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CaM localization and dynamics in NBs. (A) Prophase NBs from larvae expressing GFP-CaM stained for centrosomin (Cnn) and β-tubulin. (A′) Close up of yellow boxed region in A and line scan along the yellow line for CaM, Cnn, and Tubulin. (B) As described for A, but the cell is in metaphase. (B′) As described for A′. Note the difference in CaM localization relative to the centrosome between prophase (A) and metaphase (B). C, centrosome; P, pole. (C) Live cell imaging of mitotic NB (dotted outline) expressing GFP-CaM (Video 2). (C′) Inset of red boxed region in C. Arrows denote spindle foci. P, pole. (D) Still frames from metaphase NB (Video 3), with arrowhead denoting GFP-CaM foci moving toward the pole. Note the increase in signal intensity at pole as time progresses. (D′) Average intensity projection of metaphase (left panel) showing position of kymographs (right). Arrowheads denote foci movement. Bars: (A and B) 5 µm; (A’ and B’) 1 µm; (C) 10 µm; (C’, D, and D’) 2 µm.
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The interaction between centrosomes and mitotic spindle poles is important for efficient spindle formation, orientation, and cell polarity. However, our understanding of the dynamics of this relationship and implications for tissue homeostasis remains poorly understood. Here we report that Drosophila melanogaster calmodulin (CaM) regulates the abil...
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Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease dur...
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... The difference in size (Asp, 1954 aa; Aspm 3122 aa; ASPM 3477 aa) is mainly due to the number of isoleucine-glutamine (IQ) motifs that varies from 24 in Drosophila to 60 and 81 in mice and humans, respectively ( Figure 1) [3,[14][15][16]. The IQ motifs of Drosophila, C. elegans, mouse and human proteins bind calmodulin (CaM) ( [17][18][19][20]; see Section 5 below for Asp/ASPM-CaM interactions). SPD-2, Hydin) domain, which overlaps with the major sperm protein (MSP) domain. ...
... Early studies using antibodies against an Asp N terminal fragment of 512 amino acids showed that Asp localizes to the polar regions of the spindles and to the telophase central spindle in syncytial Drosophila embryos [2]. Subsequent studies expanded and refined these initial observations showing that Asp accumulates at the spindles poles of larval neuroblasts [23,24], epithelial cells [25], S2 tissue culture cells [18,21,26,27], and meiotic cells of both males and females [24,28,29]. Specifically, it has been reported that Asp accumulates at the transition region between the spindle and the centrosome, with Asp immunostaining partially overlapping the immunofluorescence signal elicited by γ-tubulin or Centrosomin (Cnn, the ortholog of the human centrosomal protein CDK5RAP2). ...
... In addition, the prometaphase and metaphase spindles of live larval neuroblasts and S2 cells, both expressing Asp-GFP, displayed discrete fluorescent signals along the spindle MTs. Imaging of these Asp-GFP particles revealed that they stream towards the spindle poles and are eventually incorporated into the polar Asp pool [18,21]. A poleward flow of Asp-GFP particles was also observed in the epithelial cell spindles of Drosophila pupal notum [25]. ...
The Drosophila abnormal spindle (asp) gene was discovered about 40 years ago and shown to be required for both mitotic and meiotic cell division. Subsequent studies showed that asp is highly conserved and that mutations in its human ortholog ASPM (Abnormal Spindle-like Microcephaly-associated; or MCPH5) are the most common cause of autosomal recessive primary microcephaly. This finding greatly stimulated research on ASPM and its fly and mouse (Aspm) orthologs. The three Asp orthologous proteins bind the microtubules (MTs) minus ends during cell division and also function in interphase nuclei. Investigations on different cell types showed that Asp/Aspm/ASPM depletion disrupts one or more of the following mitotic processes: aster formation, spindle pole focusing, centrosome-spindle coupling, spindle orientation, metaphase-to-anaphase progression, chromosome segregation, and cytokinesis. In addition, ASPM physically interacts with components of the DNA repair and replication machineries and is required for the maintenance of chromosomal DNA stability. We propose the working hypothesis that the asp/Aspm/ASPM genes play the same conserved functions in Drosophila, mouse, and human cells. Human microcephaly is a genetically heterogeneous disorder caused by mutations in 30 different genes that play a variety of functions required for cell division and chromosomal DNA integrity. Our hypothesis postulates that ASPM recapitulates the functions of most human microcephaly genes and provides a justification for why ASPM is the most frequently mutated gene in autosomal recessive primary microcephaly.
... Given that both PLP and Kinesin-1 are required for centriole motility, and that they colocalize on the centriole, we hypothesized that PLP and Kinesin-1 directly interact. To test for direct protein-protein interactions (PPIs), we performed a yeast twohybrid (Y2H) assay using subdivided fragments of PLP and KHC Galletta et al., 2014;Schoborg et al., 2015). This same assay confirmed that the PLP-KHC interaction is conserved between human Pericentrin (PCNT) and the cargo binding domain of the human Kinesin-1 heavy chain (KIF5B; Fig. S3, B and C). ...
Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosomal microtubules (MTs) with motor proteins anchored at the cortex or the nuclear surface. However, it remains unknown how centrioles migrate in cellular contexts in which they do not nucleate MTs. Here, we demonstrate that during interphase, inactive centrioles move directly along the interphase MT network as Kinesin-1 cargo. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP. Finally, our studies of neural stem cell asymmetric divisions in the Drosophila brain show that the PLP-Kinesin-1 interaction is essential for the timely separation of centrioles, the asymmetry of centrosome activity, and the age-dependent centrosome inheritance.
... With progression of MT regrowth, Asp-GFP associated with the extended MT bundles and accumulated at their minus ends (Figure 9(A4); see also Figure S10). This finding is consistent with previous work showing that in normal spindles, Asp moves along the MTs towards the spindle poles [70,82]. Importantly, when the Asp-GFP signal was located at the center of the tubulin clusters/asters, the Cid (CENPA) centromeric signals were not at the center of these structures but were instead surrounding them ( Figure 9B). ...
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.
... To gather information on the specific roles of the proteins that regulate KDMTR, we examined their behavior during MTR after colcemid-induced depolymerization. The cell lines carrying Cherry-tubulin and the GFP-tagged protein of interest, both under the control of a copper-inducible promoter, were treated for 16-22 h with copper sulfate, exposed to colcemid [60,73]). Importantly, when the Asp-GFP signal was located at the center of the tubulin clusters/asters, the Cid (CENPA) centromeric signals were not at the center of these structures but were instead surrounding them ( Figure 4B). ...
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.
... Currently, 25 genetic loci are associated with primary autosomal recessive microcephaly (MCPH) (Jayaraman et al., 2018;Naveed et al., 2018) in the Online Mendelian Inheritance in Man (OMIM) database (Amberger et al., 2015). In recent years, the molecular function of some of these MCPH genes has been elucidated using Drosophila (Gambarotto et al., 2019;Lucas and Raff, 2007;Poulton et al., 2017;Ramdas Nair et al., 2016;Rujano et al., 2013;Schoborg et al., 2015;Schoborg et al., 2019;Singh et al., 2014). Below, we outline how studies of Drosophila have played an important role in elucidating some of the pathogenic mechanisms associated with microcephaly and in aiding microcephaly disease diagnosis. ...
Next-generation sequencing has greatly accelerated the discovery of rare human genetic diseases. Nearly 45% of patients have variants associated with known diseases but the unsolved cases remain a conundrum. Moreover, causative mutations can be difficult to pinpoint because variants frequently map to genes with no previous disease associations and, often, only one or a few patients with variants in the same gene are identified. Model organisms, such as Drosophila, can help to identify and characterize these new disease-causing genes. Importantly, Drosophila allow quick and sophisticated genetic manipulations, permit functional testing of human variants, enable the characterization of pathogenic mechanisms and are amenable to drug tests. In this Spotlight, focusing on microcephaly as a case study, we highlight how studies of human genes in Drosophila have aided our understanding of human genetic disorders, allowing the identification of new genes in well-established signaling pathways.
... In humans, heritable microcephaly is most commonly associated with mutations in ASPM [90]. Loss of the Drosophila homolog abnormal spindle (asp) also results in microcephaly, as well as centrosome segregation and spindle orientation defects similar to the defects observed in centrosome asymmetry mutants [91]. Expression of a full-length asp transgene recues brain size and microtubule defects in asp mutants. ...
... Expression of a full-length asp transgene recues brain size and microtubule defects in asp mutants. In contrast, expression of an N-terminal asp fragment or a full-length transgene lacking a domain required for interaction with Calmodulin (asp ΔIQ ) rescues the microcephaly phenotype without rescuing the spindle morphology defects, suggesting the bent, unfocused spindles typical of asp mutants are insufficient to cause microcephalyother mechanisms are at play [91]. Given that both centrosome asymmetry mutants and animals rescued of asp-dependent microcephaly have morphologically wild-type brains, it appears that Drosophila neurogenesis is resistant to certain perturbations of centrosome activity to which human neurogenesis may be more sensitive. ...
Microcephaly is a rare, yet devastating, neurodevelopmental condition caused by genetic or environmental insults, such as the Zika virus infection. Microcephaly manifests with a severely reduced head circumference. Among the known heritable microcephaly genes, a significant proportion are annotated with centrosome-related ontologies. Centrosomes are microtubule-organizing centers, and they play fundamental roles in the proliferation of the neuronal progenitors, the neural stem cells (NSCs), which undergo repeated rounds of asymmetric cell division to drive neurogenesis and brain development. Many of the genes, pathways, and developmental paradigms that dictate NSC development in humans are conserved in Drosophila melanogaster. As such, studies of Drosophila NSCs lend invaluable insights into centrosome function within NSCs and help inform the pathophysiology of human microcephaly. This mini-review will briefly survey causative links between deregulated centrosome functions and microcephaly with particular emphasis on insights learned from Drosophila NSCs.
... To demonstrate the utility of µ-CT when combined with the genetic power of Drosophila, we applied the technique to investigate brain defects in two models of human microcephaly, which is characterized by reduced brain size, cognitive function, and lifespan (O'Neill et al., 2018). We previously showed that mutations in abnormal spindle (asp), the fly ortholog of abnormal spindle-like microcephalyassociated (ASPM), leads to adult flies displaying a reduction in head and brain size (Schoborg et al., 2015). We used µ-CT to explore heterozygous adult control (asp t25 /+; Fig. 8A-A″) and asp mutant (asp t25 /Df; Fig. 8B-B″) animals. ...
... As a final highlight of the capabilities of μ-CT, we used our asp dataset to perform phenotyping analysis to identify additional tissue defects and provide a more complete description of asp function. In addition to the well-characterized small brain phenotype (Fig. 8, Figs S6-S8; Rujano et al., 2013;Schoborg et al., 2015), our analysis revealed severe defects in the visual circuit. The size and morphology of the lamina and the retina were severely compromised, and the ocelli were reduced in size, extremely disorganized or completely absent in asp mutant animals. ...
... We attempted to measure Asp transgene levels using standard western blotting techniques with anti-GFP antibodies; however, this approach consistently failed despite numerous attempts to optimize lysis and running conditions (Rujano et al., 2013;Schoborg et al., 2015). Instead, we directly measured GFP fluorescence from dissected larval brains that were imaged live to determine expression levels. ...
Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology, and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and use of sophisticated genetic, biochemical, and imaging tools. Here we present an accessible method for non-invasive imaging of Drosophila melanogaster at high resolution using micro-computed tomography (µ-CT). We show how rapid processing of intact animals at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use µ-CT imaging to study growth defects in the Drosophila brain through the characterization of Abnormal spindle (asp) and WD Repeat Domain 62 (wdr62), orthologs of the two most commonly mutated genes in human microcephaly patients. Our work demonstrates the power of combining µ-CT with traditional genetic, cellular, and developmental biology tools available in model organisms to address novel biological mechanisms that control animal development and disease.
... It localizes to spindle poles and is required for spindle organization, spindle positioning and cytokinesis (Higgins et al., 2010;Saunders et al., 1997;van der Voet et al., 2009;Wakefield et al., 2001). For example, in Drosophila, Asp is essential for spindle pole focusing, likely due to its microtubule minus-end binding and crosslinking activities (Ito and Goshima, 2015;Schoborg et al., 2015). In mammals, ASPM affects spindle architecture in more subtle ways, as its activity appears to be somewhat redundant with centrosomal components (Higgins et al., 2010;Jiang et al., 2017;Tungadi et al., 2017). ...
Microtubules are cytoskeletal filaments essential for numerous aspects of cell physiology. They are polarized polymeric tubes with a fast growing plus end and a slow growing minus end. In this Cell Science at a Glance article and the accompanying poster, we review the current knowledge on the dynamics and organization of microtubule minus ends. Several factors, including the γ-tubulin ring complex, CAMSAP/Patronin, ASPM/Asp, SPIRAL2 (in plants) and the KANSL complex recognize microtubule minus ends and regulate their nucleation, stability and interactions with partners, such as microtubule severing enzymes, microtubule depolymerases and protein scaffolds. Together with minus-end-directed motors, these microtubule minus-end targeting proteins (-TIPs) also control the formation of microtubule-organizing centers, such as centrosomes and spindle poles, and mediate microtubule attachment to cellular membrane structures, including the cell cortex, Golgi complex and the cell nucleus. Structural and functional studies are starting to reveal the molecular mechanisms by which dynamic -TIP networks control microtubule minus ends.
... In this regard, the functions of spastin are relatively well known in mature neurons, whereas it is not so clear whether spastin has a role in neurogenesis and brain development at early developmental stages. Based on the previous reports showing that the fine tuning of microtubule dynamics is important for the regulation of neural stem cell (NSC) proliferation, asymmetric division, neurogenesis in animal models including Drosophila and mice (Derivery et al., 2015;Mora-Bermudez and Huttner, 2015;Schoborg et al., 2015;Kannan et al., 2017), we hypothesized that spastin could have a role in the regulation of brain development by controlling microtubules in NSCs. ...
Spastin is a microtubule-severing enzyme encoded by SPAST, which is broadly expressed in various cell types originated from multiple organs. Even though SPAST is well known as a regulator of the axon growth and arborization in neurons and a genetic factor of hereditary spastic paraplegia, it also takes part in a wide range of other cellular functions including the regulation of cell division and proliferation. In this study, we investigated a novel biological role of spastin in developing brain using Spast deficient mouse embryonic neural stem cells (NSCs) and perinatal mouse brain. We found that the expression of spastin begins at early embryonic stages in mouse brain. Using Spast shRNA treated NSCs and mouse brain, we showed that Spast deficiency leads to decrease of NSC proliferation and neuronal lineage differentiation. Finally, we found that spastin controls NSC proliferation by regulating microtubule dynamics in primary cilia. Collectively, these data demonstrate that spastin controls brain development by the regulation of NSC functions at early developmental stages.
... In Drosophila, the protein Mud shares both sequence identity and a similar a role in spindle orientation with NuMA, but does not substantially contribute to spindle pole focusing; although recent live imaging experiments do show a focusing role in a minority of epithelial cells [36,37]. Instead, the direct minus-end binding protein Abnormal spindle (Asp) has been proposed to be a 'functional homolog' of NuMA in this role [23,32,[38][39][40]. Indeed, Drosophila Mud is transported to spindle poles by Asp [37], whereas MT minus-end localization of mammalian NuMA does not require the mammalian Asp homolog, ASPM [28]. ...
... Asp has been proposed to function by cross-linking spindle MTs with the newly generated intra-spindle minus-ends of Augmin-nucleated MTs [39]. The observed Asp-decorated MT minus-end poleward streaming was further shown by Schoborg et al. [40] to depend on regulation by calmodulin, which binds the C-terminus of Asp and either aids localized Asp oligomerization or enhances a weak Asp C-term-MT interaction that functions additionally to its strong N-term MT binding, regulating the ability of Asp to 'bridge and zipper' neighbouring MTs. The relationship between MT cross-linking by minus-end binding proteins and MT minus-end stabilization is not fully understood. ...
... Evidence supports higher order structures of both Asp/ASPM and NuMA [26,40], and NuMA was long ago proposed to form 'insoluble spindle pole matrix' [18,22]. Certainly protein aggregates contribute to specifically retaining motors at MT minus-ends allowing pole focusing: indeed, if the plus-end motor, Eg5, is artificially oligomerized, then these will form a plus-end pole [67]. ...
The formation of a robust, bi-polar spindle apparatus, capable of accurate chromosome segregation, is a complex process requiring the co-ordinated nucleation, sorting, stabilization and organization of microtubules (MTs). Work over the last 25 years has identified protein complexes that act as functional modules to nucleate spindle MTs at distinct cellular sites such as centrosomes, kinetochores, chromatin and pre-existing MTs themselves. There is clear evidence that the extent to which these different MT nucleating pathways contribute to spindle mass both during mitosis and meiosis differs not only between organisms, but also in different cell types within an organism. This plasticity contributes the robustness of spindle formation; however, whether such plasticity is present in other aspects of spindle formation is less well understood. Here, we review the known roles of the protein complexes responsible for spindle pole focusing, investigating the evidence that these, too, act co-ordinately and differentially, depending on cellular context. We describe relationships between MT minus-end directed motors dynein and HSET/Ncd, depolymerases including katanin and MCAK, and direct minus-end binding proteins such as nuclear-mitotic apparatus protein, ASPM and Patronin/CAMSAP. We further explore the idea that the focused spindle pole acts as a non-membrane bound condensate and suggest that the metaphase spindle pole be treated as a transient organelle with context-dependent requirements for function.
