vestnik-bio-msuВестник Московского университета. Серия 16. БиологияVestnik Moskovskogo universiteta. Seriya 16. Biologiya0137-0952Lomonosov Moscow State University, School of Biology10.55959/MSU0137-0952-16-80-3S-3vestnik-bio-msu-1535Research ArticleОбзорыИзучение гибких элементов структуры микротрубочек с помощью криоэлектронной микроскопии и молекулярного моделирования: миссия выполнима?Studying flexible elements of microtubule structure using cryo-electron microscopy and molecular modeling: mission possible?https://orcid.org/0000-0002-9397-1548ФедоровВ. А.FedorovV. A.

Федоров Владимир Андреевич – канд. физ.-мат. наук, науч. сотр. кафедры биофизики биологического факультета

119234, г. Москва, Ленинские горы, д. 1, стр. 24

Тел.: 8-495-939-11-16

1–24 Leninskie gory, Moscow, 119234

xbgth@yandex.ruhttps://orcid.org/0000-0002-5918-5084ХолинаЕ. Г.KholinaE. G.

Холина Екатерина Георгиевна – канд. биол. наук, ст. преп. кафедры биофизики биологического факультета

119234, г. Москва, Ленинские горы, д. 1, стр. 24

Тел.: 8-495-939-11-16

1–24 Leninskie gory, Moscow, 119234

tenarra@mail.ruhttps://orcid.org/0000-0002-7283-8959ГудимчукН. Б.GudimchukN. B.

Гудимчук Никита Борисович – докт. физ.-мат. наук, ст. науч. сотр. кафедры биофизики физического факультета

119991, г. Москва, Ленинские горы, д. 1, стр. 2

Тел.: 8-495-938-00-06

1–2 Leninskie gory, Moscow, 119991

nikita_gb@mail.ruhttps://orcid.org/0000-0002-4949-6591КоваленкоИ. Б.KovalenkoI. B.

Коваленко Илья Борисович – докт. физ.-мат. наук, вед. науч. сотр. кафедры биофизики биологического факультета

119234, г. Москва, Ленинские горы, д. 1, стр. 24

Тел.: 8-495-939-11-16

1–24 Leninskie gory, Moscow, 119234

ikovalenko78@gmail.comКафедра биофизики, биологический факультет, Московский государственный университет имени М.В. ЛомоносоваРоссияDepartment of Biophysics, School of Biology, Lomonosov Moscow State UniversityRussian FederationКафедра биофизики, физический факультет, Московский государственный университет имени М.В. ЛомоносоваРоссияDepartment of Biophysics, School of Physics, Lomonosov Moscow State UniversityRussian Federation202510112025803S914Copyright © Федоров В.А., Холина Е.Г., Гудимчук Н.Б., Коваленко И.Б., 20252025Федоров В.А., Холина Е.Г., Гудимчук Н.Б., Коваленко И.Б.Fedorov V.A., Kholina E.G., Gudimchuk N.B., Kovalenko I.B.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestnik-bio-msu.elpub.ru/jour/article/view/1535

Микротрубочки являются базовыми элементами цитоскелета эукариотических клеток. Благодаря своей многофункциональности, уникальной структуре и высокой механической жесткости, они остаются излюбленным объектом исследований с использованием различных методов микроскопии, включая криоэлектронную. Несмотря на впечатляющие успехи в визуализации решетки микротрубочек, гибкие элементы их структуры – отдельные протофиламенты на собирающихся или разбирающихся плюс-концах, а также регуляторные неструктурированные пептиды, известные как C-концевые «хвосты», – до сих пор слабо поддаются визуализации. В этой статье мы обсуждаем прогресс в применении криоэлектронной микроскопии и томографии к изучению этих участков, а также роль и потенциал методов молекулярного моделирования для анализа и интерпретации полученных экспериментальных данных.

Microtubules are the basic elements of the cytoskeleton of eukaryotic cells. Due to their multifunctionality, unique structure and high mechanical rigidity, they remain a favorite object of research using various microscopy techniques, including cryo-electron microscopy. Despite impressive advances in visualizing the microtubule lattice, flexible elements of their structure – individual protofilaments at the assembling or disassembling ends, as well as regulatory unstructured peptides known as C-terminal “tails” – are still poorly visualized. In this paper, we discuss progress in the application of cryo-electron microscopy and tomography to the study of these structural elements, as well as the role and potential of molecular modeling methods for the analysis and interpretation of the obtained experimental data.

микротрубочкитубулинкриоэлектронная микроскопиямолекулярная динамикагибкие элементы структурыmicrotubulestubulincryo-electron microscopymolecular dynamicsflexible structural elementsРабота выполнена при финансовой поддержке Российского научного фонда (проект № 24-74-00002 (https://rscf.ru/project/24-74-00002/) и проведена без использования животных и без привлечения людей в качестве испытуемых.This work was supported by the Russian Science Foundation, project no. 24-74-00002 (https://rscf.ru/project/24-74-00002/).ReferencesDesai A., Mitchison T.J. Microtubule polymerization dynamics. Annu. Rev. Cell. Dev. Biol. 1997;13:83–117.Desai A., Mitchison T.J. Microtubule polymerization dynamics. Annu. Rev. Cell. Dev. Biol. 1997;13:83–117.Gudimchuk N.B., McIntosh J.R. Regulation of microtubule dynamics, mechanics and function through the growing tip. Nat. Rev. Mol. Cell. Biol. 2021;22(12):777–795.Gudimchuk N.B., McIntosh J.R. Regulation of microtubule dynamics, mechanics and function through the growing tip. Nat. Rev. Mol. Cell. Biol. 2021;22(12):777–795.Roll-Mecak A. The tubulin code in microtubule dynamics and information encoding. Dev. Cell. 2020;54(1):7–20.Roll-Mecak A. The tubulin code in microtubule dynamics and information encoding. Dev. Cell. 2020;54(1):7–20.McKenna E.D., Sarbanes S.L., Cummings S.W., Roll-Mecak A. The tubulin code, from molecules to health and disease. Annu. Rev. Cell. Dev. Biol. 2023;39:331–361.McKenna E.D., Sarbanes S.L., Cummings S.W., Roll-Mecak A. The tubulin code, from molecules to health and disease. Annu. Rev. Cell. Dev. Biol. 2023;39:331–361.Janke C., Magiera M.M. The tubulin code and its role in controlling microtubule properties and functions. Nat. Rev. Mol. Cell. Biol. 2020;21(6):307–326.Janke C., Magiera M.M. The tubulin code and its role in controlling microtubule properties and functions. Nat. Rev. Mol. Cell. Biol. 2020;21(6):307–326.Wagstaff J.M., Planelles-Herrero V.J., Sharov G., Alnami A., Kozielski F., Derivery E., Löwe J. Diverse cytomotive actins and tubulins share a polymerization switch mechanism conferring robust dynamics. Sci. Adv. 2023;9(13):eadf3021.Wagstaff J.M., Planelles-Herrero V.J., Sharov G., Alnami A., Kozielski F., Derivery E., Löwe J. Diverse cytomotive actins and tubulins share a polymerization switch mechanism conferring robust dynamics. Sci. Adv. 2023;9(13):eadf3021.Alushin G.M., Lander G.C., Kellogg E.H., Zhang R., Baker D., Nogales E. High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Cell. 2014;157(5):1117–1129.Alushin G.M., Lander G.C., Kellogg E.H., Zhang R., Baker D., Nogales E. High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Cell. 2014;157(5):1117–1129.Manka S.W., Moores C.A. The role of tubulin–tubulin lattice contacts in the mechanism of microtubule dynamic instability. Nat. Struct. Mol. Biol. 2018;25(7):607–615.Manka S.W., Moores C.A. The role of tubulin–tubulin lattice contacts in the mechanism of microtubule dynamic instability. Nat. Struct. Mol. Biol. 2018;25(7):607–615.Zhang R., LaFrance B., Nogales E. Separating the effects of nucleotide and EB binding on microtubule structure. Proc. Natl. Acad. Sci. U.S.A. 2018;115(27):E6191–E6200.Zhang R., LaFrance B., Nogales E. Separating the effects of nucleotide and EB binding on microtubule structure. Proc. Natl. Acad. Sci. U.S.A. 2018;115(27):E6191–E6200.Zhang R., Alushin G.M., Brown A., Nogales E. Mechanistic origin of microtubule dynamic instability and its modulation by EB proteins. Cell. 2015;162(4):849–859.Zhang R., Alushin G.M., Brown A., Nogales E. Mechanistic origin of microtubule dynamic instability and its modulation by EB proteins. Cell. 2015;162(4):849–859.Debs G.E., Cha M., Liu X., Huehn A.R., Sindelar C.V. Dynamic and asymmetric fluctuations in the microtubule wall captured by high-resolution cryoelectron microscopy. Proc. Natl. Acad. Sci. U.S.A. 2020;117(29):16976–16984.Debs G.E., Cha M., Liu X., Huehn A.R., Sindelar C.V. Dynamic and asymmetric fluctuations in the microtubule wall captured by high-resolution cryoelectron microscopy. Proc. Natl. Acad. Sci. U.S.A. 2020;117(29):16976–16984.LaFrance B.J., Roostalu J., Henkin G., Greber B.J., Zhang R., Normanno D, McCollum C.O., Surrey T., Nogales E. Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules. Proc. Natl. Acad. Sci. U.S.A. 2022;119(2):e2114994119.LaFrance B.J., Roostalu J., Henkin G., Greber B.J., Zhang R., Normanno D, McCollum C.O., Surrey T., Nogales E. Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules. Proc. Natl. Acad. Sci. U.S.A. 2022;119(2):e2114994119.Estévez-Gallego J., Blum T.B., Ruhnow F., Gili M., Speroni S., García-Castellanos R., Steinmetz M.O., Surrey T. Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap. Nat. Commun. 2025;16(1):2396.Estévez-Gallego J., Blum T.B., Ruhnow F., Gili M., Speroni S., García-Castellanos R., Steinmetz M.O., Surrey T. Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap. Nat. Commun. 2025;16(1):2396.Wang Q., Crevenna A.H., Kunze I., Mizuno N. Structural basis for the extended CAP-Gly domains of p150(glued) binding to microtubules and the implication for tubulin dynamics. Proc. Natl. Acad. Sci. U.S.A. 2014;111(31):11347–11352.Wang Q., Crevenna A.H., Kunze I., Mizuno N. Structural basis for the extended CAP-Gly domains of p150(glued) binding to microtubules and the implication for tubulin dynamics. Proc. Natl. Acad. Sci. U.S.A. 2014;111(31):11347–11352.Benoit M.P.M.H., Rao L., Asenjo A.B., Gennerich A., Sosa H. Cryo-EM unveils kinesin KIF1A’s processivity mechanism and the impact of its pathogenic variant P305L. Nat. Commun. 2024;15(1):5530.Benoit M.P.M.H., Rao L., Asenjo A.B., Gennerich A., Sosa H. Cryo-EM unveils kinesin KIF1A’s processivity mechanism and the impact of its pathogenic variant P305L. Nat. Commun. 2024;15(1):5530.Alushin G.M., Musinipally V., Matson D., Tooley J., Stukenberg P.T., Nogales E. Multimodal microtubule binding by the Ndc80 kinetochore complex. Nat. Struct. Mol. Biol. 2012;19(11):1161–1167.Alushin G.M., Musinipally V., Matson D., Tooley J., Stukenberg P.T., Nogales E. Multimodal microtubule binding by the Ndc80 kinetochore complex. Nat. Struct. Mol. Biol. 2012;19(11):1161–1167.Ustinov N.B., Korshunova A.V., Gudimchuk N.B. Protein complex NDC80: properties, functions, and possible role in pathophysiology of cell division. Biochemistry (Mosc.). 2020;85(4):448–462.Ustinov N.B., Korshunova A.V., Gudimchuk N.B. Protein complex NDC80: properties, functions, and possible role in pathophysiology of cell division. Biochemistry (Mosc.). 2020;85(4):448–462.Zehr E.A., Roll-Mecak A. Cryo-EM structures of human α1B/βI+βIVb microtubules shed light on isoform specific assembly. BioRxiv. 2023:2023.12.01.569594.Zehr E.A., Roll-Mecak A. Cryo-EM structures of human α1B/βI+βIVb microtubules shed light on isoform specific assembly. BioRxiv. 2023:2023.12.01.569594.Chen J., Kholina E., Szyk A., Fedorov V.A., Kovalenko I., Gudimchuk N., Roll-Mecak A. α-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics. Dev. Cell. 2021;56(14):2016-2028.e4.Chen J., Kholina E., Szyk A., Fedorov V.A., Kovalenko I., Gudimchuk N., Roll-Mecak A. α-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics. Dev. Cell. 2021;56(14):2016-2028.e4.McIntosh J.R., Grishchuk E.L., Morphew M.K., Efremov A.K., Zhudenkov K., Volkov V.A., Cheeseman I.M., Desai A., Mastronarde D.N., Ataullakhanov F.I. Fibrils connect microtubule tips with kinetochores: a mechanism to couple tubulin dynamics to chromosome motion. Cell. 2008;135(2):322–333.McIntosh J.R., Grishchuk E.L., Morphew M.K., Efremov A.K., Zhudenkov K., Volkov V.A., Cheeseman I.M., Desai A., Mastronarde D.N., Ataullakhanov F.I. Fibrils connect microtubule tips with kinetochores: a mechanism to couple tubulin dynamics to chromosome motion. Cell. 2008;135(2):322–333.Höög J.L., Huisman S.M., Sebö-Lemke Z., Sandblad L., McIntosh J.R., Antony C., Brunner D. Electron tomography reveals a flared morphology on growing microtubule ends. J. Cell Sci. 2011;124(Pt. 5):693–698.Höög J.L., Huisman S.M., Sebö-Lemke Z., Sandblad L., McIntosh J.R., Antony C., Brunner D. Electron tomography reveals a flared morphology on growing microtubule ends. J. Cell Sci. 2011;124(Pt. 5):693–698.McIntosh J.R., O’Toole E., Morgan G., Austin J., Ulyanov E., Ataullakhanov F., Gudimchuk N.B. Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments. J. Cell Biol. 2018;217(8):2691–2708.McIntosh J.R., O’Toole E., Morgan G., Austin J., Ulyanov E., Ataullakhanov F., Gudimchuk N.B. Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments. J. Cell Biol. 2018;217(8):2691–2708.Atherton J., Stouffer M., Francis F., Moores C.A. Microtubule architecture in vitro and in cells revealed by cryo-electron tomography. Acta Crystallogr. D Struct Biol. 2018;74(Pt. 6):572–584.Atherton J., Stouffer M., Francis F., Moores C.A. Microtubule architecture in vitro and in cells revealed by cryo-electron tomography. Acta Crystallogr. D Struct Biol. 2018;74(Pt. 6):572–584.Gudimchuk N.B., Ulyanov E.V., O’Toole E., Page C.L., Vinogradov D.S., Morgan G., Li G., Moore J.K., Szczesna E., Roll-Mecak A., Ataullakhanov F.I., McIntosh J.R. Mechanisms of microtubule dynamics and force generation examined with computational modeling and electron cryotomography. Nat. Commun. 2020;11(1):3765.Gudimchuk N.B., Ulyanov E.V., O’Toole E., Page C.L., Vinogradov D.S., Morgan G., Li G., Moore J.K., Szczesna E., Roll-Mecak A., Ataullakhanov F.I., McIntosh J.R. Mechanisms of microtubule dynamics and force generation examined with computational modeling and electron cryotomography. Nat. Commun. 2020;11(1):3765.Buchholz T.-O., Jordan M., Pigino G., Jug F. Cryo- CARE: content-aware image restoration for cryo-transmission electron microscopy data. 2019 IEEE 16th International Symposium on Biomedical Imaging (ISBI 2019). IEEE; 2019:502–506.Buchholz T.-O., Jordan M., Pigino G., Jug F. Cryo- CARE: content-aware image restoration for cryo-transmission electron microscopy data. 2019 IEEE 16th International Symposium on Biomedical Imaging (ISBI 2019). IEEE; 2019:502–506.Kalutskii M., Grubmüller H., Volkov V.A., Igaev M. Microtubule dynamics are defined by conformations and stability of clustered protofilaments. Proc. Natl. Acad. Sci. U.S.A. 2025;122(22):e2424263122.Kalutskii M., Grubmüller H., Volkov V.A., Igaev M. Microtubule dynamics are defined by conformations and stability of clustered protofilaments. Proc. Natl. Acad. Sci. U.S.A. 2025;122(22):e2424263122.Iyer S.S., Chen F., Ogunmolu F.E. et al. Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends. J. Cell Biol. 2025;224(3):e202406061.Iyer S.S., Chen F., Ogunmolu F.E. et al. Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends. J. Cell Biol. 2025;224(3):e202406061.Garnham C.P., Vemu A., Wilson-Kubalek E.M., Yu I., Szyk A., Lander G.C., Milligan R.A., Roll-Mecak A. Multivalent microtubule recognition by tubulin tyrosine ligase- like family glutamylases. Cell. 2015;161(5):1112–1123.Garnham C.P., Vemu A., Wilson-Kubalek E.M., Yu I., Szyk A., Lander G.C., Milligan R.A., Roll-Mecak A. Multivalent microtubule recognition by tubulin tyrosine ligase- like family glutamylases. Cell. 2015;161(5):1112–1123.Fera A., Reese T., Zimmerberg J., Sackett D. Radial carboxyterminal peptides of tubulin directly imaged in microtubules by electron microscopy tomography exploiting amplitude contrast. ResearchGate preprint. 2021;10.21203/rs.3.rs-409249/v1.Fera A., Reese T., Zimmerberg J., Sackett D. Radial carboxyterminal peptides of tubulin directly imaged in microtubules by electron microscopy tomography exploiting amplitude contrast. ResearchGate preprint. 2021;10.21203/rs.3.rs-409249/v1.Trabuco L.G., Villa E., Schreiner E., Harrison C.B., Schulten K. Molecular dynamics flexible fitting: A practical guide to combine cryo-electron microscopy and X-ray crystallography. Methods. 2009;49(2):174–180.Trabuco L.G., Villa E., Schreiner E., Harrison C.B., Schulten K. Molecular dynamics flexible fitting: A practical guide to combine cryo-electron microscopy and X-ray crystallography. Methods. 2009;49(2):174–180.Igaev M., Kutzner C., Bock L.V., Vaiana A.C., Grubmüller H. Automated cryo-EM structure refinement using correlation-driven molecular dynamics. Elife. 2019;8:e43542.Igaev M., Kutzner C., Bock L.V., Vaiana A.C., Grubmüller H. Automated cryo-EM structure refinement using correlation-driven molecular dynamics. Elife. 2019;8:e43542.Eshun-Wilson L., Zhang R., Portran D., Nachury M.V., Toso D.B., Löhr T., Vendruscolo M., Bonomi M., Fraser J.S., Nogales E. Effects of α-tubulin acetylation on microtubule structure and stability. Proc. Natl. Acad. Sci. U.S.A. 2019;116(21):10366–10371.Eshun-Wilson L., Zhang R., Portran D., Nachury M.V., Toso D.B., Löhr T., Vendruscolo M., Bonomi M., Fraser J.S., Nogales E. Effects of α-tubulin acetylation on microtubule structure and stability. Proc. Natl. Acad. Sci. U.S.A. 2019;116(21):10366–10371.

The authors declare that there are no conflicts of interest present.