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The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization [1]

['Umut Batman', 'Department Of Molecular Biology', 'Genetics', 'Koç University', 'Istanbul', 'Jovana Deretic', 'Elif Nur Firat-Karalar', 'Koç University School Of Medicine']

Date: 2022-07

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

In this study, we examined the localization, interactions, functions, and mechanisms of CCDC66 during cell division. We showed that CCDC66 is required for recruitment of core machinery of centrosome maturation to the centrosomes and acts as a bundling protein in vitro and in cells. Its association with centrosomes and MTs is required for spindle assembly and organization, K-fiber and midbody integrity, chromosome alignment, and cytokinesis. Our findings unravel nonciliary functions for CCDC66 during cell division and provide insight into the integrated activity of centrosomes and MAPs during spatiotemporal regulation of MT nucleation and organization in mitosis and cytokinesis.

We previously characterized coiled coil protein 66 (CCDC66) as a MAP and a regulator of primary cilium formation and composition in quiescent cells [ 47 , 48 ]. It was originally described as a gene mutated in retinal degeneration and later characterized for its retinal and olfactory functions using CCDC66 −/− mouse [ 49 – 52 ]. Recently, CCDC66 was identified as part of the Joubert syndrome interaction network consisting of other MAPs such as CSPP1, TOGARAM1, and CEP290 [ 53 ]. Consistent with its link to ciliopathies, we and others previously showed that retinal degeneration mutations disrupt its ciliary functions and interactions [ 48 , 51 ]. In addition to its ciliary functions, following lines of evidence suggest that CCDC66 might function as a regulator of cell division: CCDC66 mRNA was identified in the MT-interacting transcriptome of Xenopus tropicalis, indicative of its functions during MT-based cellular processes [ 54 ]. Additionally, CCDC66 localized to centrosomes and MTs in dividing cells and its depletion led to disorganized poles in mitotic cells [ 48 , 54 ]. Finally, CCDC66 proximity interactome generated from asynchronous cells revealed interactions with regulators of cell division [ 48 , 55 , 56 ]. However, the full extent of CCDC66 functions during different stages of cell division and the underlying molecular mechanisms are not known. Addressing these key unknowns will uncover the relationship of CCDC66 with other components of the mitotic and cytokinetic machinery and also provide insight into whether and if so, how its nonciliary functions contribute to its disease mechanisms.

The drastic remodeling of the MT network during cell division requires precise regulation of when and where MAPs are activated. A subset of MAPs is active during mitosis but inactive during interphase. Such regulation is achieved by modulation of their affinity to MTs, regulation of their cellular abundance, and localization and posttranslational modifications [ 37 ]. Importantly, there is also a group of MAPs with dual functions in dividing and nondividing cells [ 38 – 40 ]. For example, End Binding 1 (EB1) regulates MT plus-end dynamic and targets other MAPs to the plus ends both in interphase and mitosis [ 41 – 44 ]. Recently, a critical regulator of primary cilium assembly in nondividing cells, intraflagellar transport protein 88 (IFT88), has been described for its functions during mitotic spindle orientation and central spindle organization [ 45 , 46 ]. Importantly, the discovery of nonciliary functions of IFT88 unraveled that its mutations might contribute to polycystic kidneys with both impaired ciliary function and aberrant cell division [ 45 , 46 ]. Despite the progress made in the characterization of MAPs with dual roles in cycling and noncycling cells, questions remain about their functions, mechanisms, and modes of regulation in different stages of the cell cycle.

Organization of MTs into highly ordered mitotic and cytokinetic arrays play critical roles for cell division. For example, 20 to 40 kinetochore MTs in human cells form parallel bundles termed K-fibers, which run from spindle poles to kinetochores and are essential for chromosome alignment and segregation [ 26 – 29 ]. Bridging fibers, composed of antiparallel bundles of interpolar MTs, connect 2 sister K-fibers and push them apart to separate spindle poles [ 29 , 30 ]. Similarly, in anaphase, an antiparallel MT bundle forms the central spindle/midzone that pushes the spindle poles to opposite side of the cell and directs the localization of ingression furrow important for the division of cytoplasm [ 8 ]. Antiparallel MT bundles at the spindle midzone are cross-linked by the evolutionarily conserved Protein Translocator of Cytokinesis 1 (PRC1)/Ase1/MAP65 family [ 31 – 34 ]. Central spindle shortens to form the midbody during cytokinesis, which will direct the membrane abscission site [ 35 ]. Moreover, astral MTs, which emanate from the centrosomes, interact with the cell cortex to position the spindle within a cell and determine the initial cleavage plane through communication with the equatorial cortex [ 36 ]. Although multiple MAPs involved in the formation and stabilization of the distinct spindle bundles have been identified, the full extent of MAPs involved in MT cross-linking and stability as well as their mechanism of action have yet to be determined in future studies.

In animal somatic cells, MT nucleation is initiated at the centrosomes, the preexisting spindle MTs, and the chromatin, with centrosomes being the major MT-organizing centers [ 9 , 11 ]. The mechanisms by which these distinct pathways work, their relative contributions to formation of diverse MT arrays in cells, and the extent of their crosstalk have been an area of active investigation. As cells enter mitosis, pericentriolar material (PCM) around centrioles expands in a process called centrosome maturation, which increases its MT-nucleation capacity [ 12 , 13 ]. Centrosome maturation is initiated by PLK1-dependent phosphorylation of pericentrin and CDK5RAP2, which promotes recruitment of additional PCM proteins including Cep152, Cep192, and gamma-tubulin [ 14 – 21 ]. Acentrosomal MT nucleation during mitosis is triggered at the chromosomes in RanGTP, Op8/Stathmin, and the chromosomal passenger complex (CPC)-dependent pathways, and at the spindle MTs in a HAUS/augmin complex-dependent way [ 9 , 22 – 25 ].

Faithful segregation of genetic and cytoplasmic material during cell division is essential for growth and development of multicellular organisms. Deregulation of the molecular processes that regulate cell division leads to aneuploidy and chromosomal instability and thereby to the initiation and progression of various human cancers [ 1 , 2 ]. As such, mitosis and cytokinesis are highly regulated, multistep processes involving dynamic regulation and coordinated activity of multiple cellular structures and signaling pathways [ 3 – 5 ]. In particular, microtubule (MT) cytoskeleton undergoes a series of morphological changes to form diverse MT arrays such as the bipolar spindle, central spindle, and midbody. Precise spatiotemporal control of the assembly, maintenance, and dynamic remodeling of these MT arrays requires a diverse group of the MT-associated proteins (MAPs), which bind to MTs and regulate their dynamic properties, organization, and stability as well as their interactions with other proteins and cellular structures [ 6 , 7 ]. Thereby, MAPs play essential roles during numerous cell cycle processes including MT nucleation, formation, and organization of the mitotic spindle and central spindle, chromosome capture, alignment and segregation, and cleavage furrow formation and abscission [ 7 – 9 ]. Proteomic profiling of MT-based structures of dividing cells, functional screens, and loss-of-function studies have identified hundreds of MAPs as regulators of mitosis and cytokinesis [ 7 , 10 ]. However, key questions remain about their functions, mechanisms, and links to disease as well as how they cooperate with different cellular structures (i.e., centrosomes), protein complexes, and signaling pathways to modulate the parameters that ultimately define the size, shape, and dynamics of MT arrays.

Results

CCDC66 localizes to centrosomes and MTs during mitosis and cytokinesis Based on its previously reported localization and interactions, we hypothesize that CCDC66 plays important roles during mitosis and cytokinesis [48,54,55]. To test this, we first examined localization of endogenous and mNeonGreen-CCDC66 fusion proteins at different cell cycle stages in mammalian cell lines. Antibody against endogenous CCDC66 revealed its localization to the centrosome throughout the cell cycle (Figs 1A and S1A). In dividing cells, CCDC66 also localized to multiple MT-based structures including the spindle MTs in prometaphase and metaphase, the central spindle in anaphase, and the midbody in cytokinesis in human osteosarcoma (U2OS) cells (Figs 1A and S1A). To examine the dynamic localization of CCDC66 during cell cycle, we generated cell lines that stably express mNeonGreen (mNG)-CCDC66 using lentiviral transduction. mNG protein was chosen over green fluorescent protein (GFP) as the fluorescent tag due to its higher fluorescent intensity and stability [57]. Stable expression of the fusion protein in U2OS and RPE1 cells was validated by immunoblotting using mNG antibody and immunofluorescence using anti-CCDC66 antibody (S1B–S1D Fig). In fixed U2OS and RPE1 stable cells, mNG-CCDC66 localized to the centrosome and centriolar satellites in interphase cells (Figs 1B and S1E) and to the centrosomes and MT-based structures of mitosis and cytokinesis in dividing cells (Figs 1B and S1E). Although CCDC66 localization to the astral MTs and midzone was apparent in cells stably expressing mNG-CCDC66, it was very weak in cells stained for endogenous CCDC66 (Figs 1A and S1A). This might be due to the high cytoplasmic and punctate background associated with CCDC66 antibody staining and/or the relatively lower abundance of CCDC66 at the astral microtubules and spindle midzone. In agreement with its localization in fixed cells, time-lapse imaging of mNG-CCDC66 cells stained with SIR-tubulin showed that CCDC66 dynamically localized to the centrosome, centriolar satellites, and MT-based structures in different cell states (S1F and S1G Fig and S1 and S2 Movies). PPT PowerPoint slide

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TIFF original image Download: Fig 1. CCDC66 localizes to centrosomes and microtubule-based structures during cell division. (A) Localization of CCDC66 at different stages of the cell cycle. U2OS were fixed with methanol followed by acetone and stained for CCDC66, gamma-tubulin, and DAPI. Scale bar: 5 μm, insets show 4× magnifications of the boxed regions. (B) Localization of mNeonGreen-CCDC66 at different stages of the cell cycle. U2OS cells stably expressing mNeonGreen-CCDC66 fusion (U2OS::mNG-CCDC66) were fixed with 4% PFA and stained for alpha-tubulin and DAPI. Scale bar: 5 μm, insets show 4× magnifications of the boxed regions. (C) Schematic representation of FL CCDC66 domain organization. CC1 and CC2 indicate coiled-coil domains. The C-terminal region was previously described as a microtubule-binding region [48]. Still confocal images show U2OS cells transfected with mNeonGreen-CCDC66 C-terminal construct (mNG-CCDC66570-948). Approximately 24 h post-transfection, cells were fixed with 4% PFA and stained for alpha-tubulin and DAPI. mNG-CCDC66570-948 localizes to centrosomes and spindle microtubules during metaphase and to midbody during cytokinesis. Scale bar: 5 μm. (D) mNG-CCDC66570-948 sequesters gamma-tubulin to cytoplasmic aggregates. U2OS cells were transfected with mNG-CCDC66570-948, fixed with 4% PFA, and stained for gamma-tubulin and DAPI. Top panel shows a lower expressing cell in which mNG-CCDC66570-948 is restricted mostly to centrosomes. Middle panel shows a high-expressing cell with multiple cytoplasmic aggregates of mNG-CCDC66570-948 that co-localize with gamma-tubulin. Bottom panel shows mNG-CCDC66570-948 transfected cells that are treated with 5 μg/ml nocodazole for 1 h at 37°C and incubated for 10 min after nocodazole washout. Scale bar: 5 μm, insets show 4× magnifications of the boxed regions. CC, coiled-coil domain; CCDC66, coiled-coil domain-containing protein 66; DAPI, 4′,6-diamidino-2-phenylindole; FL, full length; PFA, paraformaldehyde. https://doi.org/10.1371/journal.pbio.3001708.g001 Previously, we showed that CCDC66 and its N-terminal 1–570 and C-terminal 570–948 amino acid residue fragments localize to MTs in cells [48]. Given that C-terminal fragment binds to MTs directly, we tested whether this C-terminal fragment recapitulates the localization of full-length CCDC66 during cell division. Like mNG-CCDC66, mNG-CCDC66 (570–948) localized to the centrosomes, bipolar spindle, and central spindle during mitosis and midbody during cytokinesis in U2OS cells (Fig 1C). When overexpressed, mNG-CCDC66 (570–948) formed cytoplasmic aggregates that recruited gamma-tubulin, suggesting a putative interaction between them (Fig 1D). To investigate the functional significance of this recruitment during MT nucleation, we performed MT regrowth experiments and found that the cytoplasmic aggregates nucleated MTs 10 min after nocodazole washout (Fig 1D). Together, these data indicate that the C-terminal 379 residues of CCDC66 are sufficient for its cellular localization to the centrosome and MTs in dividing cells.

CCDC66 is required for spindle assembly and orientation The localization of CCDC66 to spindle microtubules suggests that mitotic progression defects associated with its depletion might be a consequence of defective spindle assembly and orientation. To investigate this, we analyzed various spindle properties in control and CCDC66-depleted cells. First, we measured the angle between the spindle axis and the substratum, which revealed an increase from 7.7 ± 0.3° in control cells to 13.1 ± 1.5° in CCDC66-depleted cells (Fig 4A). By taking into account the changes into spindle angle, we quantified spindle length and found that it was not altered upon CCDC66 depletion (Fig 4A). Likewise, centrosome width at metaphase was comparable between control and CCDC66-depleted cells (Fig 4A). The essential role of astral MTs in spindle positioning and orientation led us to investigate CCDC66 functions during astral MT assembly and stability [62]. To this end, we quantified the astral MT fluorescence intensity and length in control and CCDC66-depleted cells and found that they were both reduced upon CCDC66 loss (Fig 4B and 4C). Likewise, the tubulin fluorescence intensity at the spindle in metaphase cells decreased about 0.6-fold in CCDC66-depleted cells relative to control cells (Fig 4B and 4C). Immunoblotting of lysates prepared from control and CCDC66 siRNA-transfected cells showed that the intensity changes in spindle MTs were not due to altered cellular abundance of alpha-tubulin (S4A Fig). Together, these results show that CCDC66 is required for spindle MT assembly and stability. PPT PowerPoint slide

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TIFF original image Download: Fig 4. CCDC66 regulates spindle organization and orientation. (A) Effects of CCDC66 depletion on spindle angle, length, and pole width. U2OS cells were transfected with control and CCDC66 siRNA, fixed with methanol and stained for gamma-tubulin; z on still images indicates the stack where the centrosome is found. Spindle angle is calculated by the formula α = 180*tan−1(h/L)/π where h represents the stack difference between 2 centrosomes, L represents the distance between centrosomes. SL is calculated by the formula SL = √(h2+L2) where h represents the stack difference between 2 centrosomes, L represents the distance between centrosomes when projected onto 1 z plane. Centrosome width at metaphase is calculated by measuring the length of the PCM of the centrosome. Data represent the mean ± SEM of 3 independent experiments. (**p < 0.01, ns: not significant). Scale bar: 5 μm. (B) Spindle microtubule density and astral microtubule length is reduced in CCDC66-depleted cells. U2OS cells were transfected with siRNA, then fixed with methanol, and stained for alpha-tubulin and DAPI. Representative images are shown. Inverted image is shown to emphasize astral microtubules better. Scale bar: 5 μm. (C) Quantification of (B). Astral microtubule and spindle microtubule intensity were measured on ImageJ by taking several points on the spindle to measure the intensity and subtracting the background mean intensity. Data represent the mean ± SEM of 3 independent experiments. (****p < 0.0001). (D) CCDC66 depletion reduces K-fiber intensity. Cells were transfected with siRNA then 48 h after transfection, cells were incubated in ice for 10 min. Cells were fixed with methanol and stained for alpha-tubulin, gamma-tubulin, and DAPI. K-fiber intensity was measured as described for (C). Data represent the mean ± SEM of 2 independent experiments. (****p < 0.0001). Scale bar: 5 μm. (E) CCDC66 localizes to K-fibers. RPE1::mNG-CCDC66 stable cell line was incubated in ice for 10 min and fixed with MeOH then stained for mNG and ACA. Images represent a single stack and were captured with the same camera settings from the same coverslip. Scale bar: 5 μm. The data underlying the graphs shown in the figure can be found in S1 Data. ACA, anticentromeric antibody; CCDC66, coiled-coil domain-containing protein 66; DAPI, 4′,6-diamidino-2-phenylindole; K-fiber, kinetochore-fiber; L, length; SEM, standard error of mean; siRNA, small interfering RNA; SL, spindle length. https://doi.org/10.1371/journal.pbio.3001708.g004 Proper formation and organization of K-fibers is essential for chromosome alignment and segregation, suggesting that chromosome-related defects in CCDC66-depleted cells could be due to impaired K-fibers [29,63]. To determine whether CCDC66 is specifically required for the K-fiber stability, we performed cold stability assay in control and CCDC66-depleted cells. In this assay, K-fibers are visualized by selective depolymerization of less stable interpolar and astral MTs by cold treatment of cells for 10 min at 4°C [64,65]. The tubulin fluorescence intensity of cold-stable K-fibers was reduced in CCDC66-depleted cells relative to control cells (Fig 4D), which identify CCDC66 as a regulator of K-fiber stability. Notably, mNG-CCDC66 and endogenous CCDC66 still localized to the spindle microtubules in cold-treated cells, which confirms its localization to K-fibers (Figs 4E and S4C). Collectively, our findings indicate that CCDC66 regulates spindle assembly and orientation to ensure proper mitotic progression.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001708

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