Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends
EB1 is a key cellular protein that delivers regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher...
| Main Authors: | , , , , , |
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| Format: | Article |
| Jezik: | English |
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eLife Sciences Publications Ltd
2024-02-01
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| Serija: | eLife |
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| Online dostop: | https://elifesciences.org/articles/91719 |
| _version_ | 1827345142988668928 |
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| author | Samuel J Gonzalez Julia M Heckel Rebecca R Goldblum Taylor A Reid Mark McClellan Melissa K Gardner |
| author_facet | Samuel J Gonzalez Julia M Heckel Rebecca R Goldblum Taylor A Reid Mark McClellan Melissa K Gardner |
| author_sort | Samuel J Gonzalez |
| collection | DOAJ |
| description | EB1 is a key cellular protein that delivers regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher affinity to GTP-tubulin subunits at the growing microtubule tip, relative to GDP-tubulin along the microtubule length. However, it is unclear whether this difference in affinity alone is sufficient to explain the tip-tracking of EB1 at growing microtubule tips. Previously, we found that EB1 binds to exposed microtubule protofilament-edge sites at a ~70 fold faster rate than to closed-lattice sites, due to diffusional steric hindrance to binding. Thus, we asked whether rapid protofilament-edge binding could contribute to efficient EB1 tip tracking. A computational simulation with differential EB1 on-rates based on closed-lattice or protofilament-edge binding, and with EB1 off-rates that were dependent on the tubulin hydrolysis state, robustly recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with a Designed Ankyrin Repeat Protein (DARPin) that binds exclusively to protofilament-edge sites, and whose binding site partially overlaps with the EB1 binding site. We found that DARPin blocked EB1 protofilament-edge binding, which led to a decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites contributes to robust EB1 tip tracking at the growing microtubule plus-end. |
| first_indexed | 2024-03-07T23:02:07Z |
| format | Article |
| id | doaj.art-6032f75d380e495b9a0e30f81c2e60d2 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-03-07T23:02:07Z |
| publishDate | 2024-02-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-6032f75d380e495b9a0e30f81c2e60d22024-02-22T12:53:55ZengeLife Sciences Publications LtdeLife2050-084X2024-02-011310.7554/eLife.91719Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-endsSamuel J Gonzalez0https://orcid.org/0000-0002-5372-8068Julia M Heckel1https://orcid.org/0000-0002-5953-1717Rebecca R Goldblum2Taylor A Reid3Mark McClellan4Melissa K Gardner5https://orcid.org/0000-0001-5906-7363Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United StatesDepartment of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United StatesDepartment of Biophysics, Molecular Biology, and Biochemistry, University of Minnesota, Minneapolis, United States; Medical Scientist Training Program, University of Minnesota, Minneapolis, United StatesDepartment of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United StatesDepartment of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United StatesDepartment of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, United StatesEB1 is a key cellular protein that delivers regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher affinity to GTP-tubulin subunits at the growing microtubule tip, relative to GDP-tubulin along the microtubule length. However, it is unclear whether this difference in affinity alone is sufficient to explain the tip-tracking of EB1 at growing microtubule tips. Previously, we found that EB1 binds to exposed microtubule protofilament-edge sites at a ~70 fold faster rate than to closed-lattice sites, due to diffusional steric hindrance to binding. Thus, we asked whether rapid protofilament-edge binding could contribute to efficient EB1 tip tracking. A computational simulation with differential EB1 on-rates based on closed-lattice or protofilament-edge binding, and with EB1 off-rates that were dependent on the tubulin hydrolysis state, robustly recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with a Designed Ankyrin Repeat Protein (DARPin) that binds exclusively to protofilament-edge sites, and whose binding site partially overlaps with the EB1 binding site. We found that DARPin blocked EB1 protofilament-edge binding, which led to a decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites contributes to robust EB1 tip tracking at the growing microtubule plus-end.https://elifesciences.org/articles/91719microtubuleEB1modelingtubulinprotofilamentdiffusion |
| spellingShingle | Samuel J Gonzalez Julia M Heckel Rebecca R Goldblum Taylor A Reid Mark McClellan Melissa K Gardner Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends eLife microtubule EB1 modeling tubulin protofilament diffusion |
| title | Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends |
| title_full | Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends |
| title_fullStr | Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends |
| title_full_unstemmed | Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends |
| title_short | Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends |
| title_sort | rapid binding to protofilament edge sites facilitates tip tracking of eb1 at growing microtubule plus ends |
| topic | microtubule EB1 modeling tubulin protofilament diffusion |
| url | https://elifesciences.org/articles/91719 |
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