Biology subjects

Ebine, K.

Publications and source records attributed to Ebine, K..

6 recordsLinked to original sources

bioRxiv2025

Comparative Proteomic Profiling of Receptor Kinase Signaling Reveals Key Trafficking Components Enforcing Plant Stomatal Development

Receptor kinases are pivotal for growth, development, and environmental response of plants. Yet, their regulatory mechanisms and spatial dynamics are still underexplored. The ERECTA-family receptor kinases coordinate diverse developmental processes, including stomatal development. To understand the proteomic landscape of the ERECTA-mediated signaling pathways, we here report comparative analyses of the ERECTA interactome and proximitome by epitope-tagged affinity-purification (ET-AP) and TurboID-based proximity labeling (TbID-PL) mass-spectrometry, respectively. While ET-AP successfully recovered receptor complex components (e.g., TOO MANY MOUTHS), TbID-PL effectively captured transient associations with the components of endosomal trafficking, i.e., clathrin-mediated endocytosis (CME) machinery. We further identify that specific subfamily members of phosphatidylinositol-binding clathrin assembly proteins (PICALMs) interact with and synergistically regulate ERECTA internalization. Mutations of these PICALMs impair ERECTA endocytosis and lead to excessive stomatal clustering by dampening the downstream signaling output. Taken together, our work provides a proteomic atlas of the ERECTA signaling network and suggests that timely removal of receptor kinase by the endocytosis machinery is essential for active signal transduction enforcing stomatal patterning.

plant biology↗

bioRxiv2025

A new corrector rescues F508del-CFTR folding through stabilization of the TMD1-NBD1 linker

While Trikafta has turned many CF patients into people that lead relatively healthy lives, additional modulators are still needed. We developed an acylsulfonamide-type C2 corrector X307810 that is potent and shows a distinctive mode of action in rescuing F508del-CFTR from degradation at the endoplasmic reticulum. We tracked the de-novo folding and assembly of each CFTR domain with a radio-active pulse chase - limited proteolysis folding assay and determined when and where X307810 works on the newly synthesized protein. X307810 rescued the Golgi form of F508del-CFTR to 80% of wild-type values. To our knowledge, such a dramatic increase has never been attained with a single corrector. Zooming in on X307810 mode of action revealed that the corrector changed protease resistance of the linker between TMD1 and NBD1 during late stages of CFTR folding. X307810 is the first corrector that added a unique new fragment to the typical limited-proteolysis pattern. Other C2 correctors do not change fragments, but portray an increase in fragment quantity characteristic of domain assembly in wild-type and corrected F508del-CFTR. X307810 instead extended protection of TMD1 from intracellular loop 2 to the unstructured regulatory insertion of NBD1. These results provide new and important insights into the impact of acylsulfonamide correctors on CFTR structure and function. SIGNIFICANCE STATEMENTCFTR is an anion channel, mutations in which cause cystic fibrosis (CF). Corrector compounds allow misfolded missense CFTR mutants to escape from degradation in the endoplasmic reticulum, which is the basis for CF therapy, in combination with a potentiator. How correctors improve folding is incompletely understood. We here report on X307810 having a novel mode of action on post-translational domain assembly of newly synthesized CFTR. Our in-cell folding assay shows that X307810 stabilizes the linker between the first transmembrane domain (TMD1) and the first nucleotide binding domain (NBD1) into the Regulatory Insertion; it strengthens the TMD1-NBD1-ICL4 interface. The action of X307810 boosts egress of F508del-CFTR from the ER to an unprecedented 80% for a single modulator.

biochemistry↗

bioRxiv2021

Identification of a divergent basal body assembly protein involved in land plant spermatogenesis

Oogamy is a form of sexual reproduction and evolved independently in animals, fungi, and plants. In streptophyte plants, Charophyceae, Coleochaetophyceae, bryophytes, lycophytes, ferns (monilophytes), and some gymnosperms (Cycads and Ginkgo) utilize spermatozoids as the male gamete. Plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning plant spermatogenesis remain to be elucidated. To identify the genes involved in plant spermatogenesis, we performed computational analyses and successfully found deeply divergent BLD10s by combining multiple methods and omics-data. We then validated the functions of candidate genes in the liverwort Marchantia polymorpha and the moss Physcomitrium patens and found that MpBLD10 and PpBLD10 are required for normal basal body and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. Streptophyte BLD10s are orthologous to BLD10/CEP135 family proteins, which function in basal body assembly, but we found that BLD10s evolved especially fast in land plants and MpBLD10 might obtain additional functions in spermatozoid formation through the fast molecular evolution. This study provides a successful example of combinatorial study from evolutionary and molecular genetic perspectives that elucidated a function of the key protein of the basal body formation that fast evolved in land plants.

evolutionary biology↗

bioRxiv2021

Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha

Gametogenesis is an essential biological event for sexual reproduction in various organisms. Bryophytes and some other plants employ motile sperms (spermatozoids) as male gametes, which self-locomote to the egg cells to accomplish fertilization. Spermatozoids of bryophytes harbor distinctive morphological characteristics, including the cell body with a helical slender shape and two motile flagella at the anterior edge. During transformation from a spermatid to spermatozoid (spermiogenesis), the shape and cellular contents of spermatids are dynamically reorganized. However, how each organelle is reorganized during plant spermiogenesis remains obscure. In this study, we classified the developmental processes during spermiogenesis in the liverwort Marchantia polymorpha according to cellular and nuclear shapes and flagella development. We then examined the remodeling of microtubules and reorganization of endomembrane organelles during spermiogenesis. The results indicate that the state of post-translational modification of tubulin is dynamically changed during the formation of the flagella and spline, and the plasma membrane and endomembrane organelles are drastically reorganized in a precisely regulated manner during spermiogenesis. These findings are expected to provide useful indexes to classify developmental and subcellular processes of spermiogenesis in bryophytes. Summary statementWe classified developmental processes of spermatozoids into 1 + 5 stages and characterized remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha.

plant biology↗

bioRxiv2020

The SYP123-VAMP727 SNARE complex is involved in the delivery of inner cell wall components to the root hair shank in Arabidopsis

A root hair is a long tubular protrusion from a root hair cell established via tip growth, which is accomplished by the polarized deposition of membranous and cell wall components at the root hair apex accompanied by simultaneous hardening of the shank. The polarized secretion of materials to the root hair apex is well investigated; however, little is known about the deposition of inner cell wall materials at the root hair shank. We have previously reported that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2)/ROP10 signaling is required for the regulation of cortical microtubule construction and the deposition of inner cell wall components at the root hair shank during hardening. To unravel the alternate secretion mechanism for delivery of the inner cell wall components to root hair shank, here, we demonstrate that root hair-specific Qa-SNARE, SYP123, localizes to the subapical zone and shank of elongating root hairs in Arabidopsis. SYP123-mediated root hair elongation was inhibited by the FAB1 inhibitor YM201636, and inhibition of PtdIns(3,5)P2 production impaired the plasma membrane localization of SYP123. We also showed that SYP123 forms a SNARE complex with VAMP727 on the plasma membrane, and syp123 and vamp727 mutants exhibited lower cell wall stiffness in the root hair shank because of impaired deposition of inner cell wall components. These results indicate that SYP123/VAMP727-mediated secretion is involved in the transport of inner cell wall components for hardening of the root hair shank.

plant biology↗

bioRxiv2020

Switching secretory pathway direction for organelle acquisition in plants

Eukaryotic cells acquired novel organelles during evolution through mechanisms that remain largely obscure. The existence of the unique oil body compartment is a synapomorphy of liverworts that represents lineage-specific acquisition of this organelle during evolution, although its origin, biogenesis, and physiological function are yet unknown. We found that two Syntaxin 1 paralogs in the liverwort, Marchantia polymorpha, are distinctly targeted to forming cell plates and the oil body, suggesting these structures share some developmental similarity. Oil body formation is under the regulation of an ERF/AP2-type transcription factor and loss of the oil body increased M. polymorpha herbivory. These findings highlight a common strategy for the acquisition of organelles with distinct functions in plants, via periodical switching in secretion direction depending on cellular phase transition.

plant biology↗