function, and (ii) kinetochore function. It is also emerging that, after NE breakdown, specific Ran network components assemble in local combinations at crucial sites of the mitotic apparatus. In the light of these findings, the original notion that nucleotide-bound forms of the Ran GTPase are distributed along a unique “gradient” in mitotic cells should be re-examined. Available data also suggest that the Ran system is deregulated in certain cellular contexts: this may represent a favoring condition for the onset and propagation of mitotic errors that can predispose cells to become genetically unstable and facilitate neoplastic growth.

Importin β is transported to spindle poles during mitosis and regulates Ran-dependent spindle assembly factors in mammalian cells

Spatial control is a key issue in cell division. The Ran GTPase regulates several fundamental processes for cell life, largely acting through importin molecules. The best understood of these is protein import through the nuclear envelope in interphase, but roles in mitotic spindle assembly are also established. In mammalian cells, in which centrosomes are major spindle organizers, a link is emerging between the Ran network, centrosomes and spindle poles. Here, we show that, after nuclear envelope breakdown, importin β is transported to the spindle poles in mammalian cells. This localization is temporally regulated from prometaphase until anaphase, when importin β dissociates from poles and is recruited back around reforming nuclei. Importin β sediments with mitotic microtubules in vitro and its accumulation at poles requires microtubule integrity and dynamics in vivo. Furthermore, RNA interference-dependent inactivation of TPX2, the major Ran-dependent spindle organizer, abolishes importin β accumulation at poles. Importin β has a functional role in spindle pole organization, because overexpression yields mitotic spindles with abnormal, fragmented poles. Coexpression of TPX2 with importin β mitigates these abnormalities. Together, these results indicate that the balance between importins and spindle regulators of the TPX2 type is crucial for spindle formation. Targeting of TPX2/importin-β complexes to poles is a key aspect in Ran-dependent control of the mitotic apparatus in mammalian cells.

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

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.

The GTPase Ran: regulation of cell life and potential roles in cell transformation.

The GTPase Ran plays a crucial role in nucleo-cytoplasmic transport of tumor suppressors, proto-oncogenes, signaling molecules and transcription factors. It also plays direct roles in mitosis, through which it regulates faithful chromosome segregation and hence the generation of genetically stable cells. Ran operates through a group of effector proteins. In this review we summarize growing evidence suggesting that deregulated activity of Ran or its effectors can contribute to pathways of cell transformation and facilitate tumor progression.

The SOCS3-Independent Expression of IDO2 Supports the Homeostatic Generation of T Regulatory Cells by Human Dendritic Cells

Dendritic cells (DCs) are professional APCs that have a role in the initiation of adaptive immune responses and tolerance. Among the tolerogenic mechanisms, the expression of the enzyme IDO1 represents an effective tool to generate T regulatory cells. In humans, different DC subsets express IDO1, but less is known about the IDO1-related enzyme IDO2. In this study, we found a different pattern of expression and regulation between IDO1 and IDO2 in human circulating DCs. At the protein level, IDO1 is expressed only in circulating myeloid DCs (mDCs) and is modulated by PGE2, whereas IDO2 is expressed in both mDCs and plasmacytoid DCs and is not modulated by PGE2. In healthy subjects, IDO1 expression requires the presence of PGE2 and needs continuous transcription and translation, whereas IDO2 expression is constitutive, independent from suppressor of cytokine signaling 3 activity. Conversely, in patients suffering from inflammatory arthritis, circulating DCs express both IDO1 and IDO2. At the functional level, both mDCs and plasmacytoid DCs generate T regulatory cells through an IDO1/IDO2-dependent mechanism. We conclude that, in humans, whereas IDO1 provides an additional mechanism of tolerance induced by proinflammatory mediators, IDO2 is stably expressed in steady-state conditions and may contribute to the homeostatic tolerogenic capacity of DCs.

Indoleamine 2,3-dioxygenase 1 (IDO1) activity correlates with immune system abnormalities in multiple myeloma

BackgroundMultiple myeloma (MM) is a plasma cell malignancy with a multifaceted immune dysfunction. Indoleamine 2,3-dioxygenase 1 (IDO1) degrades tryptophan into kynurenine (KYN), which inhibits effector T cells and promote regulatory T-cell (Treg) differentiation. It is presently unknown whether MM cells express IDO1 and whether IDO1 activity correlates with immune system impairment.MethodsWe investigated IDO1 expression in 25 consecutive patients with symptomatic MM and in 7 patients with either monoclonal gammopathy of unknown significance (MGUS; n=3) or smoldering MM (SMM; n=4). IDO1-driven tryptophan breakdown was correlated with the release of hepatocyte growth factor (HGF) and with the frequency of Treg cells and NY-ESO-1-specific CD8+ T cells.ResultsKYN was increased in 75% of patients with symptomatic MM and correlated with the expansion of CD4+CD25+FoxP3+ Treg cells and the contraction of NY-ESO-1-specific CD8+ T cells. In vitro, primary MM cells promoted the differentiation of allogeneic CD4+ T cells into bona fide CD4+CD25hiFoxP3hi Treg cells and suppressed IFN-γ/IL-2 secretion, while preserving IL-4 and IL-10 production. Both Treg expansion and inhibition of Th1 differentiation by MM cells were reverted, at least in part, by d,l-1-methyl-tryptophan, a chemical inhibitor of IDO. Notably, HGF levels were higher within the BM microenvironment of patients with IDO+ myeloma disease compared with patients having IDO- MM. Mechanistically, the antagonism of MET receptor for HGF with SU11274, a MET inhibitor, prevented HGF-induced AKT phosphorylation in MM cells and translated into reduced IDO protein levels and functional activity.ConclusionsThese data suggest that IDO1 expression may contribute to immune suppression in patients with MM and possibly other HGF-producing cancers.

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

Mitotic microtubule (MT)-targeting drugs are widely used to treat cancer. The GTPase Ran regulates multiple processes, including mitotic spindle assembly, spindle pole formation and MT dynamics; Ran activity is therefore essential to formation of a functional mitotic apparatus. The RanBP1 protein, which binds Ran and regulates its interaction with effectors, is overexpressed in many cancer types. Several observations indicate that RanBP1 contributes to regulate the function of the mitotic apparatus: RanBP1 inactivation yields hyperstable MTs and induces apoptosis during mitosis, reminiscent of the effects of the MT-stabilizing drug taxol. Here we have investigated the influence of RanBP1 on spontaneous and taxol-induced apoptosis in transformed cells. We report that RanBP1 downregulation by RNA interference activates apoptosis in several transformed cell lines regardless of their p53 status, but not in the caspase-3-defective MCF-7 breast cancer cell line. Furthermore, RanBP1-interfered cells show an increased apoptotic response to taxol compared to their counterpart with normal or high RanBP1 levels, and this response is caspase-3 dependent. These results indicate that RanBP1 can modulate the outcome of MT-targeting therapeutic protocols.

The role of the immunosuppressive microenvironment in acute myeloid leukemia development and treatment

Functional interplay between acute myeloid leukemia (AML) cells and the bone marrow microenvironment is a distinctive characteristic of this hematological cancer. Indeed, a large body of evidence suggests that proliferation, survival and drug resistance of AML are sustained and modulated by the bone marrow immunosuppressive microenvironment, where both innate and adaptive immune responses are profoundly deregulated. Furthermore, the presence of a number of different immunosuppressive mechanisms results in massive immune deregulation, which causes the eventual escape from natural immune control. Modulating the immune system, as documented by 40 years of stem cell transplantation, may improve survival of AML patients, as the immune system is clearly able to recognize and attack leukemic cells. The understanding of the factors responsible for the escape from immune destruction in AML, which becomes more prominent with disease progression, is necessary for the development of innovative immunotherapeutic treatment modalities in AML.

Nuclear reformation after mitosis requires downregulation of the Ran GTPase effector RanBP1 in mammalian cells

The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.