Biology subjects

Lee, Y.-R. J.

Publications and source records attributed to Lee, Y.-R. J..

8 recordsLinked to original sources

bioRxiv2025

A role of villin-dependent F-actin organization in peroxisome motility in Arabidopsis cells

Actin microfilaments (F-actin) serve as the track for directional movement of organelles in plants. To understand how the F-actin network is employed for the movement of peroxisomes, essential organelles in plant metabolism, we analyzed mutants of three villin (VLN) genes, which encode the primary actin-bundling factor and are most actively expressed in vegetative tissues in Arabidopsis thaliana. We found that the vln4 mutation greatly exacerbated the growth and subcellular defects in vln2 vln3. Compared to the wild-type cells, the double and triple vln mutants exhibit progressive reduction of stable F-actin bundles and rapid remodeling of the fine filaments. The defective F-actin network did not prevent peroxisomes from taking on both rapid and slow movements along the tracks but caused significantly reduced speed of movement and displacement distance of peroxisomes. Using a correlation analysis method, we classified the complex heterogeneous peroxisome movement patterns into clusters reflecting distinct movement directionalities. The vln triple mutant had significantly reduced number of peroxisomes with long-range and linear movement. Our results provide insights into how VLN-dependent F-actin organization is coupled with the complex pattern of peroxisome movement.

plant biology↗

bioRxiv2025

Cryo-EM structures of the Plant Augmin reveal its intertwined coiled-coil assembly, antiparallel dimerization and NEDD1 binding mechanisms

Microtubule (MT) branch nucleation is fundamental for building parallel MT networks in eukaryotic cells. In plants and metazoans, MT branch nucleation requires Augmin and NEDD1 proteins which bind along MTs and then recruit and activate the gamma-tubulin ring complex ({gamma}-TuRC). Augmin is a fork-shaped assembly composed of eight coiled-coil subunits, while NEDD1 is a WD40 {beta}-propellor protein that bridges across MTs, Augmin, and {gamma}-TuRC during MT branch nucleation. Here, we reconstitute hetero-tetrameric and hetero-octameric Arabidopsis thaliana Augmin assemblies, resolve their subunit interactions using crosslinking mass spectrometry and determine 3.7 to 7.3-[A] cryo-EM structures for the V-junction and extended regions of Augmin. These structures allowed us to generate a complete de novo plant Augmin model that reveals the long-range multi coiled-coil interfaces that stabilize its 40-nm hetero-octameric fork-shaped organization. We discovered the dual calponin homology (CH) domain forming its MT binding site at the end of its V-junction undertake open and closed conformations. We determined a 12-[A] dimeric Augmin cryo-EM structure revealing Augmin undergoes anti-parallel dimerization through two conserved surfaces along Augmins extended region. We reconstituted the NEDD1 WD40 {beta}-propellor with Augmin revealing it directly binds on top its V-junction and enhances Augmin dimerization. Our studies suggest that cooperativity between the Augmin dual CH domains and NEDD1 WD40 binding site may regulate Augmin V-junction dual binding to MT lattices. This unique V-shaped dual binding and organization anchors Augmins along MTs generating a platform to recruit {gamma}-TuRC and activate branched MT nucleation.

biochemistry↗

bioRxiv2023

The Microspherule protein 1 (MCRS1) homolog interacts with the Myb-like transcription factor DRMY1 and is essential for embryogenesis in Arabidopsis thaliana

The evolutionarily conserved Microspherule protein 1 (MCRS1) has diverse functions from transcriptional regulation to stabilization of microtubule minus ends in acentrosomal spindles in mammals. A previous study suggested that in the model plant Arabidopsis thaliana, inactivation of an MCRS1 homolog gene led to aborted embryogenesis. To test whether this lethality was caused by defects associated with transcription or mitosis, we used the heterozygous mcrs1 mutant to examine whether the gene was required for mitosis during gametogenesis. Results of reciprocal crosses between the mcrs1 mutant and the wild-type plant showed that the MCRS1 gene was dispensable for mitotic cell divisions associated with the development of both male and female gametophytes. An MCRS1-GFP fusion protein was expressed in the mcrs1 mutant and suppressed the mutation as reported by restored growth. This functional fusion protein exclusively localized to interphase nuclei and became undetectable during mitosis before returning to the reforming daughter nuclei. Affinity purification of the MCRS1-GFP protein resulted in consistent recovery of the Myb-like transcription factor DRMY1 (Developmentally Regulated Myb-like1) but not microtubule-associated factors. The association was further supported by the evidence of a direct interaction in living cells. Hence, the plant MCRS1 was concluded to play a role in the gene transcription in sporophyte development.

plant biology↗

bioRxiv2023

The Arabidopsis BUB1/MAD3 family protein BMF3 requires BUB3.3 to recruit CDC20 to kinetochores in spindle assembly checkpoint signaling

Mitosis is monitored by the spindle assembly checkpoint (SAC) which remains active until all chromosomes have their kinetochores attached to microtubules originated from opposite spindle poles. Plants produce both highly conserved and sequence-diverged SAC components, so it has been largely unknown how SAC activation leads to the assembly of these proteins at the unattached kinetochores to prevent anaphase onset in plants. In Arabidopsis thaliana, the noncanonical BUB3.3 protein was detected at kinetochores throughout mitosis, unlike MAD1 and the plant specific BUB1/MAD3 family protein BMF3 that associated with unattached chromosomes only. However, BUB3.3 was required to arrest mitotic progression when one or more chromosomes did not congress at the metaphase plate. Nevertheless, BUB3.3 was not required for the kinetochore localization of other SAC components and vice versa. BUB3.3 specifically interacted with BMF3 in a novel region containing two internal repeats that were not required for the kinetochore localization of BMF3. This interaction was important for BMF3 to recruit the CDC20 protein to the unattached kinetochores. Our results showed that the Arabidopsis BUB3.3 protein functioned in the activation of BMF3 for CDC20 recruitment, rather than the recruitment of BMF proteins as what has been found in fungi and animals, in order to arrest mitosis at prometaphase. Therefore, activated SAC resulted in BUB3.3-independent localization of BMF3 and MAD1 to kinetochores and BUB3.3-dependent licensing of BMF3 for CDC20 recruitment in A. thaliana. SIGNIFICANCE STATEMENTMitotic progression into anaphase is monitored by spindle assembly checkpoint that is poorly understood in plants. Using Arabidopsis thaliana as a reference system, we discovered a novel interaction pattern centered at the BUB1 and MAD3 protein BMF3 at kinetochores. A noncanonical isoform of the evolutionarily conserved BUB3 family protein BUB3.3 interacted with two novel internal repeats in BMF3 for recruiting the CDC20 protein to unattached kinetochores in order to inhibit its function in anaphase onset. Hence, our work sheds light on how spindle assembly checkpoint operates in flowering plants that produce the highly conserved BUB3.3 protein but highly divergent BMF proteins.

plant biology↗

bioRxiv2022

Phase transition of tensin-1 during the focal adhesion disassembly and cell division

Biomolecular condensates are non-membranous structures that are mainly formed through liquid-liquid phase separation. Tensins are focal adhesion (FA) proteins linking the actin cytoskeleton to integrin receptors. Here we report that GFP-tagged tensin-1 (TNS1) proteins at physiological levels phase separate to form biomolecular condensates in TNS1 knockout cells. Live cell imaging showed that new TNS1 condensates are budding from the disassembling ends of FAs, and presence of these condensates is cell cycle dependent. TNS1 condensates dissolve immediately prior to mitosis and rapidly reappear while post-mitotic daughter cells establish new FAs. TNS1 condensates contain selected FA proteins and signaling molecules such as pT308Akt but not pS473Akt, suggesting previously unknown roles of TNS1 condensates in disassembling FAs, as the storage of core FA components and the signaling intermediates.

cell biology↗

bioRxiv2022

The microtubular preprophase band recruits Myosin XI to the division site for plant cytokinesis

Plant growth is dependent on oriented cell divisions that employ the microtubular preprophase band (PPB) to position the cell plate. It has been intriguing how this transient cytoskeletal array imprints the spatial information to be read by the cytokinetic phragmoplast at later stages of mitotic cell division. In Arabidopsis thaliana, we discovered that the PPB recruited the Myosin XI motor MYA1 to the cortical division site where it joined microtubule-associated proteins and motors to form a ring of prominent cytoskeletal assemblies which received the expanding phragmoplast. This regulatory function of MYA1 in phragmoplast guidance is dependent on intact actin filaments. The discovery of these assemblies revealed the mechanism underlying how two dynamic cytoskeletal networks govern PPB-dependent division plane orientation during vegetative growth in flowering plants. ONE-SENTENCE SUMMARYMyosin XI joins microtubule-associated proteins and motors to form cortical assemblies to demarcate the cell division site.

plant biology↗

bioRxiv2022

CWLP and PRP940 form plasma-membrane nanodomain complexes with aquaporins, interact with PP2A and contribute to dehydration tolerance

The C-type hybrid-proline-rich protein (HyPRP) AtCWLP and its homolog AtPRP940 are referred as cell wall (CW)-plasma-membrane (PM) linker proteins, but little is known about their functions. Here we show that N-terminal proline-rich domains of CWLP and PRP940, containing glycosylated hydroxyproline residues, contact the CW, while their C-terminal 8CM domains function as PM-scaffolds. Both proteins are detected in PM nanodomains (PM-ND) and show co-localization and co-immunoprecipitation with aquaporins PIP2;1 and PIP2;7. Inhibition of actin polymerization by latrunculin B promotes CWLP-endosome appearance, while blocking the actomyosin-based transport by a truncated form of myosin XI-K relaxes lateral boundaries of CWLP-PIP2;1 PD-NDs. Mass spectrometry data indicate that CWLP co-purifies with dynamins implicated in fission of endocytic PD-ND invaginations. Lack of co-localization and co-immunoprecipitation with aquaporin-binding flotillin (FLOT2) indicates that CWLP and PRP940 mark a new distinct type of PM-ND. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that CWLP and PRP940 interact with multiple aquaporins and several protein phosphatase PP2A-B regulatory subunits. By preventing irreversible separation of CW and PM, and likely assisting PP2A-mediated dephosphorylation of aquaporins and closure of their water channels, overexpression of CWLP confers tolerance to plasmolysis, dehydration and freezing in Arabidopsis and to water shortage in potato plants. Summary StatementArabidopsis Hybrid-Proline-Rich Proteins CWLP and PRP940 occur in association with dynamins, recruit PP2A protein phosphatases to aquaporin water channels in plasma-membrane (PM) nanodomains and elevate tolerance to cellular dehydration.

plant biology↗

bioRxiv2017

The preprophase band-associated kinesin-14 OsKCH2 is a processive minus-end-directed microtubule motor

In animals and fungi, cytoplasmic dynein is a processive motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1 but not CC2 forms the coiled-coil for OsKCH2 dimerization. Instead, CC2 functions to enable OsKCH2 processivity by enhancing its binding to microtubules. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.

biophysics↗