Biology subjects

Fukuma, T.

Publications and source records attributed to Fukuma, T..

8 recordsLinked to original sources

bioRxiv2026

CAMSAP2 condensates drive a γ-TuRC-independent pathway for non-centrosomal microtubule nucleation

Microtubule nucleation is commonly viewed as a {gamma}-tubulin ring complex ({gamma}-TuRC)-templated process 1-3, yet many differentiated cells build extensive non-centrosomal microtubule arrays of unclear origin 4-6. Although spontaneous tubulin nucleation has long been observed in vitro as a {gamma}-TuRC-independent nucleation process 7-10, its mechanism and cellular relevance have remained unclear. Here we show that CAMSAP2, a microtubule minus-end-binding protein, links spontaneous nucleation to non-centrosomal microtubule organization. Cryo-electron microscopy (cryo-EM), high-speed atomic force microscopy (hsAFM), and molecular dynamics simulations reveal that CAMSAP2 lowers the nucleation barrier by straightening tubulin oligomers and promoting lateral protofilament interactions that drive sheet formation and closure into microtubules. hsAFM captures stepwise nucleation and early growth, revealing that tubulin rings can serve as productive intermediates rather than dead-end depolymerization products. CAMSAP2 further self-organizes through liquid-liquid phase separation (LLPS) to concentrate tubulin and assembly intermediates, thereby promoting efficient microtubule formation at non-centrosomal microtubule-organizing centers. In HeLa cells, dispersed CAMSAP2 condensates act as {gamma}-TuRC-independent microtubule-organizing centers alongside centrosomal asters, supporting a model in which spontaneous nucleation contributes to non-centrosomal microtubule biogenesis.

cell biology↗

bioRxiv2025

The increase in cell volume and nuclear number of the koji-fungus Aspergillus oryzae contributes to its high enzyme productivity

While the ratio of nuclei to cell volume is well regulated, it remains largely unexplored in multinucleate organisms. The koji-fungus Aspergillus oryzae, traditionally used in Japanese brewing and fermentation for over a thousand years, is now widely utilized in modern biotechnology as a host for enzyme production. We discovered that, over time in culture, hyphae become thicker, resulting in a tenfold increase in cell volume, and the number of nuclei in hyphal cells also increases tenfold, exceeding 200. The increase in cell volume and nuclear number is unique among the investigated Aspergillus species and correlates with its high enzyme production capabilities. Since nuclear number and cell volume are correlated, both must increase simultaneously for either to expand. Our analyses identified genetic factors and nutritional environmental signals involved in each of these increases. Increases in nuclear number and cell volume were also observed in other fungi bred for industrial use. This study not only deepens our understanding of the evolutionary processes that promote high enzyme productivity through fungal breeding, but also provides insights into the molecular mechanisms regulating cell volume and nuclear number in multinucleate organisms.

microbiology↗

bioRxiv2024

Epithelium Stratifies via Nucleation and Growth Induced by Foam-Geometric Instability

The epithelium undergoes stratification, transitioning from a monolayer to a multilayer structure, across broad phenomena. Recent studies have identified several cell behaviors as triggers, including junctional tension, cell density and geometry, and topological defects. However, how these factors drive stratification throughout the entire epithelium remains poorly understood. Here, we report a mechanism underlying epithelial stratification that mirrors the physics of phase transition. Combining cell culture with three-dimensional vertex modeling, we demonstrate that epithelial stratification is analogous to a structural phase transition driven by the nucleation-growth process, i.e., multilayer origins dispersedly appear and expand across the epithelium via unordered intermediate states. This transition is induced by a mechanical instability inherent in the foam-like geometry of the epithelium. Moreover, the nucleation-growth concept applies to embryonic skin development and intestinal cancer transformation. These findings conceptualize epithelial stratification as a form of a phase transition governed by foam mechanics, offering a physical perspective on various epithelial developments.

biophysics↗

bioRxiv2024

Probing nanomechanics by direct indentation using Nanoendoscopy-AFM reveals the nuclear elasticity transition in cancer cells

The assessment of nuclear structural changes is considered a potential biomarker of metastatic cancer. However, accurately measuring nuclear elasticity remains challenging. Traditionally, nuclear elasticity has been measured by indenting the cell membrane with a bead-attached atomic force microscopy (AFM) probe or aspirating isolated nuclei with a micropipette tip. However, indentation using a bead-attached probe is influenced by the cell membrane and cytoskeleton, while measurements of isolated nuclei do not reflect their intact state. In this study, we used Nanoendoscopy-AFM, a technique in which a nanoneedle probe is inserted into a living cell to directly measure nuclear elasticity and map its distribution. Our findings show that nuclear elasticity increases under serum depletion but decreases when serum-depleted cells are treated with TGF-{beta}, which induces epithelial-mesenchymal transition (EMT). Furthermore, we found that changes in nuclear elasticity correlate positively with trimethylation levels of histone H4 at lysine 20, rather than with nuclear lamins expression levels. These findings suggest that alterations in chromatin structure underlie changes in nuclear elasticity during cancer progression.

biophysics↗

bioRxiv2024

Dihydroxyhexanoic acid biosynthesis controls turgor in pathogenic fungi

Many plant pathogenic fungi penetrate host surfaces mechanically, using turgor pressure generated by appressoria, specialized infection cells. These appressoria develop semipermeable cell walls and accumulate osmolytes internally to create turgor by osmosis. While melanin is known to be important for turgor generation, the mechanism for wall semipermeability has remained unclear. Here we identify PKS2 and PBG13, by reverse genetics, as crucial for forming the semipermeable barrier in anthracnose and rice blast fungi. These genes encode enzymes that synthesize 3,5-dihydroxyhexanoic acid polymers essential for the cell wall properties. Deleting these enzymes impairs cell wall porosity, abolishing turgor and pathogenicity without affecting melanization. Our findings uncover a novel mechanism of turgor generation, linking enzyme function to pathogen penetration and disease potential, presenting new targets for disease control.

plant biology↗

bioRxiv2023

Comprehensive analysis of A-type lamin tail region for repairing the nuclear lamina

The nuclear lamina (NL) lines the nuclear envelope (NE) to maintain nuclear structure in metazoan cells. The major NL components, the nuclear lamins contribute to the protection against NE rupture induced by mechanical stress. Lamin A (LA) and a short form of the splicing variant lamin C (LC) are diffused from the nucleoplasm to sites of NE rupture in immortalized mouse embryonic fibroblasts (MEFs). LA localization to the rupture sites is significantly slow and weak compared to LC because of its relatively small pool in the nucleoplasm, but the precise mechanism remains unknown. In this study, we induce NE rupture in wild-type and LA/C-knockout MEFs, and Hutchinson-Gilford Progeria syndrome (HGPS) knock-in MEFs that express progerin, a LA mutant lacking the second proteolytic cleavage site, by laser microirradiation and AFM indentation. The farnesylation at the CaaX motif of unprocessed LA and the inhibition of the second proteolytic cleavage decrease the nucleoplasmic pool and slow the localization to the rupture sites in a long-time window (60-70 min) after the induction of NE rupture. Our data could explain the defective repair of NE rupture in HGPS through the farnesylation at the CaaX motif of unprocessed progerin. In addition, unique segments in LA-specific tail region cooperate with each other to inhibit the rapid accumulation within a short-time window (3 min) that is also observed with LC. Significance StatementNuclear lamins are the major components of the nuclear lamina (NL) that lies the nuclear envelope (NE). Lamin A (LA) is slowly localized to sites of nuclear envelope (NE) rupture compared to lamin C (LC). This study reveals that the farnesylation at the CaaX motif of unprocessed LA and the inhibition of the second proteolytic cleavage decrease the nucleoplasmic pool and slow the localization to the rupture sites within a long-time window (60-70 min) after the induction of NE rupture, which could explain the defective repair of NE rupture in Hutchinson-Gilford Progeria syndrome (HGPS). Additionally, unique segments in LA-specific tail region are critical for inhibiting the rapid accumulation within a short-time window (3 min).

cell biology↗

bioRxiv2022

Rapid intuitive interpretation of heterochromatin and euchromatin through Hi-C analysis

Hi-C is a technique that provides contact frequencies between pairs of loci on chromosomes. The conventional classification of heterochromatin and euchromatin based on Hi-C data is performed by principal component analysis; however, it requires long computational times and does not provide insight into the difference in contact frequencies between heterochromatin and euchromatin. Here, we propose a simple, intuitive and rapid method named the scaled contact number (SCN), which allows the contact frequencies to be visually interpreted and heterochromatin and euchromatin to be classified based on Hi-C results in a few minutes for long chromosomes at 1-kb resolution. The robustness of SCN was validated by confirming that SCN with reduced reads gives almost the same results as the original SCN. Overall, the approach described herein thus considerably decreases the time and computing power required to analyze Hi-C and further provides mechanistic insight indicating that euchromatin has more contacts than heterochromatin.

genomics↗

bioRxiv2021

Unraveling the host-selective toxic interaction of cassiicolin with lipid membranes and its cytotoxicity

Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for corynespora leaf fall disease in rubber trees. Currently, the molecular mechanism of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane-disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more susceptible to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Cytotoxicity tests on rubber leaves of RRIV 1, RRIV 4, and PB 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryo- SEM analyses of necrotic leaf tissues exposed to Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and disrupt specific membranes. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/438527v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@17ef3f4org.highwire.dtl.DTLVardef@1c24d41org.highwire.dtl.DTLVardef@15e071aorg.highwire.dtl.DTLVardef@24740e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICas1 and Cas2 directly damage and cause necrosis in the leaves of specific rubber clones. C_LIO_LICas1 and Cas2 can form biofilm-like structures on specific lipid membranes (negative phospholipids, glycerolipids, and sterols). The biofilm-like formation of Cas toxin plays an important role in selective disruption on lipid membranes. C_LIO_LIVulnerability of the specific cytoplasmic membranes to the selective Cas toxin that is the most remarkable feature of cytotoxicity of Cas toxin on plant cells. C_LI

biochemistry↗