ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules
2016; Springer Nature; Volume: 17; Issue: 10 Linguagem: Inglês
10.15252/embr.201541823
ISSN1469-3178
AutoresMarta Gai, F. Bianchi, Cristiana Vagnoni, Fiammetta Vernı̀, Silvia Bonaccorsi, Selina Pasquero, Gaia Berto, Francesco Sgrò, Alessandra M.A. Chiotto, Laura Annaratone, Anna Sapino, Anna Bergo, Nicoletta Landsberger, Jacqueline Bond, Wieland Β. Huttner, Ferdinando Di Cunto,
Tópico(s)Cellular transport and secretion
ResumoScientific Report25 August 2016free access Transparent process ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules Marta Gai Corresponding Author Marta Gai [email protected] Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Federico T Bianchi Federico T Bianchi Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Cristiana Vagnoni Cristiana Vagnoni Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Fiammetta Vernì Fiammetta Vernì Department of Biology and Biotechnologies “C. Darwin”, Sapienza, Università di Roma, Rome, Italy Search for more papers by this author Silvia Bonaccorsi Silvia Bonaccorsi Department of Biology and Biotechnologies “C. Darwin”, Sapienza, Università di Roma, Rome, Italy Search for more papers by this author Selina Pasquero Selina Pasquero Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Gaia E Berto Gaia E Berto Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Francesco Sgrò Francesco Sgrò Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Alessandra MA Chiotto Alessandra MA Chiotto Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Laura Annaratone Laura Annaratone Department of Medical Sciences, University of Turin, Turin, Italy Search for more papers by this author Anna Sapino Anna Sapino Department of Medical Sciences, University of Turin, Turin, Italy Search for more papers by this author Anna Bergo Anna Bergo San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Nicoletta Landsberger Nicoletta Landsberger orcid.org/0000-0003-0820-3155 San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Jacqueline Bond Jacqueline Bond Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK Search for more papers by this author Wieland B Huttner Wieland B Huttner Max-Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Ferdinando Di Cunto Corresponding Author Ferdinando Di Cunto [email protected] orcid.org/0000-0001-9367-6357 Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Marta Gai Corresponding Author Marta Gai [email protected] Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Federico T Bianchi Federico T Bianchi Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Cristiana Vagnoni Cristiana Vagnoni Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Fiammetta Vernì Fiammetta Vernì Department of Biology and Biotechnologies “C. Darwin”, Sapienza, Università di Roma, Rome, Italy Search for more papers by this author Silvia Bonaccorsi Silvia Bonaccorsi Department of Biology and Biotechnologies “C. Darwin”, Sapienza, Università di Roma, Rome, Italy Search for more papers by this author Selina Pasquero Selina Pasquero Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Gaia E Berto Gaia E Berto Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Francesco Sgrò Francesco Sgrò Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Alessandra MA Chiotto Alessandra MA Chiotto Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Laura Annaratone Laura Annaratone Department of Medical Sciences, University of Turin, Turin, Italy Search for more papers by this author Anna Sapino Anna Sapino Department of Medical Sciences, University of Turin, Turin, Italy Search for more papers by this author Anna Bergo Anna Bergo San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Nicoletta Landsberger Nicoletta Landsberger orcid.org/0000-0003-0820-3155 San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Jacqueline Bond Jacqueline Bond Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK Search for more papers by this author Wieland B Huttner Wieland B Huttner Max-Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Ferdinando Di Cunto Corresponding Author Ferdinando Di Cunto [email protected] orcid.org/0000-0001-9367-6357 Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Search for more papers by this author Author Information Marta Gai *,1, Federico T Bianchi1, Cristiana Vagnoni1, Fiammetta Vernì2, Silvia Bonaccorsi2, Selina Pasquero1, Gaia E Berto1, Francesco Sgrò1, Alessandra MA Chiotto1, Laura Annaratone3, Anna Sapino3, Anna Bergo4, Nicoletta Landsberger4, Jacqueline Bond5, Wieland B Huttner6 and Ferdinando Di Cunto *,1 1Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy 2Department of Biology and Biotechnologies “C. Darwin”, Sapienza, Università di Roma, Rome, Italy 3Department of Medical Sciences, University of Turin, Turin, Italy 4San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy 5Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK 6Max-Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany *Corresponding author. Tel: +39 011 670 6410; E-mail: [email protected] *Corresponding author. Tel: +39 011 670 6409; E-mail: [email protected] EMBO Reports (2016)17:1396-1409https://doi.org/10.15252/embr.201541823 Correction(s) for this article ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules02 October 2017 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins. Synopsis The microcephaly gene ASPM recruits the cytokinesis regulator CITK to the mitotic spindle of neural progenitor cells, where it promotes the nucleation and stabilization of astral microtubules, providing a link between the two microcephaly proteins. The loss of CITK function in the developing mouse cortex results in increased cell cycle exit and generation of basal progenitors. CITK physically interacts with ASPM at the spindle pole in HeLa cells. CITK- and ASPM-depleted HeLa cells display a significant reduction in the number and length of astral microtubules. Introduction The orientation of cell division is carefully controlled in both embryonic and adult tissues, in which it may regulate cell fate, generate tissue shape, and maintain normal histological architecture 123. Studies conducted over the last two decades have established that this process may play an important role to regulate the delicate balance between proliferation and differentiation that underlies normal brain development, with particular regard to the generation of a normal number of cortical neurons 245. During embryogenesis, the cerebral cortex is first composed of a single layer of neuroepithelial (NE) cells, which initially expand through symmetric divisions 6789. As development progresses, NE progenitors give rise to radial glia (RG) cells that may further expand by dividing symmetrically or may switch to asymmetric division, producing a self-renewing progenitor and a daughter cell committed to differentiation 5101112. A characteristic feature of proliferative divisions of NE and RG cells is that cleavage occurs perpendicular to the ventricular surface of the neuroepithelium, while the switch from symmetric to asymmetric divisions is accompanied by a deviation of the cleavage plane 131415. Oriented cell division is achieved through the proper positioning of the mitotic spindle 161718, which depends on the formation of molecular links between actin-rich cell cortex and astral microtubules (MT) emanating from centrosome-derived spindle poles 151920. Indeed, either loss of doublecortin (DCX), which destabilizes MT 21, or mutations of LIS1, NDE1, and NDEL1, which disrupt dynein–dynactin function at the cell cortex 222324, randomize the mitotic spindle in NE progenitors and lead to their early exhaustion. A similar depletion of progenitor cells, correlated with randomized spindle orientation, can be produced by disruption of proteins involved in centrosome function 25262728. Whether the relationship between spindle orientation and cell fate choice in cortical development is causal or only correlative is currently debated, on the basis of recent studies suggesting that spindle orientation may not be essential for cortical neurogenesis 262930. Mutation of many proteins contributing to centriole biogenesis, centrosome maturation, and spindle organization 3132 has been associated with human primary microcephaly (MCPH). MCPH is characterized by reduced head circumference, accompanied by relatively preserved brain architecture, resulting in mild-to-moderate intellectual disability and few associated symptoms 3334. At least thirteen MCPH loci (MCPH1–MCPH13) have been mapped to date 353637, and it has been established that most of the encoded proteins are capable of localizing at the centrosome, at the spindle poles, or at the spindle. Nevertheless, our understanding of how these proteins may affect spindle orientation and cell fate determination is still very limited. ASPM (abnormal spindle-like microcephaly-associated, MCPH5) is the most frequently mutated gene in MCPH 2838. ASPM is a conserved protein that associates with the MT minus ends, is recruited at the spindle poles during mitosis, and controls spindle MT organization, spindle function, and cytokinesis from insects to mammals 394041. In mammals, NE cells with reduced ASPM expression fail to orient the mitotic spindle perpendicular to the ventricular surface of the neuroepithelium and show increased frequency of asymmetric divisions, with a reduction of the pool of neuronal precursors 2542. Recent evidence indicates that ASPM may control cell fate choice by regulating the activity of Cdk2/cyclin E complex 43. However, it is presently unknown how ASPM regulates spindle orientation. Previous studies 44 showed that ASPM physically interacts with the cytokinesis regulator citron kinase (CITK) 4546. CITK is a conserved protein prominently localized at the cleavage furrow and at the midbody of mitotic cells 4647 and is involved in abscission control at the end of cytokinesis 4849. In rodents, CITK inactivation leads to severe microcephaly and lethal epilepsy 4550. This phenotype is believed to result from apoptosis due to cytokinesis failure. Delayed metaphase–anaphase transition in CITK-deficient neuronal progenitors has also been described 51. Although ASPM and CITK are both associated with microcephaly and may form a complex in HeLa cells and in developing neural tissue 44, it remained unclear whether their interaction could have functional relevance. In this report, we show that in addition to its role in cytokinesis, CITK plays a phylogenetically conserved role in the control of mitotic spindle orientation, by promoting the nucleation and stabilization of astral MT. Moreover, we show that CITK is recruited to the spindle by ASPM and that CITK overexpression rescues the spindle orientation defect elicited by ASPM knockdown. These results provide new insight into the mechanisms by which ASPM loss may cause microcephaly and suggest that a spindle orientation defect may contribute to the CITK microcephaly phenotype. Results and Discussion CITK is a phylogenetically conserved determinant of spindle orientation In light of the previously reported association between CITK and ASPM 44 and of the role played by ASPM in regulating spindle orientation 2539, we asked whether CITK may also play a role in controlling spindle orientation, besides promoting abscission 49. To address this question, we first examined whether CITK contributes to maintaining mitotic spindle perpendicular to the apical–basal axis of mouse NE progenitors. To do so, we immunostained fixed cryosections of E14.5 mice neocortices for spindle-pole protein γ-tubulin and DNA, and we measured the angle formed by the cleavage plan of apical progenitors (AP) with the apical surface of cortex 13142530 (Fig 1A). This angle was defined as vertical when it was comprised between 76° and 90° and oblique when it was in the range from 0 to 75°. Most AP (75%) divide vertically in CitK+/+ cortices, while vertical divisions occurred in only 24% of AP in CitK−/− mice (Fig 1B), indicating that spindle orientation is perturbed by CITK loss. Figure 1. CITK controls mitotic spindle orientation in developing mouse and fly brains WT and CITK−/− embryonic (E14.5) mouse cerebral cortex was stained for γ-tubulin (green) and DNA (gray). The ventricular plane is marked by a red dashed line, and the spindle axis of apical progenitors is indicated by a white dashed line. The angle between these two lines represents the mitotic angle. Scale bars, 5 μm. Quantification of vertical divisions of apical progenitors in WT and CITK−/− mice. n = 3 per each genotype. Pregnant CITK+/− females, crossed with CITK+/− males, were injected with BrdU at E13.5; 24 h later, WT and CITK−/− embryonic (E14.5) mouse cerebral cortex was fixed in 4% PFA, cryosectioned, and stained for Ki67 (red), BrdU (green), Tbr2 (blue), and DAPI (gray). The different cortical regions are indicated: CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars, 10 μm. White arrows in the inset indicate two Ki67− cells positive for BrdU and Tbr2. Quantification of cell cycle exit (ratio of cells BrdU+/Ki67−) in the proliferative regions (VZ/SVZ) and in the neuronal layers (IZ and CP) of sections prepared as in panel (C) (n = 4 per each genotype). Sections prepared as in (C) were stained for TUBB3 (TuJ) to reveal post-mitotic neurons and with DAPI. Red arrowheads indicate apoptotic neurons (defined by pyknotic nuclei). Quantification of neuronal distribution in VZ/SVZ versus total neurons population is shown on the right. Quantification of the percentage of Tbr2-positive cells in the BrdU-positive population of the same sections analyzed for panel (D) (n = 4 per each genotype). Neuroblasts of wild-type and dck alleles immunostained for α-tubulin and Miranda. Note that whereas in wild-type cells there is a tight coupling of the mitotic spindle with the polarity axis, in dck alleles the spindle shows a more oblique orientation with respect to Mira crescent. Scale bar, 5 μm. Distribution of spindle angle amplitude (°) in neuroblasts of wild-type and ck alleles (n = 58). Data information: Data shown in histograms are means ± SEM. Statistical significance was assessed using a two-tailed Student's t-test. **P < 0.01; *P < 0.05. Download figure Download PowerPoint To evaluate whether this phenotype correlates with increased commitment to differentiation, we pulse-labeled E13.5 embryos with BrdU and analyzed how many cells had left the cell cycle 24 h later, measuring the percentage of cells positive for BrdU, but negative for the cell cycle marker Ki67 52. A significant increase in BrdU+/Ki67− cells was observed in the proliferative regions and in the intermediate zone (Fig 1C and D), suggesting a shift from proliferative to differentiative divisions. Indeed, the increase in cells exited from cell cycle in these regions could not be explained by neuron migration defects, because the percentage of neurons in VZ/SVZ relative to the total neuron population is the same in WT and CITK−/− mice (Fig 1E). Interestingly, the percentage of BrdU+/Tbr2+ cells in the proliferative regions was significantly increased (Fig 1F). In contrast, the total number of Tbr2+ cells was not different in control and knockout cortices (55.4 ± 4.6 and 52.2 ± 4.1 per 100-μm-wide cortical column, respectively). It must be noted that the number of neurons produced in CITK−/− may be strongly underestimated, because of their apoptotic death (Fig 1E). Indeed, we observed a strong absolute reduction of BrdU-positive cells in the neuronal layers of CitK−/− mice (Fig 1C), consistent with the previous finding that in this model apoptosis occurs especially in post-mitotic neuroblasts and neurons 53. Altogether, these results indicate that CitK−/− developing cortex is characterized by increased cell cycle exit and increased generation of basal progenitors. Since the sequence of CITK is well conserved between Drosophila and mammals, and since the loss of CITK in Drosophila produces a cytokinesis-failure phenotype remarkably similar to the phenotype detected in mammalian cells 47545556, we asked whether the role of CITK in spindle orientation is conserved as well. To address this question, we analyzed neuroblast (NB) divisions in larval brains from individuals homozygous for either dck1 or dck2, two presumptive null alleles at the locus encoding the Drosophila orthologue of CITK 47. Drosophila NBs are stem cells that divide asymmetrically, to give rise to another NB and to a smaller ganglion mother cell (GMC) committed to differentiation. To ensure a correct asymmetric division, NB spindle must be aligned to the cell polarity axis determined by the differential apico-basal concentration of several proteins. The basal cortex is enriched in proteins that are preferentially segregated into the GMC at the end of division and whose localization is in turn mediated by a large multiprotein complex that concentrates at the apical cortex 57. We immunostained wild-type and dck mutant larval brains for tubulin and for the basal marker Miranda (Mira) and measured the angle between a line connecting the two spindle poles and a line bisecting the crescent formed by Mira in metaphase NBs (Fig 1G). The angle ranged between 0° and 5° in 88% of control NBs (Fig 1H), indicating a tight coupling of the mitotic spindle with the polarity axis. Conversely, although both dck1 and dck2 mutant NBs consistently displayed well-formed Mira crescents, the majority of the spindles showed more oblique orientations, ranging from 6° to 45° (n = 33 for dck1, and 45 for dck2, respectively; Fig 1H). These results indicate that besides its role in abscission control, CITK plays a phylogenetically conserved role earlier in mitosis, to ensure correct positioning of the mitotic spindle. To better characterize this function, we resorted to HeLa cells, which are sensitive to CITK depletion 4958 and have extensively been used to study spindle positioning mechanisms 59. We depleted CITK in these cells by RNAi using validated sequences 49 (Fig EV1A) and analyzed cell division through phase-contrast time-lapse microscopy. Interestingly, divisions of cells treated with CITK-specific siRNAs were misoriented, with the two forming daughters partially overlapping rather than being adjacent and with one daughter cell dividing outside of the focal plan, thus delaying adherence to the substrate (Fig 2A–C and Movies EV1 and EV2). We then analyzed, in fixed samples, the angles formed by metaphase spindles with the culture dish. CITK-depleted cells displayed angles distribution skewed toward high values and significant increase in angles average (Fig 2D and E), whereas spindle length was not affected by CITK depletion (Fig EV4A). A similar phenotype was observed by inducing CITK depletion with a second, independent siRNA sequence (Fig EV1B and C). The phenotype was rescued by restoring CITK levels through the expression of an RNAi-resistant construct (Fig EV1C), further confirming that it is due to CITK depletion rather than to off-target effects of the siRNA sequences used. The phenotype observed in CITK-depleted cells was similar to the phenotype produced by ASPM depletion 39 (Fig 2F). Moreover, the defect on mitotic spindle orientation caused by CITK and ASPM simultaneous knockdown was the same as single knockdown (Fig 2F), indicating that these two proteins are on the same pathway. Click here to expand this figure. Figure EV1. Specificity of the CITK-RNAi experiments Western blot analysis performed with the indicated antibodies on lysates of HeLa cells transfected with control or with two different CITK-specific siRNAs (CITK1 and CITK2). The results of experiments shown in the main figures were obtained with the CITK1 sequence. Distribution of spindle angles (°) in HeLa cells depleted of CITK using the CITK2 sequence or transfected with control sequence. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip (n ≥ 150 cells in three independent experiments). Quantification of average and distribution of spindle angles (°) in HeLa cells depleted of CITK using the CITK2 (n ≥ 50 cells in three independent experiments). Quantification of angles average in HeLa cells transfected with either control or CITK1 siRNAs and cotransfected with expression plasmids expressing mCherry alone or fused to RNAi-resistant (mouse) CITK wild-type sequence (n ≥ 50 cells in three independent experiments). Data information: Data presented are means ± SEM. Statistical significance was assessed using a two-tailed Student's t-test. ***P < 0.001; **P < 0.01; *P < 0.05. Download figure Download PowerPoint Figure 2. CITK controls mitotic spindle orientation in HeLa cells Selected frames from time-lapse imaging experiments (see Movies EV1 and EV2) showing two dividing cells transfected with either control (CTRL) or CITK-specific siRNAs, respectively. Note one of the two daughter cells in the lower panel (red arrow) going out of focus as a consequence of oblique division. Graphical representation of vertical and oblique divisions. The cleavage plane is indicated by a black dashed line. Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis (oblique division) in CITK-siRNA-treated HeLa cells compared to control (n > 50 cells, three independent experiments). Control or CITK-depleted cells were immunostained for γ-tubulin (red) and DNA (blue) and imaged in z (0.3-μm-thick sections). Upper panel: maximum-intensity projections of confocal z-stacks are shown. Lower panel: cross section (XZ) through the two poles of the same cell. Scale bars, 5 μm. Distribution of spindle angles (°) in control and in CITK-depleted cells. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip (n ≥ 150 cells, six independent experiments). Quantification of spindle angles average in control cells and in cells depleted of CITK or ASPM or codepleted of the two proteins (n ≥ 150 cells, in at least three independent experiments). Data information: Data shown in histograms are means ± SEM. Statistical significance was assessed using a two-tailed Student's t-test. ***P < 0.001, **P < 0.01. Download figure Download PowerPoint Altogether, these results show that CITK is a conserved determinant of spindle orientation and demonstrate that it acts in mitosis even before becoming enriched at the cleavage furrow and midbody during cytokinesis. CITK is associated with mitotic spindle through ASPM Several proteomic studies reported that CITK is associated with the mitotic spindle 606162, but this localization was not confirmed by other techniques. Previous immunolocalization studies have shown that CITK is localized to the nucleus in interphase cells, accumulates in the cytoplasm before anaphase, and becomes enriched at the cleavage furrow and at the midbody during cytokinesis 4663. A similar pattern was detected by live cell imaging in HeLa cells expressing physiological levels of GFP-tagged CITK from a stably integrated BAC transgene 646566 (Movie EV3). As expected, during mitosis the protein appears to be evenly distributed in the cytoplasm and no enrichment is visible at the spindle or at the cell cortex (Movie EV3). Although these data confirmed the previous reports 4663, they did not exclude the possibility that a pool of the protein may associate with mitotic spindle. To address this issue, we performed mild detergent extraction before fixation, which removes cytosolic proteins and facilitates the visualization of cytoskeleton-associated proteins. Under these conditions, immunofluorescence with anti-CITK antibodies detected a clear enrichment of the protein on both the spindle and the spindle poles, as it partially overlapped with the γ-tubulin signal (Fig 3A). This signal disappears in cells treated with CITK siRNA (Fig 3A). Similar results were obtained using anti-GFP antibodies in HeLa cells expressing GFP-tagged CITK from a BAC transgene 66 (Fig 3B). Specific association of CITK with spindle poles was further validated through biochemistry, as we found that CITK is enriched in centrosomal preparations obtained from HeLa cells synchronized in metaphase (Fig 3C). In addition, we observed that the signals of both CITK endogenous protein and CITK-GFP extensively overlap with ASPM immunoreactivity at the spindle poles (Fig 3D and E). Figure 3. CITK is associated with mitotic spindle poles through ASPM HeLa cells pre-extracted for 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), γ-tubulin (red), and DNA (blue). HeLa cells expressing CITK-GFP (green) from a BAC transgene treated as in (A) and immunostained for γ-tubulin (red) and DNA (blue). Western blot of centrosome-containing fractions from HeLa cells, showing that CITK copurifies with γ-tubulin. HeLa cells pre-extracted 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), ASPM (red), and DNA (blue). HeLa cells expressing CITK-GFP treated as in (A) and immunostained for GFP (green), ASPM (red), and DNA (blue). Close physical proximity between CITK and ASPM, revealed by PLA on ASPM-GFP-expressing HeLa cells, incubated with GFP and CITK antibodies and developed with PLA-specific secondary reagents. Control or CITK-depleted cells immunostained for ASPM (green), γ-tubulin (red), and DNA (blue). Control or ASPM-depleted cells treated as in (A) and immunostained for CITK (green), γ-tubulin (red), and DNA (blue). Quantification of CITK-positive centrosomes and of the ratio of CITK spindle pole intensity versus total cell mean intensity, in control and ASPM-depleted cells (n ≥ 75 cells, four independent experiments). Data presented are means ± SEM. ***P < 0.001; **P < 0.01. Statistical significance was assessed using a two-tailed Student's t-test. Data information: All cell images represent maximum-intensity projections of confocal z-stacks. Scale bars: 10 μm (A, B), 5 μm (D, E, G and H), and 3 μm (F). Download figure Download PowerPoint To confirm the physical interaction between CITK and ASPM, we performed in situ proximity ligation assay (PLA) which can reveal whether two proteins are at a distance < 40 nm 67. In mitotic cells expressing physiological levels of ASPM-GFP 66 and immunostained for GFP and CITK, we observed a strong signal, which was completely absent in normal HeLa cells, not expressing tagged ASPM (Fig 3F). Fluorescen
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