A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice

Ford, Matthew J.; Yeyati, Patricia L; Mali, Girish R.; Keighren, Margaret; Waddell, Scott H.; Mjoseng, Heidi K.; Douglas, Adam; Hall, Andrew G.; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Meehan, Richard R.; Boulter, Luke; Jackson, Ian J.; Mill, Pleasantine; Mort, Richard L.

doi:10.1016/j.devcel.2018.10.027

Cited by 70 publications

(83 citation statements)

References 81 publications

(116 reference statements)

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“…As the primary cilium is an important signaling hub, the control of cilia assembly and disassembly is critical. In most differentiated somatic cells, the primary cilium is fully disassembled before mitosis before being reassembled following division in G1 of the next cell cycle (Pugacheva et al, 2007;Ford et al, 2018). By contrast, certain neuronal stem cells only partially disassemble the cilium in mitosis.…”

Section: Introductionmentioning

confidence: 99%

Nek2 kinase displaces distal appendages from the mother centriole prior to mitosis

Viol

Hata

Pastor-Peidro

et al. 2020

Journal of Cell Biology

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Distal appendages (DAs) of the mother centriole are essential for the initial steps of ciliogenesis in G1/G0 phase of the cell cycle. DAs are released from centrosomes in mitosis by an undefined mechanism. Here, we show that specific DAs lose their centrosomal localization at the G2/M transition in a manner that relies upon Nek2 kinase activity to ensure low DA levels at mitotic centrosomes. Overexpression of active Nek2A, but not kinase-dead Nek2A, prematurely displaced DAs from the interphase centrosomes of immortalized retina pigment epithelial (RPE1) cells. This dramatic impact was also observed in mammary epithelial cells with constitutively high levels of Nek2. Conversely, Nek2 knockout led to incomplete dissociation of DAs and cilia in mitosis. As a consequence, we observed the presence of a cilia remnant that promoted the asymmetric inheritance of ciliary signaling components and supported cilium reassembly after cell division. Together, our data establish Nek2 as an important kinase that regulates DAs before mitosis.

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Section: Introductionmentioning

confidence: 99%

Nek2 kinase displaces distal appendages from the mother centriole prior to mitosis

Viol

Hata

Pastor-Peidro

et al. 2020

Journal of Cell Biology

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“…Various cell cycle sensors have been developed, starting with FUCCI in 2008 (Sakaue-Sawano et al, 2008), each optimized for different research paradigms (Abe et al, 2013;Bouldin and Kimelman, 2014b;Fukuhara et al, 2014;Ogura et al, 2011;Pineda et al, 2016;Ridenour et al, 2012;Sakaue-Sawano et al, 2017;Sugiyama et al, 2009;Zielke et al, 2016). While FUCCI-style biosensors have been developed to better assess G0/G1 in zebrafish using inactivated p27 (Oki et al, 2014), and in tissue culture by assessing the primary cilium cycle (Ford et al, 2018), they require multiple transgenes to readout cell cycle states accurately. As DHB responds to CDK levels, it is likely to function broadly across a wide range of organisms.…”

Section: Discussionmentioning

confidence: 99%

“…An additional limitation of the original FUCCI-based sensors is an inability to precisely distinguish between S and G2. This has been addressed with newer FUCCI variants, which utilize additional fluorescently labeled cell cycle genes that are able to distinguish extra cell cycle states (Bajar et al, 2016;Ford et al, 2018;Grant et al, 2018;Sakaue-Sawano et al, 2017); however, these variants require spectrally separable fluorophores for each discrete cell cycle state to be measured (Zielke and Edgar, 2015). As few labs have the equipment necessary to separate more than three or four fluorophores simultaneously, the requirement of several fluorophores to visualize cell cycle state can impede the researcher's ability to study additional cell biological behaviors by fluorescence microscopy, limiting the scope and complexity of cell cycle studies.…”

Section: Introductionmentioning

confidence: 99%

Visualizing the metazoan proliferation-terminal differentiation decisionin vivo

Kohrman

Adikes

Martinez

et al. 2019

Preprint

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During organismal development, differential regulation of the cell cycle is critical to many cell biological processes, including cell fate specification and differentiation. While the mechanisms of cell cycle regulation are well studied, how control of the cell cycle is linked to differentiated cellular behavior remains poorly understood, mostly due to our inability to directly and precisely measure cell cycle state. In order to characterize cell cycle state live, we adapted a cyclindependent kinase (CDK) biosensor for in vivo use in the roundworm nematode, Caenorhabditis elegans. The CDK biosensor measures the cytoplasmic-to-nuclear localization of a portion of human DNA Helicase B (DHB) fused to a fluorescent protein to assess cell cycle state. The dynamic localization of DHB results from phosphorylation of the biosensor by CDKs, thereby allowing for quantitative assessment of cell cycle state. We demonstrate here the use of this biosensor to quantify lineage-specific differences between cycling cells and to examine the proliferation-differentiation decision. Unlike other live cell imaging tools (e.g., FUCCI), we show that DHB can be used to distinguish between actively cycling cells in the G1 phase of the cell cycle and terminally differentiated cells exited in G0. Thus, we provide here a new resource to study the control and timing of the metazoan cell cycle during cell fate specification and differentiation.

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“…Specifically, we found that Cyclin D1-CDK4/6 accelerates the G1/S transition in DFCs, while also facilitates cilia formation via stabilization of zFoxj1a. Ciliary dynamics appear to be precisely coordinated with cell cycle progression [59]. It has been suggested previously that cell quiescence is essential for the formation of mouse nodal cilia [60].…”

Section: Discussionmentioning

confidence: 99%

Chemokine signaling links cell cycle progression and cilia formation for left-right symmetry breaking

Zhu

Guan

et al. 2019

Preprint

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23 Zebrafish dorsal forerunner cells (DFCs) undergo vigorous proliferation during 24 epiboly and then exit cell cycle to generate Kupffer's vesicle (KV), a ciliated organ 25 necessary for establishing left-right (L-R) asymmetry. DFC proliferation defects are 26 often accompanied by impaired cilia elongation in KV, but the functional and 27 molecular interaction between cell-cycle progression and cilia formation remains 28 unknown. Here we show that chemokine receptor Cxcr4a is required for L-R laterality 29 by controlling DFC proliferation and KV ciliogenesis. Functional analysis revealed 30 that Cxcr4a accelerates G1/S transition in DFCs and stabilizes Foxj1a, a master 31 regulator of motile cilia, by stimulating Cyclin D1 expression through ERK1/2 32 signaling. Mechanistically, Cyclin D1-CDK4/6 drives G1/S transition during DFC 33 proliferation and phosphorylates Foxj1a, thereby disrupting its association with 34 Psmd4b, a 19S regulatory subunit. This prevents the ubiquitin-independent 35 proteasomal degradation of Foxj1a. Our study uncovers a role for Cxcr4 signaling in 36 L-R patterning and provides fundamental insights into the molecular linkage between 37 cell-cycle progression and ciliogenesis. 38 39 D1-CDK4/6, Foxj1a, Psmd4b, ubiquitin-independent proteasomal degradation.41 42 43 44 3 Author summary 45 During the organogenesis of zebrafish L-R organizer named KV, DFCs 46 proliferate rapidly during epiboly and then exit the cell cycle to differentiate into 47 ciliated epithelial KV cells. Cell cycle defects in DFCs are often accompanied by an 48 alteration in KV cilia elongation. However, whether the cell cycle and cilia formation 49 are mechanistically linked remains as an open question. In this study, we report that 50 Cxcr4 signaling is required for DFC proliferation and KV ciliogenesis. We reveal that 51 Cxcl12b/Cxcr4a signaling activates ERK1/2, which then promotes Cyclin D1 52 expression. Cyclin D1-CDK4/6 accelerates the G1/S transition in DFCs, while also 53 facilitates cilia formation via stabilization of Foxj1a. Notably, Foxj1 undergoes 54 proteasomal degradation via Ub-independent pathway during KV organogenesis. Our 55 study further demonstrates that CDK4 phosphorylates and stabilizes Foxj1a by 56 disrupting its association with Psmd4b, a 19S regulatory subunit. In summary, 57 Cxcl12b/Cxcr4a chemokine signaling links cell cycle progression and cilia formation 58 for L-R symmetry breaking via regulating Cyclin D1 expression. 59 60 61 62 63 64 65 66 67 Vertebrates exhibit striking left-right (L-R) asymmetries in the structure and 68 position of their cardiovascular and gastrointestinal systems. Initially, early embryos 69 develop symmetrically along the prospective body midline. This embryonic symmetry 70 is broken during somite stages when an asymmetric fluid flow is generated by motile 71 cilia within the L-R organizer (LRO), a transient structure located at the posterior end 72 of the notochord [1]. Specifically, in zebrafish, the ciliated LRO is referred to as 73 Kupffer's vesicle (KV), which ...

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A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice

Cited by 70 publications

References 81 publications

Nek2 kinase displaces distal appendages from the mother centriole prior to mitosis

Nek2 kinase displaces distal appendages from the mother centriole prior to mitosis

Visualizing the metazoan proliferation-terminal differentiation decisionin vivo

Chemokine signaling links cell cycle progression and cilia formation for left-right symmetry breaking

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