Biology subjects

Hotta, T.

Publications and source records attributed to Hotta, T..

7 recordsLinked to original sources

bioRxiv2025

Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

Microtubules are cytoskeletal filaments that self-assemble from the protein tubulin, a heterodimer of -tubulin and {beta}-tubulin, and are important for cell mechanics, migration, and division. Much work has focused on how the nucleotide state of {beta}-tubulin regulates the structure and dynamics of microtubules. In contrast, less is known about the structure and function of the C-terminal tails (CTTs) of - and {beta}-tubulin which are thought to freely protrude from the surface of the microtubule. To study the CTT of -tubulin, we developed three different biosensors that bind the tyrosinated -tubulin CTT (Y-CTT). Surprisingly, live imaging of the probes indicates that the Y-CTT is minimally accessible along the microtubule lattice under normal cellular conditions. Lattice binding of the Y-CTT probes can be increased by three different ways of changing the tubulin conformational state: the drug Taxol, expression of microtubule-associated proteins (MAPs) that recognize or promote an expanded tubulin conformation, or expression of tubulin that cannot hydrolyze GTP. Molecular dynamics simulations indicate that the Y-CTT undergoes numerous transient interactions with the bodies of -tubulin and {beta}-tubulin in the lattice, and that the frequency of these interactions is regulated by the tubulin nucleotide state. These findings suggest that accessibility of the Y-CTT is locally governed by nucleotide- and MAP-dependent conformational changes to tubulin subunits within the microtubule lattice.

cell biology↗

bioRxiv2025

MATCAP1 preferentially binds an expanded tubulin conformation to generate detyrosinated and ?C2 a-tubulin

Microtubules are cytoskeletal filaments with critical roles in cell division, cell motility, intracellular trafficking, and cilium function. In cells, subsets of microtubules are selectively marked by posttranslational modifications (PTMs), which control the ability of microtubule-associated proteins (MAPs) and molecular motors to engage microtubules. Detyrosination ({Delta}Y) and {Delta}C2 are PTMs of -tubulin, wherein one or two residues, respectively, are enzymatically removed from the C-terminus of the protein. How specific patterns of PTMs are generated in cells is not understood. Here, we use in vitro reconstitution assays to investigate the microtubule binding behavior of metallopeptidase MATCAP1 and the mechanism by which it generates {Delta}Y and {Delta}C2 modifications of -tubulin. We demonstrate that MATCAP1 preferentially binds to microtubules composed of tubulin subunits in an expanded conformation, which can be induced by preventing {beta}-tubulin GTP hydrolysis, Taxol treatment, or kinesin-1 stepping. MATCAP1 binds to expanded microtubule lattices with long dwell time and sequentially removes the terminal tyrosine residue to generate {Delta}Y-microtubules and the penultimate glutamate residue to generate {Delta}C2-microtubules. Thus, the lattice conformation of microtubules is a key factor that gates the binding and activity of MATCAP1.

cell biology↗

bioRxiv2023

Thermo-sensitive condensation of SUF4 tunes flowering time in Arabidopsis thaliana

Intrinsically disordered proteins (IDP) lack stable tertiary structures, which allows them to change conformation and function under different physicochemical conditions. This may be highly advantageous for plants, which often use changes in their environment to elicit a variety of responses, including developmental events. For instance, some plants delay flowering in the fall and require exposure to winter temperatures as a cue to initiate flowering the following spring. Many of the genes involved in temperature-dependent flowering have been extensively studied in Arabidopsis, yet how plants coordinate their molecular states with seasonal temperature change is poorly understood. Here, we explore the role of temperature-sensitive phase separation of the IDP and flowering-time regulator, SUPPRESSOR OF FRIGIDA 4 (SUF4), in modulating flowering time. SUF4 has a well-defined role in regulating temperature-dependent flowering time by activating the master floral suppressor FLOWERING LOCUS C (FLC). We show that in plant nuclei, SUF4 is a temperature-sensitive protein that assembles into biomolecular condensates in warm temperatures (20{degrees}C). When temperatures cool (4{degrees}C), SUF4 nuclear condensates disassemble, causing SUF4 to disperse within the nucleoplasm. Additionally, we demonstrate that the number of SUF4 condensates quantitatively correlates with flowering time. Progressive alterations to the amino acid composition of SUF4s disordered region cause likewise progressive changes in temperature-dependent condensation both in vitro and in vivo, FLC transcription, and the onset of flowering. Lastly, we observe that SUF4 condensates coincide with the accumulation of other key flowering-time proteins (FRIGIDA and ELF7). These findings indicate that condensation of SUF4 likely plays a pivotal role in promoting flowering, possibly by concentrating and stabilizing regulatory factors needed for the transcriptional activation of FLC through temperature-dependent phase separation. This research suggests that in plants, IDPs can sense environmental cues and regulate critical developmental processes. One-Sentence SummaryEvidence suggest that Arabidopsis uses temperature-sensitive condensation to regulate FLC transcription and flowering time.

plant biology↗

bioRxiv2023

KIF1C, an RNA transporting kinesin-3, undergoes liquid-liquid phase separation through its C-terminal disordered domain

The spatial distribution of mRNA is critical for local control of protein production. Recent studies have identified the kinesin-3 family member KIF1C as an RNA transporter. However, it is not clear how KIF1C interacts with RNA molecules. Here, we show that KIF1Cs C-terminal tail domain is an intrinsically disordered region (IDR) containing a prion-like domain (PLD) that is unique compared to the C-terminal tails of other kinesin family members. In cells, KIF1C constructs undergo reversible formation of dynamic puncta that display physical properties of liquid condensates and incorporate RNA molecules in a sequence-selective manner. The IDR is necessary and sufficient for driving liquid-liquid phase separation (LLPS) but the condensate properties can be modulated by adjacent coiled-coil segments. The purified KIF1C IDR domain undergoes LLPS in vitro at near-endogenous nM concentrations in a salt-dependent manner. Deletion of the IDR abolished the ability of KIF1C to undergo LLPS and disrupted the distribution of mRNA cargoes to the cell periphery. Our work thus uncovers an intrinsic correlation between the LLPS activity of KIF1C and its role as an RNA transporter. In addition, as the first kinesin motor reported to undergo LLPS, our work reveals a previously uncharacterized mode of motor-cargo interaction that extends our understanding of the behavior of cytoskeletal motor proteins.

cell 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↗

bioRxiv2020

A Cytoskeletal Function for PBRM1 Reading Methylated Microtubules

The chromatin modifier SETD2 was recently shown to be a dual-function methyltransferase that "writes" methyl marks on both chromatin and the mitotic spindle, revealing -tubulin methylation as a new posttranslational modification of microtubules. Here, we report the first cytoskeletal "reader" for this SETD2 methyl mark: the polybromo protein PBRM1. We found PBRM1 directly binds the -Tub-K40me3 mark on tubulin, and localizes to the mitotic spindle and spindle pole during cell division. PBRM1 can assemble a PBAF complex in the absence of chromatin as revealed by mass spectrometry, and can recruit other PBAF complex components including SMARCA4 and ARID2 to -tubulin. In addition to PBRM1, other PBAF components were also localized to the mitotic spindle and spindle pole. This PBAF localization was dependent on recruitment to microtubules by PBRM1, and loss of spindle-associated PBRM1/PBAF led to genomic instability as assessed by increased formation of micronuclei. These data reveal a previously unknown function for PBRM1 beyond its role remodeling chromatin, and expand the repertoire of chromatin remodelers involved in writing and reading methyl marks on the cytoskeleton. The results of this study lay the foundation for a new paradigm for the epigenetic machinery as chromatocytoskeletal modifiers, with coordinated nuclear and cytoskeletal functions.

cell biology↗

bioRxiv2018

Regulation of KIF1A motility via polyglutamylation of tubulin C-terminal tails

Axonal transport is a highly regulated cellular process responsible for site-specific neuronal cargo delivery. This process is mediated in part by KIF1A, a member of the kinesin-3 family of molecular motors. It is imperative that KIF1As highly efficient, superprocessive motility along microtubules is tightly regulated as misregulation of KIF1A cargo delivery is observed in many neurodegenerative diseases. However, the regulatory mechanisms responsible for KIF1As motility, and subsequent proper spatiotemporal cargo delivery, are largely unknown. One potential regulatory mechanism of KIF1A motility is through the posttranslational modifications (PTMs) of axonal microtubules. These PTMs, often occurring on the C-terminal tails of the microtubule tracks, act as molecular \"traffic signals\" helping to direct kinesin motor cargo delivery. Occurring on neuronal microtubules, C-terminal tail polygutamylation is known to be important for KIF1A cargo transport. KIF1As initial interaction with microtubule C-terminal tails is facilitated by the K-loop, a positively charged surface loop of the KIF1A motor domain. However, the K-loops role in KIF1A motility and response to perturbations in C-terminal tail polyglutamylation is underexplored. Using single-molecule imaging, we present evidence of KIF1As previously unreported pausing behavior on multiple microtubule structures. Further analysis revealed that these pauses link multiple processive segments together, contributing to KIF1As characteristic superprocessive run length. We further demonstrate that KIF1A pausing is mediated by a K-loop/polyglutamylated C-terminal tail interaction and is a regulatory mechanism of KIF1A motility. In summary, we introduce a new mechanism of KIF1A motility regulation, providing further insight into KIF1As role in axonal transport.

biophysics↗