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The role of Ran in mitosis. (A) The Drosophila syncytial embryo as a tool for understanding mitosis. In the Drosophila early embryo, the first 13 rounds of mitosis occur rapidly and take place in a shared cytoplasm. Unlike vertebrate cells, which undergo open mitosis and disassemble the nuclear envelope during mitosis, Drosophila undergoes semi-open mitosis, only disassembling the nuclear envelope at the spindle poles. Red, centrosomes; green, MTs; blue, chromosomes. In both vertebrates and Drosophila, Ran.GTP is generated in the vicinity of the chromatin, resulting in a gradient (shown in gray), which is strongest around the chromosomes and weakest at the poles and cortex. (B) Ran mediates mitotic functions via release of Spindle Assembly Factors (SAFs). During mitosis, Ran.GTP is generated around the chromosomes by the chromatin-bound RanGEF RCC1, facilitating the release of SAFs, which are otherwise sequestered by Importins (Imp-β/Imp-α). SAFs have critical roles in, amongst other things, MT anchoring to the kinetochores and centrosomes, in spindle growth from the chromatin, in MT bundling and stabilization, and in anchoring of astral MTs to the cell cortex.
Source publication
Over the last two decades, the small GTPase Ran has emerged as a central regulator of both mitosis and meiosis, particularly in the generation, maintenance, and regulation of the microtubule (MT)-based bipolar spindle. Ran-regulated pathways in mitosis bear many similarities to the well-characterized functions of Ran in nuclear transport and, as wi...
Citations
... This finding provides a possible explanation for the observation that in some organisms, such as Drosophila, branched nucleation is dependent on the Ran-GTP pathway (Chen et al., 2015), but independent of TPX2 (Verma and Maresca, 2019), although TPX2 is an important SAF in Xenopus laevis eggs with well-established (A) Schematic of the augmin complex (PDB 7SQK) and the importin-regulated microtubule binding site of the HAUS8 subunit. (B) Sequences of synthetic peptides derived from the N terminus of HAUS8 (amino acids 1-50). ...
Mitotic spindle assembly during cell division is a highly regulated process. Ran-GTP produced around chromosomes controls the activity of a multitude of spindle assembly factors by releasing them from inhibitory interaction with importins. A major consequence of Ran-GTP regulation is the local stimulation of branched microtubule nucleation around chromosomes, which is mediated by the augmin complex (composed of the eight subunits HAUS1-HAUS8), a process that is crucially important for correct spindle assembly. However, augmin is not known to be a direct target of the Ran-GTP pathway, raising the question of how its activity is controlled. Here, we present the in vitro reconstitution of Ran-GTP-regulated microtubule binding of the human augmin complex. We demonstrate that importins directly bind to augmin, which prevents augmin from binding to microtubules. Ran-GTP relieves this inhibition. Therefore, the augmin complex is a direct target of the Ran-GTP pathway, suggesting that branching microtubule nucleation is directly regulated by the Ran-GTP gradient around chromosomes in dividing cells.
... It has been shown that the Ran GTP concentration in the nucleus controls mitotic progression. The cell's microtubule system is modified during mitosis to generate the spindle apparatus [59]. Ran GTP has been demonstrated to induce the production of microtubule asters surrounding centrosomes in vitro, whereas RanT24N, which is predominantly coupled to GDP, lacks this capacity [60]. ...
... Ran GTP has been demonstrated to induce the production of microtubule asters surrounding centrosomes in vitro, whereas RanT24N, which is predominantly coupled to GDP, lacks this capacity [60]. In addition, mutations in RCC1 and RanBP1 have been reported to cause cell cycle arrest due to microtubule misalignment [17,59]. Normal conditions result in the formation of a bipolar spindle in cells with duplicate centrosomes, which is characterized by the nucleation of numerous longer microtubules by the centrosomes, which have a strong affinity for chromosomes and are therefore oriented towards them until only a bipolar spindle remains [61,62]. ...
Ran is a member of the Ras superfamily of proteins, which primarily regulates nucleocytoplasmic trafficking and mediates mitosis by regulating spindle formation and nuclear envelope (NE) reassembly. Therefore, Ran is an integral cell fate determinant. It has been demonstrated that aberrant Ran expression in cancer is a result of upstream dysregulation of the expression of various factors, such as osteopontin (OPN), and aberrant activation of various signaling pathways, including the extracellular-regulated kinase/mitogen-activated protein kinase (ERK/MEK) and phosphatidylinositol 3-kinase/Protein kinase B (PI3K/Akt) pathways. In vitro, Ran overexpression has severe effects on the cell phenotype, altering proliferation, adhesion, colony density, and invasion. Therefore, Ran overexpression has been identified in numerous types of cancer and has been shown to correlate with tumor grade and the degree of metastasis present in various cancers. The increased malignancy and invasiveness have been attributed to multiple mechanisms. Increased dependence on Ran for spindle formation and mitosis is a consequence of the upregulation of these pathways and the ensuing overexpression of Ran, which increases cellular dependence on Ran for survival. This increases the sensitivity of cells to changes in Ran concentration, with ablation being associated with aneuploidy, cell cycle arrest, and ultimately, cell death. It has also been demonstrated that Ran dysregulation influences nucleocytoplasmic transport, leading to transcription factor misallocation. Consequently, patients with tumors that overexpress Ran have been shown to have a higher malignancy rate and a shorter survival time compared to their counterparts.
... Although S2 tissue culture cells form a RanGTP gradient around the chromosomes, RNAi-mediated depletion of >95% of RCC1 does not affect spindle assembly and functioning, and, consistently, it does not result in defective kinetochore-driven MT growth [28]. However, Drosophila embryos injected with a dominant negative form of Ran are severely defective in chromosome-driven MT regrowth after cold-induced depolymerization [19,29]. Thus, it is currently unclear whether chromosome-associated MT polymerization in S2 cells requires a minimal concentration of RanGTP, or whether it is RanGTP-independent. ...
Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.
... Another kinesin-like protein, KIF18A, corresponds to a microtubule depolymerase with a role in chromosome congression to form the metaphase plate during mitosis (54). Furthermore, LOC101738390 is a homolog of the mitotic spindle and nuclear protein Mink in Drosophila (55). No clear function was revealed for uncharacterized protein LOC101740936, but HHPred analysis (56) revealed the existence of a short sequence (ENTPPHSH) that is also present in the linker sequence of a peptide that interacts with the catalytic and substrate recognition sites in the CDK2/cyclin A complex, a kinase that is active in the cell cycle S phase (57). ...
Within the hemolymph, insect hemocytes constitute a heterogeneous population of macrophage-like cells that play important roles in innate immunity, homeostasis and development. Classification of hemocytes in different subtypes by size, morphology and biochemical or immunological markers has been difficult and only in Drosophila extensive genetic analysis allowed the construction of a coherent picture of hemocyte differentiation from pro-hemocytes to granulocytes, crystal cells and plasmatocytes. However, the advent of high-throughput single cell technologies, such as single cell RNA sequencing (scRNA-seq), is bound to have a high impact on the study of hemocytes subtypes and their phenotypes in other insects for which a sophisticated genetic toolbox is not available. Instead of averaging gene expression across all cells as occurs in bulk-RNA-seq, scRNA-seq allows high-throughput and specific visualization of the differentiation status of individual cells. With scRNA-seq, interesting cell types can be identified in heterogeneous populations and direct analysis of rare cell types is possible. Next to its ability to profile the transcriptomes of individual cells in tissue samples, scRNA-seq can be used to propose marker genes that are characteristic of different hemocyte subtypes and predict their functions. In this perspective, the identities of the different marker genes that were identified by scRNA-seq analysis to define 13 distinct cell clusters of hemocytes in larvae of the silkworm, Bombyx mori, are discussed in detail. The analysis confirms the broad division of hemocytes in granulocytes, plasmatocytes, oenocytoids and perhaps spherulocytes but also reveals considerable complexity at the molecular level and highly specialized functions. In addition, predicted hemocyte marker genes in Bombyx generally show only limited convergence with the genes that are considered characteristic for hemocyte subtypes in Drosophila.
... The Ran has a key role in the generation, maintenance, and regulation of the microtubule-based bipolar spindle during mitosis and meiosis. 47 Different types of cell division occur throughout the development of an insect embryo. In Drosophila melanogaster, polarized mitosis occurs in the ovarian epithelium, meiosis happens in the oocytes, and syncytial mitosis occurs in early embryos. ...
... Dysfunction of Ran and its related protein partners affects cell division and embryo development. [47][48][49] Therefore, it appears that dsLdRan might act as an ovicide. We need to perform further experiments in the future to evaluate the influence of dsLdRan on developing embryos. ...
BACKGROUND
RNA interference (RNAi) is a breakthrough technology in pest control. It is highly efficient to Coleopteran pests such as the Colorado potato beetle Leptinotarsa decemlineata, a serious pest defoliator mainly attacking potatoes worldwide. The first step for effective pest control by RNAi is the development of effective and reliable target genes.
RESULTS
Our results revealed that continuous ingestion of dsLdRan for 3 days successfully silenced the target gene, inhibited larval growth and killed 100% L. decemlineata larvae. When the bioassay began at the second‐, third/fourth‐instar larval stages, the larval lethality mainly occurred at the fourth larval instar and prepupal stages, respectively. Importantly, consumption of dsLdRan for 3 days by the newly‐emerged males and females effectively knocked down the target transcript, reduced fresh weights and caused 100% of lethality within a week. The LdRan females possessed underdeveloped ovaries.
CONCLUSION
Considering that the larvae, adults and eggs are simultaneously sited on the potato plants, bacterially‐expressed dsLdRan is a potential RNAi‐based strategy for managing L. decemlineata in the potato field. © 2022 Society of Chemical Industry.
... Although S2 tissue culture cells form a RanGTP gradient around the chromosomes, RNAi-mediated depletion of >95% of RCC1 does not affect spindle assembly and functioning, and, consistently, it does not result in defective kinetochore-driven MT growth [27]. However, Drosophila embryos injected with a dominant negative form of Ran are severely defective in chromosome-driven MTR after cold-induced depolymerization [18,28]. Thus, it is currently unclear whether chromosome-associated MT polymerization in S2 cells requires a minimal concentration of RanGTP, or whether it is RanGTP-independent. ...
Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but allows KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast / orbit / chb ( CLASP1 ), mei-38 ( TPX2 ), mars ( HURP ), dgt6 ( HAUS6 ), Eb1 ( MAPRE1/EB1 ), Patronin ( CAMSAP2 ), asp ( ASPM ) and Klp10A ( KIF2A ). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6 and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1 and Patronin positively regulate polymerization, bundling and stabilization of regrowing MTs until a bipolar spindle is reformed.
Author summary
The mitotic spindle is a microtubule (MT)-based molecular machine that mediates precise chromosome segregation during cell division. Both Drosophila and human cells assemble their spindles exploiting two main classes of MTs: MTs nucleated by the centrosomes (MT nucleating organelles) and MTs generated at or near the kinetochores (the chromosome-associated structures that bind the spindle MTs). Cells of both species can assemble a functional mitotic spindle in the complete absence of centrosomes, but the mechanisms underlying this process are still poorly understood. We used Drosophila S2 cells as model system to analyze spindle reassembly following colcemid-induced MT depolymerization. MT regrowth (MTR) after colcemid treatment was particularly informative as this drug disrupts the MT nucleating ability of the centrosomes but allows kinetochore-driven MTR (KDMTR). We analyzed KDMTR in normal cells and in cells subjected to RNA interference (RNAi)- mediated depletion of 8 different evolutionarily conserved proteins involved in spindle assembly, and identified proteins that either promote or delay KDMTR. These results coupled with the analysis of proteins localization during spindle reassembly allowed us to integrate the current model on the role of kinetochore-driven MT growth in spindle formation.
... However, both share similarities in the molecular mechanism behind MT-nucleation. A first body of literature suggests that this involves the Ras-related nuclear (Ran) pathway (Ran pathway in Drosophila reviewed in (Chen et al., 2015)). Briefly, Ran, a nuclear transport protein can switch from an inactive Ran-GDP to an active Ran-GTP. ...
Most animal cells are diploid, containing two copies of each chromosome. Establishment of proper bipolar mitotic spindle containing two centrosomes, one at each pole contributes to accurate chromosome segregation. This is essential for the maintenance of genome stability, tissue and organism homeostasis. However, numerical deviations to the diploid set are observed in healthy tissues. Polyploidy is the doubling of the whole chromosome set and aneuploidy concerns the gain or loss of whole chromosomes. Importantly, whole genome duplications and aneuploidy have also been associated to pathological conditions. For example, variations to genome content are associated with chromosome instability and cancer development, however their exact contribution to cancer genome remains poorly understood.In the first part of my PhD project, I investigated the consequences of polyploidy during cell division. I found that the presence of extra DNA and extra centrosomes generated invariably multipolar spindles. Then I identified contributors to the multipolar status using in vivo approaches in Drosophila neural stem cells and in vitro culture of cancer cells. Further I combined DNA and spindle perturbations with computer modelling and found that in polyploid cells, the presence of excessive DNA acts as a physical barrier blocking spindle pole coalescence and bipolarity. Indeed, laser ablation to disrupt and increase in microtubule stability and length to bypass the DNA-barrier could rescue bipolar spindle formation. This discovery challenges the current view that suggested extra-centrosomes as only contributor to spindle multipolarity and provides a rational to understand chromosome instability typical of polyploid cells.The aim of the second part of my PhD project was to generate a novel tool to quantitively probe chromosome loss in vivo in Drosophila tissues. Aneuploidy has been observed in various physiological tissues, however the frequency of this error remained highly debatable. In addition, tools developed so far to assess aneuploidy lack a temporal dimension. To circumvent this, I used the expression of a GFP report gene driven by the GAL4/UAS system and its inhibition by GAL80. In principle, the random loss of the chromosome carrying the GAL80 sequence leads to GFP appearance in aneuploid cells that can therefore be followed in live tissues. I found that chromosome loss was extremely infrequent in most tissues of the wild type fly. This tool combined with fluorescent marker and/or tested in various genetic background, might help understanding mechanisms behind aneuploidy genesis and outcome in vivo.While developing this tool, I discovered that in the larval brain, GFP cells where not a by-product of chromosome loss but rather an unexpected mis-regulation in the expression of the GAL80 gene. These results have strong implications for the Drosophila community as it can result in false positive in clonal experiments. Further, I discovered a mosaicism and plasticity of the Drosophila brain in neural stem cells for gene expression which differs from other organs and that is influenced by environmental stimuli. This possibly reflects a certain level of plasticity in the brain necessary for neuronal diversity, adaptation and survival.
... During cargo export, the complex of exportin, RanGTP, and protein cargo is exported via NPC. RanGDP is transported from cytoplasm into the nucleus and converted to the RanGTP by the chromatin-bound Ran-GEF, forming the RanGTPase cycle [83] ( Figure 2D). ...
Nucleocytoplasmic transport (NCT) across the nuclear envelope is precisely regulated in eukaryotic cells, and it plays critical roles in maintenance of cellular homeostasis. Accumulating evidence has demonstrated that dysregulations of NCT are implicated in aging and age-related neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer’s disease (AD), and Huntington disease (HD). This is an emerging research field. The molecular mechanisms underlying impaired NCT and the pathogenesis leading to neurodegeneration are not clear. In this review, we comprehensively described the components of NCT machinery, including nuclear envelope (NE), nuclear pore complex (NPC), importins and exportins, RanGTPase and its regulators, and the regulatory mechanisms of nuclear transport of both protein and transcript cargos. Additionally, we discussed the possible molecular mechanisms of impaired NCT underlying aging and neurodegenerative diseases, such as ALS/FTD, HD, and AD.
... RanGTP contributes to MT polymerization at the chromosomes by activating spindle assembly factors (SAFs) [7,8]. Activation of SAFs involves the RanGTP-induced dissociation of inhibitory importins. ...
RepoMan is a chromosome-associated scaffold protein that integrates signaling of multiple kinases and phosphatases to coordinate spindle-kinetochore interactions, chromosome (de)condensation and nuclear envelope (dis)assembly during mitosis. Another key mitotic event is the assembly of a microtubule-based spindle, which involves redundant pathways emanating from the centrosomes, microtubules and chromosomes. Here we describe a novel mitotic function of RepoMan in regulating chromosome-dependent microtubule assembly. At limiting concentrations of microtubule-destabilizing agents, RepoMan-depleted cells showed enhanced chromosome clustering. This clustering was completely dependent on the partial inhibition of microtubule growth originating from the chromosome-dependent pathway. We also demonstrated that RepoMan interacts with prime regulators of the chromosome-dependent spindle assembly such as NuSAP1, NuMA, and TPX2. In addition, RepoMan was required to enable or maintain phosphorylation of NuSAP1 at CDK sites, thereby enabling activation of NuSAP1 through dissociation of inhibitory importin β/7. Our data identify RepoMan as an enhancer of microtubule assembly at chromosomes.
... Several genes encoding proteins that could function in phagocytosis such as Biogenesis of lysosome-related organelles complex 1, subunit 2, and Tetraspanin 42El were specifically enriched by two-fold in S. aureus versus clean injury. Several genes implicated in cell division were down-regulated in S. aureus samples compared to clean injury samples, such as mitotic spindle and nuclear protein (mink, -2.4 fold change) [119], stathmin (stai, -3.8 fold change) [120] and cyclin B (cycB, -2.2 fold change) [121] suggesting a cell cycle arrest in response to infection with this Gram-positive bacterium (S7 File) [122]. Surprisingly, the gene encoding the lipase Magro was expressed 14 times less upon systemic infection with S. aureus compared to clean injury or EcH. ...
Drosophila melanogaster’s blood cells (hemocytes) play essential roles in wound healing and are involved in clearing microbial infections. Here, we report the transcriptional changes of larval plasmatocytes after clean injury or infection with the Gram-negative bacterium Escherichia coli or the Gram-positive bacterium Staphylococcus aureus compared to hemocytes recovered from unchallenged larvae via RNA-Sequencing. This study reveals 676 differentially expressed genes (DEGs) in hemocytes from clean injury samples compared to unchallenged samples, and 235 and 184 DEGs in E. coli and S. aureus samples respectively compared to clean injury samples. The clean injury samples showed enriched DEGs for immunity, clotting, cytoskeleton, cell migration, hemocyte differentiation, and indicated a metabolic reprogramming to aerobic glycolysis, a well-defined metabolic adaptation observed in mammalian macrophages. Microbial infections trigger significant transcription of immune genes, with significant differences between the E. coli and S. aureus samples suggesting that hemocytes have the ability to engage various programs upon infection. Collectively, our data bring new insights on Drosophila hemocyte function and open the route to post-genomic functional analysis of the cellular immune response.
