Chloroplast nucleoids as a transformable network revealed by live imaging with a microfluidic device

Kamimura, Yoshitaka; Tanaka, Hitomi; Kobayashi, Yusuke; Shikanai, Toshiharu; Nishimura, Yoshiki

doi:10.1038/s42003-018-0055-1

Cited by 20 publications

(10 citation statements)

References 30 publications

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“…By 12–14 hrs after release, wild type cells undergo multiple synchronous cycles of DNA replication and mitosis within the mother cell wall, yielding multinucleate cells producing signals of DNA content of 1, 2, 4, 8 and 16C ( Fig 1A and 1B ). Chloroplast DNA replication has been reported to be uncoupled from the nuclear replication cycle, and likely only accounts for a small part of the total FACS signal [ 37 ].…”

Section: Resultsmentioning

confidence: 99%

Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii

et al. 2021

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DNA replication is fundamental to all living organisms. In yeast and animals, it is triggered by an assembly of pre-replicative complex including ORC, CDC6 and MCMs. Cyclin Dependent Kinase (CDK) regulates both assembly and firing of the pre-replicative complex. We tested temperature-sensitive mutants blocking Chlamydomonas DNA replication. The mutants were partially or completely defective in DNA replication and did not produce mitotic spindles. After a long G1, wild type Chlamydomonas cells enter a division phase when it undergoes multiple rapid synchronous divisions (‘multiple fission’). Using tagged transgenic strains, we found that MCM4 and MCM6 were localized to the nucleus throughout the entire multiple fission division cycle, except for transient cytoplasmic localization during each mitosis. Chlamydomonas CDC6 was transiently localized in nucleus in early division cycles. CDC6 protein levels were very low, probably due to proteasomal degradation. CDC6 levels were severely reduced by inactivation of CDKA1 (CDK1 ortholog) but not the plant-specific CDKB1. Proteasome inhibition did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 might upregulate CDC6 at the transcriptional level. All of the DNA replication proteins tested were essentially undetectable until late G1. They accumulated specifically during multiple fission and then were degraded as cells completed their terminal divisions. We speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. We also developed a simple assay for salt-resistant chromatin binding of MCM4, and found that tight MCM4 loading was dependent on ORC1, CDC6 and MCM6, but not on RNR1 or CDKB1. These results provide a microbial framework for approaching replication control in the plant kingdom.

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Section: Resultsmentioning

confidence: 99%

Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii

et al. 2021

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“…By 12-14 hrs after release, wild type cells undergo multiple synchronous cycles of DNA replication and mitosis within the mother cell wall, yielding multinucleate cells producing signals of DNA content of 1, 2, 4, 8 and 16C (Figure 1A and 1B). Chloroplast DNA replication occurs early and uncoupled from the nuclear replication cycle and likely only accounts for a small part of the total FACS signal [37].…”

Section: Resultsmentioning

confidence: 99%

Conservation and divergence of DNA replication control inChlamydomonas reinhardtii

Ikui

Ueki

Pecani

et al. 2020

Preprint

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We recently isolated temperature-sensitive cell cycle mutants in Chlamydomonas reinhardtii for which the causative mutations were located in genes annotated for potential involvement in DNA replication. Chlamydomonas has a very long G1 period during which cells grow up to ~10-fold without division, followed by rapid cycles of DNA replication and mitosis ('multiple fission'). All of the candidate DNA replication mutants tested were defective in completion of the first round of DNA replication, and also failed to produce mitotic spindles. For a subset of the mutants, we rescued temperature-sensitive lethality with tagged transgenes and used the resulting strains to analyze abundance and localization control of the tagged protein. All of the DNA replication proteins tested were essentially undetectable until late G1, accumulated during the period of multiple fission and then were degraded as cells completed their terminal divisions. MCM4 and MCM6 were localized to the nucleus during the division cycle except for transient cytoplasmic localization during mitosis. CDC45 showed strict protein location to the nucleus and co-localized to spindles during mitosis. In contrast, CDC6 was detected in the nucleus only transiently during early divisions within the overall multiple fission cycle. Cdc6 protein levels were very low, but increased upon treatment with MG132, a proteasome inhibitor. We also tested if these DNA replication proteins are regulated by cyclin dependent kinase (CDK). There are two main CDKs in Chlamydomonas, CDKA1 and CDKB1. We found that CDC6 protein level was severely reduced in a cdka1 mutant, but not in a cdkb1 mutant. MG132 did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 upregulates CDC6 at the transcription level. Since MCM4, MCM6 and CDC6 were all essentially undetectable during the long G1 before DNA replication cycles began, we speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. These results provide a microbial framework for approaching replication control in the plant kingdom.Once DNA replication is initiated, the pre-RC components such as Cdc6, Mcm2-7, and the ORC complex are phosphorylated by Cyclin/CDK; these multiple mechanisms prevent a second round of MCM reloading and consequent DNA replication from occurring before mitosis [13]. For example, Cdc6 is phosphorylated by Cyclin/CDK, targeting it for ubiquitin-mediated proteolysis through the SCF complex [14-17], Mcm2-7 is exported from the nucleus as noted above, and ORC function is inhibited by phosphorylation.These mechanisms collectively prevent origin relicensing within S phase.The pre-RC proteins are mostly conserved in humans, but there are significant functional differences. While ScOrc1-6 binds to specific origin DNA sequences throughout the cell cycle, HsOrc1-6 is transiently associated with chromatin during G1 phase, and no origin consensus sequence has been defined [18]. HsMcm2 and HsMcm4 are phosphorylated by Cyc...

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“…In plant science however, few microdevices have been developed so far [8]. Among them, the vast majority of the literature deals with root or pollen tube immobilization and studies [9–16], and half a dozen only address the question of the manipulation and observation of protoplasts [17–23].…”

Section: Introductionmentioning

confidence: 99%

Design of a comprehensive microfluidic and microscopic toolbox for the ultra-wide spatio-temporal study of plant protoplasts development and physiology

et al. 2019

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Background Plant protoplasts are basic plant cells units in which the pecto-cellulosic cell wall has been removed, but the plasma membrane is intact. One of the main features of plant cells is their strong plasticity, and their propensity to regenerate an organism from a single cell. Methods and differentiation protocols used in plant physiology and biology usually involve macroscopic vessels and containers that make difficult, for example, to follow the fate of the same protoplast all along its full development cycle, but also to perform continuous studies of the influence of various gradients in this context. These limits have hampered the precise study of regeneration processes. Results Herein, we present the design of a comprehensive, physiologically relevant, easy-to-use and low-cost microfluidic and microscopic setup for the monitoring of Physcomitrella patens ( P. patens ) growth and development on a long-term basis. The experimental solution we developed is made of two parts (i) a microfluidic chip composed of a single layer of about a hundred flow-through microfluidic traps for the immobilization of protoplasts, and (ii) a low-cost, light-controlled, custom-made microscope allowing the continuous recording of the moss development in physiological conditions. We validated the experimental setup with three proofs of concepts: (i) the kinetic monitoring of first division steps and cell wall regeneration, (ii) the influence of the photoperiod on growth of the protonemata, and (iii) finally the induction of leafy buds using a phytohormone, cytokinin. Conclusions We developed the design of a comprehensive, physiologically relevant, easy-to-use and low-cost experimental setup for the study of P. patens development in a microfluidic environment. This setup allows imaging of P. patens development at high resolution and over long time periods. Electronic supplementary material The online version of this article (10.1186/s13007-019-0459-z) contains supplementary material, which is available to authorized users.

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Chloroplast nucleoids as a transformable network revealed by live imaging with a microfluidic device

Cited by 20 publications

References 30 publications

Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii

Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii

Conservation and divergence of DNA replication control inChlamydomonas reinhardtii

Design of a comprehensive microfluidic and microscopic toolbox for the ultra-wide spatio-temporal study of plant protoplasts development and physiology

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