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@unpublished{Yurtsever2026,

title = {CAMSAP2 condensates drive a γ-TuRC-independent pathway for non-centrosomal microtubule nucleation},

author = {Ayhan Yurtsever and Tsuyoshi Imasaki and Ryota Kitano and Kien Xuan Ngo and Tomoaki Yagi and Shuhei Kuno and Hanjin Liu and Takaaki Kato and Hideki Shigematsu and Md Fahim Newaz and Yuji Sugita and Takeshi Fukuma and Ryo Nitta},

url = {http://biorxiv.org/lookup/doi/10.64898/2026.05.28.728596},

doi = {10.64898/2026.05.28.728596},

year  = {2026},

date = {2026-05-29},

publisher = {openRxiv},

abstract = {Abstract

                

                  Microtubule nucleation is commonly viewed as a γ-tubulin ring complex (γ-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 γ-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 γ-TuRC-independent microtubule-organizing centers alongside centrosomal asters, supporting a model in which spontaneous nucleation contributes to non-centrosomal microtubule biogenesis.

                },

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Liu2025,

title = {Human spastin functions as an ATPase-independent microtubule nucleator via ring-stack formation},

author = {Hanjin Liu and Nao Matsumoto and Toshiyuki Kaneshiro and Takaaki Kato and Hideki Shigematsu and Satoshi Kikkawa and Eriko Nitta and Tsuyoshi Imasaki and Ryo Nitta},

url = {http://biorxiv.org/lookup/doi/10.1101/2025.11.28.691112},

doi = {10.1101/2025.11.28.691112},

year  = {2025},

date = {2025-11-30},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Abstract

                Microtubule severing enzyme spastin plays pivotal roles in cytokinesis and neuronal outgrowth, and its mutations cause hereditary spastic paraplegia (HSP). Here we show that, at physiological tubulin concentrations, human spastin behaves predominantly as an ATPase- independent microtubule nucleator rather than a severase. Biochemical and structural analyses revealed that spastin assembles tubulin into stacked-ring intermediates via strategically positioned microtubule-binding domains, thereby generating nucleation-competent sites for polymerization. Tubulin subunits within these spastin-induced rings adopt a straight, microtubule-like interface, in marked contrast to the twisted tubulin spirals characteristic of depolymerizing microtubule ends. Our results redefine the mechanistic landscape of multifaceted spastin functions and provide insights that may inform the pathological basis of HSP and guide future therapeutic strategies.},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Kambara2025,

title = {Enhanced axonal transport in large vertebrates: KIF5A adaptations in giraffes and pythons},

author = {Taketoshi Kambara and Lu Rao and Yosuke Yamagishi and Kazuho Ikeda and Daisuke Taniguchi and Tsuyoshi Imasaki and Hideki Shigematsu and Naoki Sakai and Arne Gennerich and Ryo Nitta and Yasushi Okada},

url = {http://biorxiv.org/lookup/doi/10.1101/2025.05.04.652084},

doi = {10.1101/2025.05.04.652084},

year  = {2025},

date = {2025-05-05},

publisher = {Cold Spring Harbor Laboratory},

abstract = {SUMMARYEfficient axonal transport is essential for neuronal function, particularly in species with exceptionally long axons. The Kinesin-1 motor protein KIF5A plays a key role in this process, but whether and how it adapts to the transport demands of large vertebrates remains unclear. Here, we show that KIF5A from giraffes (GcKIF5A) and pythons (PbKIF5A), moves 25% faster than its mouse counterpart (MmKIF5A) on neuronal microtubulesin vitroand within axons of cultured mouse hippocampal neurons. This enhanced velocity, driven by three unique amino acid substitutions (R114Q, S155A and Y309F), facilitates long-distance transport in these species and represents a case of convergent evolution. Our structural analysis reveals that accelerated ADP release underlies the increased speed of GcKIF5A. Despite exhibiting a reduced force generation, GcKIF5A maintains efficient cargo transport under load. Furthermore, GcKIF5A exerts less drag in mixed-motor environments, an adaptation particularly beneficial for multiple motor-driven long-distance axonal transport. These findings reveal that KIF5A has evolved specific adaptations to facilitate efficient axonal transport in large vertebrates, highlighting the evolutionary plasticity of kinesin motors.},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Jiancheng2024,

title = {Dimerization GAS2 mediates microtubule and F-actin crosslinking},

author = {An Jiancheng and Tsuyoshi Imasaki and Akihiro Narita and Shinsuke Niwa and Ryohei Sasaki and Tsukasa Makino and Ryo Nitta and Masahide Kikkawa},

url = {http://biorxiv.org/lookup/doi/10.1101/2024.08.19.608523},

doi = {10.1101/2024.08.19.608523},

year  = {2024},

date = {2024-08-19},

urldate = {2024-08-19},

publisher = {Cold Spring Harbor Laboratory},

abstract = {<jats:p>GAS2 was originally identified as a growth arrest-specific protein, and recent studies have revealed its involvement in multiple cellular processes. Its dual interaction with actin filaments and microtubules highlights its essential role in cytoskeletal organization, such as cell division, apoptosis, and possibly tumorigenesis. However, the structural bases by which GAS2 regulates cytoskeletal dynamics remain unclear. In this study, we present cryo-EM structures of the GAS2-CH3 domain in complex with F-actin at 2.8 Angstrom resolution, representing the first type 3 CH domain structure bound to F-actin, confirming its actin-binding activity. We also provide the first near-atomic resolution cryo-EM structure of the GAS2-GAR domain bound to microtubules and identified conserved microtubule-binding residues. Our biochemical experiments show that GAS2 promotes microtubule nucleation and polymerization and its C-terminal region is essential for dimerization, bundling of both F-actin and microtubules, and microtubule nucleation. Based on these results, we propose how GAS2 controls cytoskeletal organization.</jats:p>},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Inaba2024,

title = {Cryo-electron tomography of the actin cytoskeleton and membrane organization during lamellipodia formation using optogenetics},

author = {Hironori Inaba and Tsuyoshi Imasaki and Kazuhiro Aoyama and Shogo Yoshihara and Hiroko Takazaki and Takayuki Kato and Hidemasa Goto and Kaoru Mitsuoka and Ryo Nitta and Takao Nakata},

url = {http://biorxiv.org/lookup/doi/10.1101/2024.08.13.607852},

doi = {10.1101/2024.08.13.607852},

year  = {2024},

date = {2024-08-14},

urldate = {2024-08-14},

publisher = {Cold Spring Harbor Laboratory},

abstract = {<jats:p>Lamellipodia are sheet-like cellular protrusions crucial for cell migration and endocytosis; their ultrastructure has been extensively studied using electron microscopy. However, the ultrastructure of the actin cytoskeleton during lamellipodia formation remains underexplored. Here, we employed the optogenetic tool PA-Rac1 combined with cryo-electron tomography (cryo-ET) to precisely control Rac1 activation and subsequent freezing via blue light irradiation. This approach enabled detailed structural analysis of newly formed lamellipodia in cells with intact plasma membranes. We successfully visualized lamellipodia with varying degrees of extension, representing different stages of lamellipodia formation. In minor extensions, several unbundled actin filaments formed "Minor protrusions" at several points along the leading edge. For moderately extended lamellipodia, cross-linked actin filaments formed small filopodia-like structures, termed "mini filopodia." In the most extended lamellipodia, filopodia matured at multiple points along the leading edge, and the number of cross-linked actin filaments running nearly parallel to the leading edge increased throughout the lamellipodia. These observations suggest that actin polymerization begins in specific plasma membrane regions, forming mini filopodia that either mature into full filopodia or detach from the leading edge to form parallel filaments. This turnover of actin structures likely drives lamellipodial protrusion and stabilizes the base, providing new insights into the structural dynamics of the actin cytoskeleton and the mechanisms driving cell migration.</jats:p>},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Shibata2024,

title = {Structural transitions in kinesin minus-end directed microtubule motility},

author = {Satoki Shibata and Matthew Y. Wang and Tsuyoshi Imasaki and Hideki Shigetmatsu and Yuanyuan Wei and Chacko Jobichen and Hajime Hagio and J Sivaraman and Sharyn A. Endow and Ryo Nitta},

url = {http://biorxiv.org/lookup/doi/10.1101/2024.07.29.605428},

doi = {10.1101/2024.07.29.605428},

year  = {2024},

date = {2024-07-29},

urldate = {2024-07-29},

publisher = {Cold Spring Harbor Laboratory},

abstract = {<jats:p>Kinesin motor proteins hydrolyze ATP to produce force for spindle assembly and vesicle transport, performing essential functions in cell division and motility, but the structural changes required for force generation are uncertain. We now report high-resolution structures showing new transitions in the kinesin mechanochemical cycle, including power stroke fluctuations upon ATP binding and a post-hydrolysis state with bound ADP + free phosphate. We find that rate-limiting ADP release occurs upon microtubule binding, accompanied by central beta-sheet twisting, which triggers the power stroke - stalk rotation and neck mimic docking - upon ATP binding. Microtubule release occurs with beta-strand-to-loop transitions, implying that beta-strand refolding induces Pi release and the recovery stroke. The strained beta-sheet during the power stroke and strand-to-loop transitions identify the beta-sheet as the long-sought motor spring.</jats:p>},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Taguchi2022.02.14.480441,

title = {Structural model of microtubule dynamics inhibition by Kinesin-4 from the crystal structure of KLP-12 –tubulin complex},

author = {Shinya Taguchi and Juri Nakano and Tsuyoshi Imasaki and Tomoki Kita and Yumiko Saijo-Hamano and Naoki Sakai and Hideki Shigematsu and Hiromichi Okuma and Takahiro Shimizu and Eriko Nitta and Satoshi Kikkawa and Satoshi Mizobuchi and Shinsuke Niwa and Ryo Nitta},

url = {https://www.biorxiv.org/content/early/2022/02/15/2022.02.14.480441},

doi = {10.1101/2022.02.14.480441},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.Competing Interest StatementThe authors have declared no competing interest.},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Okuma2022.10.31.514472,

title = {Structural basis of Irgb6 inactivation by~Toxoplasma gondii~through the phosphorylation of switch I.},

author = {Hiromichi Okuma and Yumiko Saijo-Hamano and Aalaa Alrahman Sherif and Emi Hoshizaki and Naoki Sakai and Takaaki Kato and Tsuyoshi Imasaki and Eriko Nitta and Miwa Sasai and Yoshimasa Maniwa and Hidetaka Kosako and Daron M Standley and Masahiro Yamamoto and Ryo Nitta},

url = {https://www.biorxiv.org/content/early/2022/11/01/2022.10.31.514472},

doi = {10.1101/2022.10.31.514472},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Upon infection with~Toxoplasma gondii, host cells produce immune-related GTPases (IRGs) to kill the parasite.~T. gondii~counters this response by releasing ROP18 kinase,~which inactivates IRG GTPases and inhibits their recruitment to the~T. gondii~parasitophorous vacuole (PV). However, the molecular mechanisms of this process are entirely unknown. Here we report the atomic structures of Irgb6 with a phosphomimetic mutation by ROP18. The mutant has~lower GTPase activity and is not recruited to the PV membrane (PVM). The crystal structure shows the mutant exhibit a distinct conformation from~the physiological nucleotide-free form, thus preventing GTPase cycling. This change allosterically modifies the conformation of the membrane-binding interface, preventing physiological PVM-binding.~Docking simulation of PI5P also supports the impaired binding of the mutant to PVM. We thus demonstrate the structural basis for~T. gondii~escape from host cell-autonomous defense, and provide a structural model for regulating enzymatic activity by phosphorylation.Competing Interest StatementThe authors have declared no competing interest.},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Imasaki2021.03.01.433304,

title = {CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre},

author = {Tsuyoshi Imasaki and Satoshi Kikkawa and Shinsuke Niwa and Yumiko Saijo-Hamano and Hideki Shigematsu and Kazuhiro Aoyama and Kaoru Mitsuoka and Mari Aoki and Ayako Sakamoto and Yuri Tomabechi and Naoki Sakai and Mikako Shirouzu and Shinya Taguchi and Yosuke Yamagishi and Tomiyoshi Setsu and Yoshiaki Sakihama and Takahiro Shimizu and Eriko Nitta and Masatoshi Takeichi and Ryo Nitta},

url = {https://www.biorxiv.org/content/early/2021/03/01/2021.03.01.433304},

doi = {10.1101/2021.03.01.433304},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, detachment of a considerable number of microtubules from the centrosome is known to contribute to fundamental processes in cells. Here, we present evidence that minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation from soluble αβ-tubulin independent of γ-tubulin. CAMSAP2 significantly reduces the nucleation barrier close to the critical concentration for microtubule polymerization by stabilizing the longitudinal contacts among αβ-tubulins. CAMSAP2 clusters together with αβ-tubulin to generate nucleation intermediates, from which numerous microtubules radiate, forming aster-like structures. Our findings suggest that CAMSAP2 supports microtubule growth by organizing a nucleation centre as well as by stabilizing microtubule nucleation intermediates.Competing Interest StatementThe authors have declared no competing interest.},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Hamano2021.07.01.450801,

title = {Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6},

author = {Yumiko Saijo Hamano and Aalaa Alrahman Sherif and Ariel Pradipta and Miwa Sasai and Naoki Sakai and Yoshiaki Sakihama and Masahiro Yamamoto and Daron M Standley and Ryo Nitta},

url = {https://www.biorxiv.org/content/early/2021/07/01/2021.07.01.450801},

doi = {10.1101/2021.07.01.450801},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. It is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive due to lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP-binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.Competing Interest StatementThe authors have declared no competing interest.},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{nokey,

title = {CAMSAP2 condensates drive a γ-TuRC-independent pathway for non-centrosomal microtubule nucleation},

author = {Yurtsever, Ayhan; Imasaki, Tsuyoshi; Kitano, Ryota; Ngo, Kien Xuan; Yagi, Tomoaki; Kuno, Shuhei; Liu, Hanjin; Kato, Takaaki; Shigematsu, Hideki; Newaz, Md Fahim; Sugita, Yuji; Fukuma, Takeshi; Nitta, Ryo},

doi = {10.64898/2026.05.28.728596},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Yurtsever2026,

title = {CAMSAP2 condensates drive a γ-TuRC-independent pathway for non-centrosomal microtubule nucleation},

author = {Ayhan Yurtsever and Tsuyoshi Imasaki and Ryota Kitano and Kien Xuan Ngo and Tomoaki Yagi and Shuhei Kuno and Hanjin Liu and Takaaki Kato and Hideki Shigematsu and Md Fahim Newaz and Yuji Sugita and Takeshi Fukuma and Ryo Nitta},

url = {http://biorxiv.org/lookup/doi/10.64898/2026.05.28.728596},

doi = {10.64898/2026.05.28.728596},

year  = {2026},

date = {2026-05-29},

publisher = {openRxiv},

abstract = {Abstract

                

                  Microtubule nucleation is commonly viewed as a γ-tubulin ring complex (γ-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 γ-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 γ-TuRC-independent microtubule-organizing centers alongside centrosomal asters, supporting a model in which spontaneous nucleation contributes to non-centrosomal microtubule biogenesis.

                },

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Liu2025,

title = {Human spastin functions as an ATPase-independent microtubule nucleator via ring-stack formation},

author = {Hanjin Liu and Nao Matsumoto and Toshiyuki Kaneshiro and Takaaki Kato and Hideki Shigematsu and Satoshi Kikkawa and Eriko Nitta and Tsuyoshi Imasaki and Ryo Nitta},

url = {http://biorxiv.org/lookup/doi/10.1101/2025.11.28.691112},

doi = {10.1101/2025.11.28.691112},

year  = {2025},

date = {2025-11-30},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Abstract

                Microtubule severing enzyme spastin plays pivotal roles in cytokinesis and neuronal outgrowth, and its mutations cause hereditary spastic paraplegia (HSP). Here we show that, at physiological tubulin concentrations, human spastin behaves predominantly as an ATPase- independent microtubule nucleator rather than a severase. Biochemical and structural analyses revealed that spastin assembles tubulin into stacked-ring intermediates via strategically positioned microtubule-binding domains, thereby generating nucleation-competent sites for polymerization. Tubulin subunits within these spastin-induced rings adopt a straight, microtubule-like interface, in marked contrast to the twisted tubulin spirals characteristic of depolymerizing microtubule ends. Our results redefine the mechanistic landscape of multifaceted spastin functions and provide insights that may inform the pathological basis of HSP and guide future therapeutic strategies.},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Kambara2025,

title = {Enhanced axonal transport in large vertebrates: KIF5A adaptations in giraffes and pythons},

author = {Taketoshi Kambara and Lu Rao and Yosuke Yamagishi and Kazuho Ikeda and Daisuke Taniguchi and Tsuyoshi Imasaki and Hideki Shigematsu and Naoki Sakai and Arne Gennerich and Ryo Nitta and Yasushi Okada},

url = {http://biorxiv.org/lookup/doi/10.1101/2025.05.04.652084},

doi = {10.1101/2025.05.04.652084},

year  = {2025},

date = {2025-05-05},

publisher = {Cold Spring Harbor Laboratory},

abstract = {SUMMARYEfficient axonal transport is essential for neuronal function, particularly in species with exceptionally long axons. The Kinesin-1 motor protein KIF5A plays a key role in this process, but whether and how it adapts to the transport demands of large vertebrates remains unclear. Here, we show that KIF5A from giraffes (GcKIF5A) and pythons (PbKIF5A), moves 25% faster than its mouse counterpart (MmKIF5A) on neuronal microtubulesin vitroand within axons of cultured mouse hippocampal neurons. This enhanced velocity, driven by three unique amino acid substitutions (R114Q, S155A and Y309F), facilitates long-distance transport in these species and represents a case of convergent evolution. Our structural analysis reveals that accelerated ADP release underlies the increased speed of GcKIF5A. Despite exhibiting a reduced force generation, GcKIF5A maintains efficient cargo transport under load. Furthermore, GcKIF5A exerts less drag in mixed-motor environments, an adaptation particularly beneficial for multiple motor-driven long-distance axonal transport. These findings reveal that KIF5A has evolved specific adaptations to facilitate efficient axonal transport in large vertebrates, highlighting the evolutionary plasticity of kinesin motors.},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Jiancheng2024,

title = {Dimerization GAS2 mediates microtubule and F-actin crosslinking},

author = {An Jiancheng and Tsuyoshi Imasaki and Akihiro Narita and Shinsuke Niwa and Ryohei Sasaki and Tsukasa Makino and Ryo Nitta and Masahide Kikkawa},

url = {http://biorxiv.org/lookup/doi/10.1101/2024.08.19.608523},

doi = {10.1101/2024.08.19.608523},

year  = {2024},

date = {2024-08-19},

urldate = {2024-08-19},

publisher = {Cold Spring Harbor Laboratory},

abstract = {<jats:p>GAS2 was originally identified as a growth arrest-specific protein, and recent studies have revealed its involvement in multiple cellular processes. Its dual interaction with actin filaments and microtubules highlights its essential role in cytoskeletal organization, such as cell division, apoptosis, and possibly tumorigenesis. However, the structural bases by which GAS2 regulates cytoskeletal dynamics remain unclear. In this study, we present cryo-EM structures of the GAS2-CH3 domain in complex with F-actin at 2.8 Angstrom resolution, representing the first type 3 CH domain structure bound to F-actin, confirming its actin-binding activity. We also provide the first near-atomic resolution cryo-EM structure of the GAS2-GAR domain bound to microtubules and identified conserved microtubule-binding residues. Our biochemical experiments show that GAS2 promotes microtubule nucleation and polymerization and its C-terminal region is essential for dimerization, bundling of both F-actin and microtubules, and microtubule nucleation. Based on these results, we propose how GAS2 controls cytoskeletal organization.</jats:p>},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Inaba2024,

title = {Cryo-electron tomography of the actin cytoskeleton and membrane organization during lamellipodia formation using optogenetics},

author = {Hironori Inaba and Tsuyoshi Imasaki and Kazuhiro Aoyama and Shogo Yoshihara and Hiroko Takazaki and Takayuki Kato and Hidemasa Goto and Kaoru Mitsuoka and Ryo Nitta and Takao Nakata},

url = {http://biorxiv.org/lookup/doi/10.1101/2024.08.13.607852},

doi = {10.1101/2024.08.13.607852},

year  = {2024},

date = {2024-08-14},

urldate = {2024-08-14},

publisher = {Cold Spring Harbor Laboratory},

abstract = {<jats:p>Lamellipodia are sheet-like cellular protrusions crucial for cell migration and endocytosis; their ultrastructure has been extensively studied using electron microscopy. However, the ultrastructure of the actin cytoskeleton during lamellipodia formation remains underexplored. Here, we employed the optogenetic tool PA-Rac1 combined with cryo-electron tomography (cryo-ET) to precisely control Rac1 activation and subsequent freezing via blue light irradiation. This approach enabled detailed structural analysis of newly formed lamellipodia in cells with intact plasma membranes. We successfully visualized lamellipodia with varying degrees of extension, representing different stages of lamellipodia formation. In minor extensions, several unbundled actin filaments formed "Minor protrusions" at several points along the leading edge. For moderately extended lamellipodia, cross-linked actin filaments formed small filopodia-like structures, termed "mini filopodia." In the most extended lamellipodia, filopodia matured at multiple points along the leading edge, and the number of cross-linked actin filaments running nearly parallel to the leading edge increased throughout the lamellipodia. These observations suggest that actin polymerization begins in specific plasma membrane regions, forming mini filopodia that either mature into full filopodia or detach from the leading edge to form parallel filaments. This turnover of actin structures likely drives lamellipodial protrusion and stabilizes the base, providing new insights into the structural dynamics of the actin cytoskeleton and the mechanisms driving cell migration.</jats:p>},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Shibata2024,

title = {Structural transitions in kinesin minus-end directed microtubule motility},

author = {Satoki Shibata and Matthew Y. Wang and Tsuyoshi Imasaki and Hideki Shigetmatsu and Yuanyuan Wei and Chacko Jobichen and Hajime Hagio and J Sivaraman and Sharyn A. Endow and Ryo Nitta},

url = {http://biorxiv.org/lookup/doi/10.1101/2024.07.29.605428},

doi = {10.1101/2024.07.29.605428},

year  = {2024},

date = {2024-07-29},

urldate = {2024-07-29},

publisher = {Cold Spring Harbor Laboratory},

abstract = {<jats:p>Kinesin motor proteins hydrolyze ATP to produce force for spindle assembly and vesicle transport, performing essential functions in cell division and motility, but the structural changes required for force generation are uncertain. We now report high-resolution structures showing new transitions in the kinesin mechanochemical cycle, including power stroke fluctuations upon ATP binding and a post-hydrolysis state with bound ADP + free phosphate. We find that rate-limiting ADP release occurs upon microtubule binding, accompanied by central beta-sheet twisting, which triggers the power stroke - stalk rotation and neck mimic docking - upon ATP binding. Microtubule release occurs with beta-strand-to-loop transitions, implying that beta-strand refolding induces Pi release and the recovery stroke. The strained beta-sheet during the power stroke and strand-to-loop transitions identify the beta-sheet as the long-sought motor spring.</jats:p>},

howpublished = {bioRxiv},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Taguchi2022.02.14.480441,

title = {Structural model of microtubule dynamics inhibition by Kinesin-4 from the crystal structure of KLP-12 –tubulin complex},

author = {Shinya Taguchi and Juri Nakano and Tsuyoshi Imasaki and Tomoki Kita and Yumiko Saijo-Hamano and Naoki Sakai and Hideki Shigematsu and Hiromichi Okuma and Takahiro Shimizu and Eriko Nitta and Satoshi Kikkawa and Satoshi Mizobuchi and Shinsuke Niwa and Ryo Nitta},

url = {https://www.biorxiv.org/content/early/2022/02/15/2022.02.14.480441},

doi = {10.1101/2022.02.14.480441},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.Competing Interest StatementThe authors have declared no competing interest.},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Okuma2022.10.31.514472,

title = {Structural basis of Irgb6 inactivation by~Toxoplasma gondii~through the phosphorylation of switch I.},

author = {Hiromichi Okuma and Yumiko Saijo-Hamano and Aalaa Alrahman Sherif and Emi Hoshizaki and Naoki Sakai and Takaaki Kato and Tsuyoshi Imasaki and Eriko Nitta and Miwa Sasai and Yoshimasa Maniwa and Hidetaka Kosako and Daron M Standley and Masahiro Yamamoto and Ryo Nitta},

url = {https://www.biorxiv.org/content/early/2022/11/01/2022.10.31.514472},

doi = {10.1101/2022.10.31.514472},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Upon infection with~Toxoplasma gondii, host cells produce immune-related GTPases (IRGs) to kill the parasite.~T. gondii~counters this response by releasing ROP18 kinase,~which inactivates IRG GTPases and inhibits their recruitment to the~T. gondii~parasitophorous vacuole (PV). However, the molecular mechanisms of this process are entirely unknown. Here we report the atomic structures of Irgb6 with a phosphomimetic mutation by ROP18. The mutant has~lower GTPase activity and is not recruited to the PV membrane (PVM). The crystal structure shows the mutant exhibit a distinct conformation from~the physiological nucleotide-free form, thus preventing GTPase cycling. This change allosterically modifies the conformation of the membrane-binding interface, preventing physiological PVM-binding.~Docking simulation of PI5P also supports the impaired binding of the mutant to PVM. We thus demonstrate the structural basis for~T. gondii~escape from host cell-autonomous defense, and provide a structural model for regulating enzymatic activity by phosphorylation.Competing Interest StatementThe authors have declared no competing interest.},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Imasaki2021.03.01.433304,

title = {CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre},

author = {Tsuyoshi Imasaki and Satoshi Kikkawa and Shinsuke Niwa and Yumiko Saijo-Hamano and Hideki Shigematsu and Kazuhiro Aoyama and Kaoru Mitsuoka and Mari Aoki and Ayako Sakamoto and Yuri Tomabechi and Naoki Sakai and Mikako Shirouzu and Shinya Taguchi and Yosuke Yamagishi and Tomiyoshi Setsu and Yoshiaki Sakihama and Takahiro Shimizu and Eriko Nitta and Masatoshi Takeichi and Ryo Nitta},

url = {https://www.biorxiv.org/content/early/2021/03/01/2021.03.01.433304},

doi = {10.1101/2021.03.01.433304},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, detachment of a considerable number of microtubules from the centrosome is known to contribute to fundamental processes in cells. Here, we present evidence that minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation from soluble αβ-tubulin independent of γ-tubulin. CAMSAP2 significantly reduces the nucleation barrier close to the critical concentration for microtubule polymerization by stabilizing the longitudinal contacts among αβ-tubulins. CAMSAP2 clusters together with αβ-tubulin to generate nucleation intermediates, from which numerous microtubules radiate, forming aster-like structures. Our findings suggest that CAMSAP2 supports microtubule growth by organizing a nucleation centre as well as by stabilizing microtubule nucleation intermediates.Competing Interest StatementThe authors have declared no competing interest.},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{Hamano2021.07.01.450801,

title = {Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6},

author = {Yumiko Saijo Hamano and Aalaa Alrahman Sherif and Ariel Pradipta and Miwa Sasai and Naoki Sakai and Yoshiaki Sakihama and Masahiro Yamamoto and Daron M Standley and Ryo Nitta},

url = {https://www.biorxiv.org/content/early/2021/07/01/2021.07.01.450801},

doi = {10.1101/2021.07.01.450801},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. It is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive due to lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP-binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.Competing Interest StatementThe authors have declared no competing interest.},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}

@unpublished{nokey,

title = {CAMSAP2 condensates drive a γ-TuRC-independent pathway for non-centrosomal microtubule nucleation},

author = {Yurtsever, Ayhan; Imasaki, Tsuyoshi; Kitano, Ryota; Ngo, Kien Xuan; Yagi, Tomoaki; Kuno, Shuhei; Liu, Hanjin; Kato, Takaaki; Shigematsu, Hideki; Newaz, Md Fahim; Sugita, Yuji; Fukuma, Takeshi; Nitta, Ryo},

doi = {10.64898/2026.05.28.728596},

keywords = {},

pubstate = {published},

tppubtype = {unpublished}

}