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Review
. 2019 Jan;211(1):35-73.
doi: 10.1534/genetics.118.301367.

Mitotic Cell Division in Caenorhabditis elegans

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Free PMC article
Review

Mitotic Cell Division in Caenorhabditis elegans

Lionel Pintard et al. Genetics. .
Free PMC article

Abstract

Mitotic cell divisions increase cell number while faithfully distributing the replicated genome at each division. The Caenorhabditis elegans embryo is a powerful model for eukaryotic cell division. Nearly all of the genes that regulate cell division in C. elegans are conserved across metazoan species, including humans. The C. elegans pathways tend to be streamlined, facilitating dissection of the more redundant human pathways. Here, we summarize the virtues of C. elegans as a model system and review our current understanding of centriole duplication, the acquisition of pericentriolar material by centrioles to form centrosomes, the assembly of kinetochores and the mitotic spindle, chromosome segregation, and cytokinesis.

Keywords: Cell division; Centrosome; Cytokinesis; Kinetochore; Mitosis; Spindle assembly; WormBook.

Figures

Figure 1
Figure 1
Mitotic cell division in the early C. elegans embryo. (A) Differential interference contrast (DIC) image of an adult worm. The positions of the two most mature oocytes relative to the spermatheca are indicated by white arrows and labeled -1 and -2; embryos within the uterus are also indicated. Bar, 100 μm. (B) Live image of embryos in the uterus of an adult worm expressing GFP-tagged β-tubulin (green) and mCherry-tagged H2B (red) to visualize oocyte meiotic and embryonic mitotic spindles. (C) Frames from in vivo live imaging of mitotic embryos expressing GFP-tagged β-tubulin (green) and mCherry-tagged H2B (red), or (D) GFP::NPP19 (green) and mCherry::H2B (red), to mark microtubules, chromosomes, and the nuclear envelope, respectively. In these and other figures, anterior (A) is to the left and posterior (P) is to the right. Bar, 10 μm. (E) Accompanying schematics (microtubules in green, paternal and maternal chromosomes in blue and pink, respectively). Time in minutes relative to Nuclear Envelope Breakdown NEBD at time = 0.
Figure 2
Figure 2
Centriole architecture. (A) Schematic illustration of C. elegans and human centriole architecture. (B) Spatial localization of PCM components. For C. elegans, results from immuno-gold electron microscopy staining data are shown. Most of the SPD-2 signal accumulated within a 200 nm diameter range (Pelletier et al. 2004). Human data are based on 3D-SIM images (Sonnen et al. 2012). SPD-2 and its human homolog Cep192 localize at the interface between centriole and PCM, and SPD-2 is potentially a component of the paddlewheel in C. elegans (loss of SPD-2 from centriole in sas-7 mutants coincides with the loss of paddlewheel structure). Modified from Sugioka et al. (2017). (C) Cross-section and lateral view of wild-type and sas-7 mutant centrioles. Overlay indicates an interpretation of structures. Arrows indicate wild-type and defective sas-7 mutant paddlewheel structures. Bars, 50 nm. Modified from Sugioka et al. (2017). (D) Schematics showing the steps in the assembly and maturation of the daughter centriole along with the factors required for each step. Assembly of the daughter centriole begins when a cartwheel (red) forms at a right angle to the mother centriole. In the second step, an inner tube forms and subsequently mediates the peripheral assembly of nine symmetrically arranged microtubules (light blue) forms around the cartwheel. Assembly of the paddlewheel and acquisition of the ability to duplicate again (centriole maturation: daughter to mother centriole transition) requires SAS-7. (E) Early steps of cartwheel assembly with the ninefold symmetry dictated by the structure of SAS-6. SAS-6 contains an N-terminal globular domain, a long coiled-coil, and an unstructured C-terminal region. C. elegans SAS‐6 alone forms an antiparallel tetramer, whereas binding of SAS‐5 (green) disrupts the tetrameric association of SAS‐6. The N-terminal globular domains of SAS-6 form the hub of the cartwheel with the coiled-coil dimerized spokes projecting outward.
Figure 3
Figure 3
Pathways regulating C. elegans centriole duplication and assembly. (A) Phenotypes observed when a sperm cell containing a wild-type pair of centrioles fertilizes a wild-type oocyte (left column) or an oocyte lacking a component essential for daughter centriole formation (e.g., zyg-1 or sas-4 mutants; right column). Defects in centriole duplication lead to the assembly of monopolar spindles during mitosis in two-cell stage embryos. (B) Different inputs involving E3-ligases, the cullin 1-RING E3-ligases CRL1LIN-23 and CRL1SEL-10, and the APC/CFZR-1 that regulate ZYG-1 and SAS-5 protein levels to limit centriole duplication. Red blunt arrows represent negative regulation. SZY-20 also inhibits the pathway controlling centriole duplication (blue oval), but the underlying mechanism is not known.
Figure 4
Figure 4
Centrosomes and mitotic spindle assembly in the one-cell C. elegans embryo. (A) Frames from in vivo live imaging of mitotic embryos expressing GFP-tagged β-tubulin (green) and mCherry-tagged H2B (magenta) to mark microtubules and chromosomes, respectively, from nuclear envelope breakdown to anaphase. (B) Schematic of the C. elegans centrosome containing a pair of centrioles surrounded by pericentriolar material (green) and microtubules anchored at their minus ends. (C) Schematic of the mitotic spindle in the one-cell C. elegans embryo. (D) Centrosome localized factors regulating centriole duplication (yellow) and PCM assembly (green).
Figure 5
Figure 5
Functional analysis of kinetochore assembly and function in one-cell C. elegans embryos. (A) Schematics illustrating the kinetochore null (KNL) phenotype, characterized by a failure to assemble kinetochores competent for spindle microtubule attachment. Consequently, DNA segregation is severely defective and spindle poles separate prematurely and excessively in response to the astral pulling forces mediated by the Gα pathway, as schematized in the inset. This pathway, which comprises a complex of Gα, GPR-1/2, and LIN-5, anchors dynein to the cell cortex to generate pulling forces when dynein walks toward microtubule minus ends anchored at the spindle poles. These force generators are enriched posteriorly in response to cell polarity factors such that the spindle becomes posteriorly displaced and the division is asymmetric. (B) Spindle pole tracking detects defective kinetochore-microtubule attachments. Frames from in vivo live imaging videomicrographs of control and ndc-80(RNAi) one-cell embryos expressing GFP::H2b and GFP::γ-tubulin to label chromosomes (green arrow) and spindle poles (orange arrowheads), respectively, from NEBD to anaphase. Yellow arrowheads highlight premature spindle pole separation in ndc-80(RNAi) embryos as compared to wild-type. Bar, 5 μm. Modified from Cheerambathur et al. (2017).
Figure 6
Figure 6
C. elegans kinetochore assembly: building spindle microtubule attachment sites. (A) Schematic of C. elegans kinetochore assembly. Proteins directly interacting with microtubules are highlighted in light green. Arrows indicate dependencies. (B) Schematics of kinetochore proteins and activities that interact with microtubules. (C) Domain organization of the scaffolding protein KNL-1 (red), a large multidomain and multifunctional scaffold protein required for kinetochore targeting of several other outer-domain kinetochore proteins, including, the SAC proteins BUB-1 and BUB-3, NDC-80, MIS-12, and the RZZ complex. KNL-1 also contains a docking site (RRVSF) for the PP1 phosphatase, which dephosphorylates KNL-1 on the MELT repeats and thereby eliminates the interaction between BUB-1 and KNL-1.
Figure 7
Figure 7
Kinetochores direct FZY-1/CDC-20 to the mitotic accelerator (APC/C) or brake (SAC) in response to microtubule attachment status: Schematics showing the two fates of CDC-20. CDC-20 is dynamically recruited to kinetochores where it interacts with BUB-1, via the ABBA motif. In the presence of unattached kinetochores (top panel) phosphorylated CDC-20 binds MAD-2 in the closed conformation and together with MAD-3 and BUB-3 assembles the mitotic checkpoint complex (MCC) that prevents APC/C activation. The MCC binds to a CDC-20 subunit physically associated with the APC/C and prevents it from binding substrates. It is known in mammalian cells that the MCC is continuously degraded as it binds the APC/C. When all kinetochores are properly attached to spindle microtubules and chromosomes are aligned at the metaphase plate (bottom panel), the generation of new MCC complexes is stopped. CDC-20 is dephosphorylated at the kinetochore by PP1 (and unknown cytosolic phosphatases) and then binds the APC/C after the previously bound MCC has been degraded [for review see Alfieri et al. (2017)].
Figure 8
Figure 8
A subset of kinetochore components drive central spindle assembly. (A) Schematics of the assay used to assess the mechanical integrity of the central spindle. When the mechanical integrity of the central spindle is compromised (“weak central spindle”), spindle poles separate prematurely and excessively in response to the astral pulling forces (red arrows) as compared to control embryos. Downregulation of the astral pulling forces via gpr-1/2 RNAi suppresses this phenotype. (B and C) Kymographs assessing rates of chromosome segregation due to central spindle and cortical pulling force defects in embryos of the indicated genotypes. Note that gpr-1/2 inactivation rescues the excessive and premature spindle pole separation phenotype observed in cls-2(RNAi) and spd-1(RNAi). However, in contrast to spd-1+grp-1/2(RNAi) embryos, the central spindle fails to assemble in cls-2 + grp-1/2(RNAi) embryos in these conditions, indicated by the absence of the central spindle marker GFP::AIR-2 (white arrow). (D) Schematic of the Gα pathway. gpr-1/2 inactivation (red cross) suppresses the astral pulling forces. (E) A subset of kinetochore components acting downstream of KNL-1 regulate central spindle assembly.
Figure 9
Figure 9
Contractile ring components and assembly at the cell equator during mitosis. (A) Kymographs of the equatorial region of one-cell stage embryos expressing GFP::PH to mark the plasma membrane and show furrow ingression during cytokinesis in wild type (Control) and upon mild, moderate, or severe depletion of rho-1/RhoA by RNA interference. Modified from Loria et al. (2012). (B) Schematics illustrating the role of astral microtubules and the midzone in furrow positioning. During anaphase, astral microtubules clear contractile ring proteins (orange) from the polar cortex at the anterior pole. Data supporting a role for astral microtubules in clearing contractile ring proteins at the posterior poles are less compelling. During anaphase, the spindle midzone, through centralspindlin, promotes the cortical accumulation of contractile ring proteins at the site of furrowing. In response to polar and centralspindlin signaling, the contractile ring assembles and ingresses during cytokinesis.
Figure 10
Figure 10
Signaling pathways that control cortical activity during cytokinesis. Summary of the genetic pathways that influence contractile ring assembly and ingression during cytokinesis in the early C. elegans embryo.

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