Biology subjects

Kodera, C.

Publications and source records attributed to Kodera, C..

8 recordsLinked to original sources

bioRxiv2026

A triple fluorescent marker for live imaging of plant cell morphogenesis

Live imaging of plant subcellular structures is key to deciphering the spatiotemporal bases of cellular processes, and their functional impact on growth and morphogenesis at various biological scales. Live imaging of plant cells essentially relies on expression of fluorescent markers labeling cells or subcellular structures of interest. Simultaneous multi-channel imaging of several markers is still not routine practice in plant cell biology, owing to issues linked to genetic or spectral compatibility of markers, differences in expression levels, silencing, toxicity, etc. Here we designed a three-color marker in Arabidopsis thaliana and Capsella rubella, enabling high-resolution live imaging of plant morphogenesis, including labeling of the cell membrane, the nucleus and the microtubule cytoskeleton. Detection of MT arrays involved the development of a MAP4-MBD-based microtubule marker optimized for plant cells. The three-color marker allows visualization of the three-dimensional organization and dynamics of plant microtubules within the intracellular space with unprecedented precision, in various organs including the root and shoot meristems, the leaf, anther, and gynoecium. Our results demonstrate the potential of such single-construct strategy for cell biology studies in plants.

plant biology↗

bioRxiv2024

Live-HPF CLEM using the HPM Live μ: Finding back all needles in every haystack. The last frontier in CLEM

Correlative light and electron microscopy is a scientific method that encompasses several technologies and workflows. One of the pioneering workflows consisted in vitrifying by high pressure freezing a sample, shortly after live observation1. Despite its extraordinary potential, it did not turn into a routine technique for practical reasons. We redesigned the entire tool set, from the sample carrier to the high-pressure freezing machine, to standardize and democratise the technique. In our manuscript, we present all the technological developments that lead to a routine workflow for live to HPF CLEM. We demonstrate our ability to track rapidly moving endosomes live and retrieve them at the electron microscopy level, in three dimensions, with high confidence.

cell biology↗

bioRxiv2024

Vitrification by high pressure freezing of a wide variety of sample using the HPM Live μ

This study explores the efficacy and reliability of high-pressure freezing (HPF) as a sample preparation technique for electron microscopy (EM) analysis across a diverse range of biological samples. Utilizing the HPM Live {micro} technology, based on the historical hydraulic HPM010 from BalTec, we demonstrate the reliability of our industrial equipment to achieve the critical parameters necessary for vitrification. By directly measuring physical values within the HPF chamber, we ensure the proper functioning of the equipment, contributing to the techniques reliability. A meticulous approach was adopted for each sample type, acknowledging the uniqueness of each specimen, and associating final sample analysis with its HPF curve, aiding in protocol optimization. Samples including human cell pellets, cell monolayer, mouse brain and liver biopsies, C. elegans, zebrafish, and A. thaliana root and seedlings were processed for EM analysis following HPF. The ultrastructure of each sample type was rigorously examined, revealing homogeneous preservation and minimal ice nucleation artifacts. Challenges such as plant leaf vitrification were addressed, highlighting the importance of methodological adaptation. Overall, our findings underscore the robustness and versatility of our HPM Live {micro} in preserving biological ultrastructure, offering valuable insights for researchers employing EM techniques in diverse biological studies.

cell biology↗

bioRxiv2021

KCTD19 associates with ZFP541 and HDAC1 and is required for meiotic exit in male mice

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. Immunoprecipitation-mass spectrometry identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19, indicating that KCTD19 is involved in chromatin modification. Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 were essential for meiotic exit in male mice. Author summaryMeiosis is a fundamental process that consisting of one round of genomic DNA replication and two rounds of chromosome segregation producing four haploid cells. To properly distribute their genetic material, cells need to undergo complex chromosome events such as a physical linkage of homologous chromosomes (termed synapsis) and meiotic recombination. The molecules involved in these events have not been fully characterized yet, especially in mammals. Using a CRISPR/Cas9-screening system, we identified the potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis in male mice. Further, we identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19. By observing meiosis of Zfp541 knockout germ cells, we found that Zfp541 was also essential for meiotic completion. These results show that the KCTD19/ZFP541 complex plays a critical role and is indispensable for male meiosis and fertility.

developmental biology↗

bioRxiv2021

Meiosis-specific ZFP541 repressor complex promotes meiotic prophase exit during spermatogenesis

During spermatogenesis, meiosis is accompanied by robust alteration in gene expression and chromatin status. However, it remained elusive how meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identified a novel protein complex consisting of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulator for meiotic prophase exit. Our genetic study showed that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses revealed that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrated that germ-cell specific ZFP541-KCTD19 containing complex promotes meiotic prophase exit in males, and triggers reconstruction of the transcription network and chromatin organization leading to post-meiotic development.

developmental biology↗

bioRxiv2019

Dissection of the early pollen-stigma interactions by live-cell imaging

Early events occurring at the surface of the female organ are critical for plant reproduction, especially in species with a dry stigma. Following landing on the stigmatic papilla cells, the pollen hydrates and germinates a tube, which penetrates the cell wall and grows towards the ovules to convey the male gametes to the embryo sac. In self-incompatible (SI) species within the Brassicaceae, these processes are blocked when the stigma encounters an incompatible pollen. Here, based on the generation of SI-Arabidopsis lines and by setting up a live imaging system, we showed that control of pollen hydration has a central role in pollen selectivity. The faster pollen pumps water from the papilla during an initial period of 10 minutes, the faster it germinates. Furthermore, we found that the SI barriers act to block the proper hydration of incompatible pollen and when hydration is promoted by high humidity, an additional control prevents pollen tube penetration into the stigmatic wall. In papilla cells, actin bundles focalize at the contact site with the compatible pollen but not with the incompatible one, raising the possibility that stigmatic cells react to the mechanical pressure applied by the invading growing tube. HighlightA live imaging system coupled with self-incompatible Arabidopsis lines highlight the role of stigmatic cells in controlling pollen hydration and in reacting to pollen tube intrusion by remodeling actin cytoskeleton.

plant biology↗

bioRxiv2018

KATANIN-dependent mechanical properties of the stigmatic cell wall regulate pollen tube pathfinding

Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules. Pollen tubes first grow within the cell wall of the papilla cells, applying pressure to the cell. Mechanical forces are known to play a major role in plant cell shape by controlling the orientation of cortical microtubules (CMTs), which in turn mediate deposition of cellulose microfibrils (CMFs). Here, by combining cell imaging and genetic approaches, we show that isotropic reorientation of CMTs and CMFs in aged and katanin1-5 (ktn1-5) papilla cells is accompanied by a tendency of pollen tubes to coil around the papillae. Furthermore, we uncover that aged and ktn1-5 papilla cells have a softer cell wall and provide less resistance to pollen tube growth. Our results reveal an unexpected role for KTN1 in pollen tube guidance by ensuring mechanical anisotropy of the papilla cell wall.

plant biology↗

bioRxiv2018

The molecular signatures of compatible and incompatible pollination

Fertilization in flowering plants depends on the early contact and recognition of pollen grains by the receptive papilla cells of the stigma. To identify the associated molecular pathways, we developed a transcriptomic analysis based on single nucleotide polymorphisms (SNPs) present in two Arabidopsis thaliana accessions, one used as female and the other as male. We succeeded in distinguishing 80 % of transcripts according to their parental origins and drew up a catalog of genes whose expression is modified after pollen-stigma interaction. Global analysis of our data reveals that pattern-triggered immunity (PTI)-associated transcripts are upregulated after compatible pollination. From our analysis, we predicted the activation of the Mitogen-activated Protein Kinase 3 on the female side after compatible pollination, which we confirmed through expression and mutant analysis. Our work defines the molecular signatures of compatible and incompatible pollination, highlights the active status of incompatible stigmas, and unravels a new MPK3-dependent cell wall feature associated with stigma-pollen interaction.

plant biology↗