Human Microcephaly Protein RTTN Is Required for Proper Mitotic Progression and Correct Spindle Position

Autosomal recessive primary microcephaly (MCPH) is a complex neurodevelopmental disorder characterized by a small brain size with mild to moderate intellectual disability. We previously demonstrated that human microcephaly RTTN played an important role in regulating centriole duplication during interphase, but the role of RTTN in mitosis is not fully understood. Here, we show that RTTN is required for normal mitotic progression and correct spindle position. The depletion of RTTN induces the dispersion of the pericentriolar protein γ-tubulin and multiple mitotic abnormalities, including monopolar, abnormal bipolar, and multipolar spindles. Importantly, the loss of RTTN altered NuMA/p150Glued congression to the spindle poles, perturbed NuMA cortical localization, and reduced the number and the length of astral microtubules. Together, our results provide a new insight into how RTTN functions in mitosis.


Introduction
In most animal cells, the centrosome is the major microtubule-organizing center (MTOC). It is composed of a pair of centrioles surrounded by pericentriolar material (PCM). Centrioles are barrel-shaped structures composed of microtubules (MTs), and as basal bodies, they serve as a template for the formation of cilia and flagella [1][2][3]. Autosomal recessive primary microcephaly (MCPH) is a rare genetically heterogeneous disorder characterized by small brain size with mild to severe mental retardation [4]. A number of genes that encode centriole/centrosome proteins have been identified as causal genes of MCPH, including MCPH1, WDR62, CDK5RAP2, CEP152, ASPM, CPAP/CENPJ, CEP63, and STIL [5]. Interestingly, more than half of microcephaly proteins were found to cooperate with each other and participate in centriole biogenesis and/or centrosome function.
We and others previously found that human microcephaly protein CPAP (centrosomal protein 4.1-associated protein) plays a key role in the regulation of centriole duplication, particularly in the control of centriole length [6][7][8]. We further demonstrated that various human microcephaly proteins could interact with each other, including CPAP-STIL [9], CPAP-SAS6-CEP135 [10], and RTTN-STIL [11], which are required for building a daughter centriole. In addition, it was reported that human microcephaly protein CEP152 interacts with not only CEP63 [12] but also CPAP [13], and WDR62 also forms a complex with ASPM and CEP63 that regulates centriole biogenesis [14]. These findings led us to propose that a defect in centriole biogenesis in neural progenitors is one of the key pathways leading to MCPH [5,[9][10][11].
RTTN (also known as Rotatin) is an evolutionarily conserved centrosome-associated protein found in many organisms. The RTTN gene was initially identified in a homozygous mutant mouse that shows defects in axial rotation, left-right specification, and embryonic lethality [15], and Ana 3 (a fly homologue of RTTN) was previously reported to be required for structural integrity of centrioles and basal bodies [16]. Interestingly, we found that complete loss of RTTN impairs centriole biogenesis and induces primitive procentriole bodies (PPBs) that contain the early-born centriolar proteins (e.g., SAS-6, STIL, and CPAP) but lack the later-born proteins (POC5 and POC1B), suggesting that the later process of centriole assembly is inhibited during the S phase in interphase cells [11].
Recently, a number of RTTN gene mutations have been identified in human patients with primary microcephaly associated with primordial dwarfism [17,18], lissencephaly associated with simplified gyral pattern [19,20], polymicrogyria associated with seizures [21], and pontocerebellar hypoplasia [20]. Interestingly, skin fibroblasts from patients with RTTN gene mutations exhibit mitotic defects, leading to aneuploidy and apoptosis [22]. However, the fundamental roles of RTTN in mitosis are not completely understood. Here, we reported that loss of RTTN disrupted normal cell progression, including G2/M phase arrest and increased aneuploidy cells. Additionally, RTTN deletion results in PCM dispersion and multiple mitotic abnormalities, including monopolar, abnormal bipolar, or multipolar spindles. Moreover, loss of RTTN caused the spindle positioning defects, such as mis-localization of cortical NuMA and reduction of the number and the length of astral microtubules. A possible mechanism of how RTTN participates in mitosis is discussed.

Immunofluorescence Confocal Microscopy
Cells grown on coverslips were fixed in methanol at −20 • C for 5 min. To examine the astral microtubules, cells were pre-extracted with 0.5% Triton X-100 in PHEM buffer (120 mM PIPES, 50 mM HEPES, 20 mM EGTA, 8 mM MgSO 4 ) for 1 min at 37 • C then fixed in methanol at −20 • C for 5 min. The fixed cells were then blocked with 10% normal goat serum in PBS and incubated with the indicated primary antibodies. After being washed with PBST (PBS containing 0.1% Tween-20), the cells were incubated with Alexa Fluor 488-, Alexa Fluor 568-, or Alexa Fluor 647-conjugated secondary antibodies (Invitrogen). DNA was counterstained with DAPI (4,6-diamidino-2-phenylindole). The samples were mounted in Vectashield mounting media (Vector Laboratories) and visualized on a confocal microscope (LSM 880; Carl Zeiss, Jena, Germany) with a Plan Apochromat 63x/1.4 NA oil-immersion objective. Images were acquired with the ZEN software (Carl Zeiss).

Flow Cytometry Analysis
To analyze the cell cycle, cells were fixed in ice-cold 100% ethanol for 30 min, washed with PBS, and stained with 40 µg/mL propidium iodide at room temperature for 30 min. Cells were then analyzed by flow cytometer (Attune-NxT; Thermo Fisher Scientific, Waltham, MA, USA).

Spindle Orientation Analysis
To examine whether RTTN depletion may cause the spindle orientation defect, we measured the linear distance of the xy plane and the vertical distance of the z plane between the two spindle poles (the maximum intensity of CDK5RAP2 signal was used as the spindle pole marker) of metaphase cells and calculated the spindle angles α. Z-stack images (0.95 µm per stack) of metaphase cells stained with antibodies against CDK5RAP2 and α-tubulin were analyzed using the ZEN software (Carl Zeiss).

Statistics
To determine the significance among the two or more experimental conditions, an unpaired two-tailed Student's t test or one-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 5 software) was used for analysis. Data are presented as the mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.0001 were considered statistically significant.

Loss of RTTN Altered NuMA/p150Glued Distribution during Mitosis
It was reported that, during early mitosis, the interaction of NuMA with dynein and Eg5 is required to tether the microtubules to the pole and focus the astral microtubules to the centrosome [24][25][26]. To investigate whether loss of RTTN altered the localization of NuMA and p150Glued (a subunit of dynactin complex that acts as a co-factor for the microtubule motor cytoplasmic dynein) during mitosis, we performed immunofluorescence staining on cells with antibodies against NuMA and p150Glued. Our results showed that NuMA and p150Glued signals normally exhibited an umbrella shape that congressed to the spindle poles in the control cells (RTTN +/+ ; p53 +/+ and RTTN +/+ ; p53 −/− ) during mitosis ( Figure 3A, left and middle panels), and their quantitation at the spindle poles is shown in Figure 3B (NuMA) and Figure 3C (p150Glued).
NuMA and p150Glued (a subunit of dynactin complex that acts as a co-factor for the mi-crotubule motor cytoplasmic dynein) during mitosis, we performed immunofluorescence staining on cells with antibodies against NuMA and p150Glued. Our results showed that NuMA and p150Glued signals normally exhibited an umbrella shape that congressed to the spindle poles in the control cells (RTTN +/+ ; p53 +/+ and RTTN +/+ ; p53 −/− ) during mitosis ( Figure 3A, left and middle panels), and their quantitation at the spindle poles is shown in Figure 3B (NuMA) and Figure 3C (p150Glued).  In addition to the umbrella pattern, a portion of NuMA (~30%, Figure 3D) displayed a cortical localization laterally to the spindle poles at one end or both ends in control RPE1 metaphase cells (arrowheads in RTTN +/+ ; p53 +/+ and RTTN +/+ ; p53 −/− cells, Figure 3A, left and middle panels). Interestingly, RTTN knockout caused the disruption of the umbrellashaped spindle pole to become more disorganized in RTTN −/− ; p53 −/− cells ( Figure 3A, right panel; Figure 3B,C) but without protein loss of NuMA and p150Glued ( Figure 3E). Intriguingly, the proportion of mis-localized cortical NuMA (from lateral distribution to random distribution) was significantly increased (~53%) in RTTN −/− ; p53 −/− cells compared to the control groups (arrowheads in Figure 3A, quantitation in Figure 3D). Together, our results suggest that RTTN plays an essential role in regulating the spindle orientation and positioning, in which the NuMA/Dynein complex is involved.
p53 −/− cells. (D) Histograms illustrating the percentages of metaphase cells with cortical NuMA (both lateral and mis-localized patterns). Data are represented as the mean ± SEM from three independent experiments. Data are analyzed by one-way ANOVA with Tukey's multiple comparison test. *** p < 0.0001. Scale bars: 5 μm.   d (a, b)) and the z plane vertical distance (d (z)) between two spindle poles (a and b) obtained from a series of z-stack images were used to calculate the spindle angle α.

Discussion
Recessive mutations in the RTTN gene were identified as a cause of primary microcephaly, polymicrogyria, and lissencephaly, which exhibit heterogeneous clinical phenotypes and cerebral malformations. We and others previously showed that RTTN (Rotatin) is a centriolar protein that participates in centriole elongation during interphase [11], and RTTN mutant cells exhibited severe mitotic failure with centrosome amplification and multipolar spindle formation [22]. However, the molecular mechanism of how RTTN functions in mitosis remains largely unknown. Here, we report that complete loss of RTTN resulted in PCM dispersion and multiple mitotic abnormalities (e.g., monopolar, abnormal bipolar, and multipolar spindle formation), leading to aneuploidy and apoptosis (Figures 1 and 2), consistent with a previous report [22]. Importantly, RTTN loss impeded the congression of NuMA and p150Glued at the mitotic spindle poles, caused the mis-localization of cortical NuMA (Figure 3), and reduced the number and the length of astral microtubules (Figure 4).
Proper cell division relies on accurate mitotic spindle positioning. In mammalian cells, the mitotic spindle positioning is mostly governed by the evolutionary conserved ternary complex NuMA/LGN/Gαi, which serves to anchor the microtubule minus end-directed motor protein complex dynein at the cell cortex [32]. Thus, the cortical dynein positions the spindle by exerting pulling forces on astral microtubules and/or by anchoring the plus-end of astral microtubules to the cortex [27,32,33]. In this study, we found that loss of RTTN caused PCM dispersion ( Figure 1A), altered NuMA/p150Glued congression to the spindle poles ( Figure 3A-C), induced mis-localization of cortical NuMA ( Figure 3A,D), and destabilized astral MTs ( Figure 4A). Importantly, we found that loss of RTTN causes spindle misorientation ( Figure 5). Together, our findings suggest that RTTN may have a role in maintaining spindle pole integrity and spindle position, at least in part, through a NuMA/dynein-mediated process during mitosis.
Recently, several MCPH proteins (e.g., WDR62, CDK5RAP2, CENPJ/CPAP, and STIL) were reported to be involved in regulating spindle position, centrosome duplication, centrosome integrity, and/or microtubule stabilization [6,9,14,29,34,35]. Mitotic spindle orientation is essential for cell fate decisions, particularly in neuronal progenitor cells (NPCs) that divide symmetrically or asymmetrically to produce different cell fates during neurogenesis [36,37]. Intriguingly, the loss of WDR62, CDK5RAP2, and ASPM induced the spindle misorientation of NPCs, leading to premature neuronal differentiation in the developing cerebral cortex [34,38,39]. Furthermore, our recent data demonstrated that conditional Cpap deletion in the central nervous system preferentially induces multiple mitotic abnormalities with predominantly monopolar spindles in NPCs, perturbs symmetric NPC divisions, and promotes premature neuronal differentiation [40]. Here, we found that loss of RTTN induces multiple mitotic abnormalities and perturbs cortical NuMA localization and astral microtubule stabilization. We hypothesize that such an alteration in mitotic NPCs may induce spindle positioning defects, resulting in impeding NPC division mode and promoting premature neuronal differentiation. Future investigation of the shRttn-treated developing brain or conditional RTTN knockout mice may elucidate this possibility.

Conclusions
We showed that the human microcephaly protein RTTN plays an essential role in mitosis. RTTN is required for proper mitotic progression, the maintenance of spindle pole integrity, and correct spindle position.