{"paper_id":"1aa0ffb8-2d62-4285-852d-b1f1de488b13","body_text":"MAP65-1/PRC1 reinforces microtubules through nucleation 1 \nMariana Romeiro Motta1,2*, Constantin Grisam1, Mona Grünewald1, Belinda König1, Laura 2 \nMeißner3a, Lukas Niese3, Stefan Diez3,4, Olivier Hamant2*, Laura Schaedel1* 3 \n1Experimental Physics, Center for Biophysics, Saarland University, 66123 Saarbrücken, Germany 4 \n2Laboratoire Reproduction et Développement des Plantes, Université de Lyon, École normale supérieure 5 \nde Lyon, 69364 Lyon, France 6 \n3B CUBE – Center for Molecular Bioengineering  and Cluster of Excellence Physics of Life , TUD Dresden 7 \nUniversity of Technology, 01307 Dresden, Germany 8 \n4Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany 9 \naCurrent address: Molecular Biophysics and Biochemistry Department, Yale University, 06511 New Haven, 10 \nUSA 11 \n*Corresponding authors ( mariana.romeiro-motta@uni-saarland.de, olivier.hamant@ens-lyon.fr, 12 \nlaura.aradillazapata@uni-saarland.de) 13 \n 14 \nAbstract 15 \nMicrotubules are dynamic cytoskeletal filaments that help shape cells and guide their 16 \nresponse to external cues, including mechanical stress. How cells reorganize and 17 \nreinforce microtubule arrays under stress  remains poorly understood . Here, we show 18 \nthat MAP65 -1/PRC1 promotes microtubule nucleation on pre-existing lattices in vitro , 19 \nparticularly on microtubules with lattice defects and damage sites  associated with  20 \nmechanical stress. MAP65-1 preferentially binds bent microtubules both in vitro and in 21 \ncells, likely because bending induces lattice damage, enhancing nucleation on and 22 \nbundling of these microtubules in vitro. This creates a  potential feedback loop  where 23 \nmechanical stress promotes MAP65-1 binding, which in turn stabilizes microtubules by 24 \nnucleation and bundling  and reinforces the alignment of the array with mechanical 25 \nstress. Thus, we reveal a previously unknown role for MAP65/PRC1 proteins in lattice-26 \nbased nucleation  and suggest a mechanism by which cells record and respond to 27 \nmechanical stress through microtubule reorganization. 28 \n 29 \nIntroduction 30 \nMicrotubules form highly organized arrays that  play critical roles in intracellular 31 \ntransport, cell division, and morphogenesis. Their array architecture is dynamically 32 \nsculpted through growth and disassembly 1,2, severing 3, cross -linking4, and local 33 \nnucleation5. These mechanisms allow cells to build microtubule arrays adapted to their 34 \nshape and function, and in response to external cues. 35 \nMicrotubule arrays can reorganize in response to mechanical stress 6, enabling cells to 36 \nadapt to external forces. In animal cells, mechanical cues influence microtubule 37 \norientation during processes such as epithelial remodeling, cell migration,  cell division, 38 \nor tissue folding7–10. In plants, microtubules align with the main tensile stress direction at 39 \nthe cell cortex 6,11,12, guiding the deposition of cellulose fibers and influencing tissue 40 \nmorphogenesis13. Such observations suggest that microtubules act as mechanosensors, 41 \nrelaying external cues into cytoskeletal organization. 42 \nWhile centrosomes and the g-tubulin ring complex were long considered the primary 43 \nsites of microtubule nucleation, many di^erentiated cells – including plant cells 14, 44 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nneurons15, and epithelial cells 16 – organize microtubules without centrosomes. 45 \nAlternative nucleation pathways are therefore critical for building functional microtubule 46 \narrays in these cells. Several microtubule-associated proteins (MAPs) have been shown 47 \nto reduce the critical tubulin concentration for nucleation in solution in vitro, suggesting 48 \na potential role in the generation of new microtubules 17. However, whether such 49 \nnucleation occurs under physiological conditions, and how it is spatially regulated within 50 \ncells, remains unclear. Existing microtubule lattices may serve as platforms for 51 \nmicrotubule nucleation, as observed in structures such as the spindle midzone 18 or 52 \ncortical microtubule arrays in plants19. 53 \nCritical to the formation of both the spindle midzone and plant cortical microtubule array 54 \nis the MAP65/PRC1 protein family4,20,21, which is best known for cross-linking antiparallel 55 \nmicrotubules. Beyond cross -linking, the MAP65 family has been shown to increase 56 \nmicrotubule flexibility 22. However, the role of MAP65 proteins in mechanosensitive 57 \nresponses has not been explored. 58 \nHere, we show that MAP65 -1/PRC1 promote microtubule nucleation on existing 59 \nmicrotubule lattices. This activity is enhanced at sites of structural irregularity in the 60 \nlattice, such as defects or damage  sites. These findings suggest a previously 61 \nunrecognized role of MAP65 -1/PRC1 in reinforcing microtubule arrays through lattice -62 \nbased nucleation. We propose a model in which microtubule alignment with mechanical 63 \nstress in plant cells generates lattice defects and damage that promote the recruitment 64 \nof MAP65-1. Once recruited, MAP65-1 nucleates, bundles, and  softens microtubules, 65 \nthereby supporting the alignment of the microtubule array with the principal direction of 66 \ntensile stress. 67 \n 68 \nResults 69 \nThe MAP65 family promotes microtubule nucleation on existing microtubule lattices 70 \nIn vitro microtubule reconstitution assays o^er a controlled and simplified environment 71 \nto study microtubule dynamics and protein interactions. For that reason, we examined 72 \nthe e^ect of MAP65 -1, a well -known microtubule bundler  (Fig. S1a-c), on microtubule 73 \ndynamics in vitro . In control conditions without MAP65 -1, polymerized microtubules 74 \nincubated with free tubulin at concentrations above the polymerization threshold – but 75 \nwell below the threshold for nucleation in solution – exhibit growth only from their free 76 \nends (Fig. 1a -c and Fig. S2a). Because we used a low tubulin concentration  of 4 µM, 77 \nmicrotubule tips grow slowly (Fig. 1c, control). However, in presence of 100 nM MAP65-1, 78 \nnew microtubules appeared along the existing microtubule lattices (Fig. 1c-e). These new 79 \nmicrotubules exhibited dynamic instability, growing completely along the “template” 80 \nmicrotubules (likely bundled by MAP65 -1), and their fluorescence intensity was 81 \ncomparable to the growing microtubule tips (Fig. 1d and e). Increasing the MAP65 -1 82 \nconcentration from 100 to 200 nM led to a significant decrease in the distance between 83 \nmicrotubule appearance events, which represents the inverse of spatial frequency (Fig. 84 \nS2b and c). Hence, we concluded that MAP65-1 can induce microtubule appearance in a 85 \nconcentration-dependent manner. 86 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nGiven that the function of MAP65-1 is conserved across the eukaryotic domain, we tested 87 \nif the human homolog of MAP65-1, PRC1, also promotes microtubule appearance. In the 88 \npresence of 300 pM PRC1, microtubules appeared and grew on the existing lattices 89 \nsimilarly to MAP65 -1 (Fig. 1c). Importantly, we worked with MAP concentrations well 90 \nbelow those that induce microtubule nucleation in solution. For MAP65 -1, we only 91 \nobserved microtubule nucleation in solution at concentrations as high as 500 nM MAP65-92 \n1 (Fig. S3); for PRC1, nucleation in solution was detected only above 3 nM PRC1 (Fig. S3). 93 \nAlthough MAP65 -1 shares only 24% sequence identity with PRC1, it has significantly 94 \nhigher identity with the other Arabidopsis MAP65 family members (Table S1). This 95 \nsuggests that these other MAP65 proteins likely share MAP65-1’s microtubule-generating 96 \nfunction. 97 \nPrevious studies have shown that the MAP SSNA1 promotes microtubule lattice 98 \nextensions in the form of branches that directly emerge from one or more protofilaments 99 \nof the mother microtubule23. To determine whether the microtubules that appeared in the 100 \npresence of MAP65 -1 and PRC1 were branches of the underlying lattice or appeared 101 \nfollowing genuine nucleation events, we introduced human kinesin -5 (KIF11) into our 102 \nassays after a 30-minute incubation of MAP65-1 with microtubules and free tubulin (Fig. 103 \n1f-i). Since KIF11 cross-links and slides antiparallel microtubules, the observed mobility 104 \nof most new microtubules upon KIF11 addition (75%, Fig. 1i) indicates that they were 105 \nantiparallel and structurally independent of the “template” microtubule. Thus, we 106 \nconfirmed that these microtubules derived from bona fide nucleation events. Since both 107 \nMAP65-1 and PRC1 preferentially bind to antiparallel microtubules 24, it is not surprising 108 \nthat most nucleated microtubules are oriented in an antiparallel manner (Fig. 1d). 109 \nWith a higher PRC1 concentration (1 nM), we observed ubiquitous PRC1 -mediated 110 \nmicrotubule nucleation and growth that quickly produced thick microtubule bundles 111 \n(Fig. 1j and k and S4a and b). In the control (no PRC1), after the same timeframe, only 112 \nshort polymerization stretches were observed from the microtubule ends (Fig. 1k). 113 \nHence, MAP65-1/PRC1 promote microtubule nucleation on the microtubule lattice. 114 \n 115 \nMAP65-1 reversibly binds free tubulin when associated with the microtubule lattice 116 \nTo investigate the mechanism by which MAP65 -1 and PRC1 promote microtubule 117 \nnucleation, we examined whether MAP65 -1 can recruit free tubulin to the microtubule 118 \nlattice. Since microtubule nucleation and growth require GTP , we performed assays in the 119 \nabsence of GTP to prevent polymerization and analyze free tubulin binding (Fig. 2a and 120 \nS5a and b). Under these conditions, MAP65-1 localized along the microtubule lattice and 121 \nrecruited free tubulin, as hypothesized (Fig. 2b-f). 122 \nQuantitative analysis revealed that, at 200 nM MAP65 -1, the average enrichment of free 123 \ntubulin compared to the solution was approximately 43% (Fig. 2f). However, in our 124 \nsystem, a ~3.5 -fold enrichment in tubulin concentration is typically required to initiate 125 \nnucleation in solution in the absence of MAPs. Although we occasionally observed 126 \nlocalized patches where tubulin enrichment approached this threshold, such events 127 \nwere considerably less frequent than the nucleation events observed, as reflected by the 128 \nmeasured distance between nucleation events under the same MAP65-1 concentration 129 \n(Fig. S2b and c). One possibility is that small nucleation intermediates fall well below our 130 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nresolution limit, leading us to underestimate local tubulin concentration. Yet, we cannot 131 \nexclude that mechanisms other than tubulin concentration may play a role in the 132 \nobserved microtubule nucleation. 133 \nTo determine whether MAP65 -1 and free tubulin bound to the microtubule lattice are 134 \ndynamically exchanged with molecules in solution, we performed fluorescence recovery 135 \nafter photobleaching (FRAP) experiments in the absence of GTP . Following 136 \nphotobleaching, both GFP -MAP65-1 and free tubulin signals partially recovered on the 137 \nmicrotubule lattice ( within 300 s; Fig. 2g and h), indicating that both proteins undergo 138 \ndynamic exchange with the soluble pool. 139 \n 140 \nMAP65-1 recognizes bent microtubules in cells and in vitro 141 \nOur in vitro data demonstrate that MAP65-1 promotes microtubule nucleation on existing 142 \nlattices, at least partly by recruiting free tubulin that undergoes exchange with the soluble 143 \nprotein pool. Yet, the relevance of this activity and its spatial regulation in cells remained 144 \nunclear.  145 \nBecause microtubules are sti^ polymers, they would tend to align with the lowest 146 \ncurvature cell axis by default. This is observed in non -pressurized wall-less plant cells25 147 \nor in non -growing hypocotyl cells 26. However, in turgid and growing plant cells, cortical 148 \nmicrotubules often align with the highest curvature axis instead. Because the highest 149 \ncurvature axis for a pressurized cylindrical cell also corresponds to  the maximal tensile 150 \nstress direction, it was proposed that tensile stress overrides geometrical cues 25. Since 151 \nMAP65-1 makes microtubules softer, we reasoned that microtubules would be less 152 \nresponsive to the lowest curvature cell axis and more responsive to mechanical stress in 153 \nthe presence of MAP65-1. Thus, we wondered whether MAP65-1 can specifically promote 154 \nmicrotubule nucleation on existing, bent microtubules, leading to a reinforcement of this 155 \nspecific microtubule subset. To investigate this, we observed the distribution of MAP65-156 \n1 in cells of young Arabidopsis stems containing a microtubule (GFP-MBD) and a MAP65-157 \n1 (MAP65-1-mCherry) reporter. MAP65-1 fluorescence intensity was around twice as high 158 \non bent microtubules in comparison to straight ones (Fig. 3a -c and Table S2), and we 159 \nobserved that both MAP65 -1 and MBD fluorescence intensity positively correlated with 160 \nmaximum microtubule curvature (Fig. 3d and e). Note that it remains unclear whether 161 \nMAP65-1 binds to pre-existing bundles that are more prominent in highly curved regions, 162 \nor whether it contributes to bundle formation in these regions by favoring microtubule 163 \nnucleation and bundling in highly bent microtubules. 164 \nThe analyses above in plant tissues give an indication of MAP65 -1’s ability to recognize 165 \nbent microtubules. However, due to the complexity of the tissue, including water fluxes 166 \nand di^erent turgid statuses for instance, these analyses only o^er a correlation. To get 167 \ncloser to causality, we used a much simpler system, consisting of wall -less plant cells 168 \n(protoplasts) confined in rectangular microwells (Fig. 3f). Upon osmotic pressurization, 169 \nachieved by transferring cells to a medium with lower osmolarity, microtubules have 170 \nbeen shown to reorient from their default longitudinal orientation to a transverse 171 \nalignment, which corresponds to the highest curvature and predicted highest tension 172 \naxis25. Under these conditions, MAP65 -1 preferentially accumulated on microtubules 173 \naligning transversely (Fig. 3g). This was supported by a significant, albeit weak (Spearman 174 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nr = -0.25), negative correlation between normalized mean MAP65 -1 intensity and mean 175 \nmicrotubule orientation two hours after pressurization (Fig. 3h), indicating that MAP65-1 176 \nis enriched on mechanically challenged microtubules. Microtubules may thus be able to 177 \ndisentangle geometry from mechanical stress through a feedback loop where MAP65 -1 178 \nrecruitment allows microtubule softening, which, in turn, promotes alignment with 179 \ntensile stress, bending along the highly curved axis of the cell,  and further MAP65 -1 180 \nrecruitment. 181 \nIn cells, MAP65-1 accumulation on bent microtubules may result from di^erent factors, 182 \nsuch as the curvature of the microtubule itself, mechanical stress, or the recruitment of 183 \nother MAPs. Hence, to dissect these factors, we tested in our in vitro assays if MAP65-1 184 \ncould recognize microtubule curvature alone. To do that, we bent microtubules by using 185 \nfluid flow and maintained them in a bent conformation by attaching the microtubule cap 186 \nto the coverslip. Next, we incubated microtubules with GFP -MAP65-1. We found that 187 \nMAP65-1 accumulated around 3 times more on bent microtubules in comparison to 188 \nstraight ones (Fig. 4a and b). Accordingly, mean MAP65-1 intensity significantly correlated 189 \nwith maximum microtubule curvature (Fig. 4c). 190 \nNext, we tested if MAP65-1-mediated microtubule nucleation happened more frequently 191 \non bent microtubules as well. We incubated microtubules with free tubulin and 100 nM 192 \nMAP65-1 for 30 min followed by washing free tubulin away and microtubule stabilization 193 \nwith taxol (Fig. 4d-g). The distance between nucleation events was approximately 5 times 194 \nsmaller in bent microtubules in comparison to straight microtubules (Fig. 4f). Average 195 \nspatial nucleation frequency also increased with maximum microtubule curvature (Fig. 196 \n4g). Notably, microtubules nucleated also in regions other than those of highest 197 \ncurvature along bent microtubules (Fig. S6), which suggests that additional factors likely 198 \ninfluence the site of nucleation. 199 \n 200 \nMAP65-1 does not recognize microtubule curvature through lattice expansion or 201 \ncompaction 202 \nMAPs that recognize microtubule curvature often exhibit preferential binding to either 203 \nexpanded or compacted lattice conformations because a curved microtubule has an 204 \nexpanded outer surface and a compacted inner surface27,28. 205 \nInterestingly, GMPCPP -polymerized microtubules, which adopt an expanded lattice 206 \nconformation29, were the predominant sites of nucleation in the presence of MAP65 -1, 207 \ndespite comprising only a small fraction of the microtubule length (Fig. S7a and b). To test 208 \nwhether MAP65 -1 preferentially binds to GMPCPP -containing microtubules, we 209 \nquantified GFP -MAP65-1 intensity on GMPCPP seeds versus GDP lattice regions. We 210 \nobserved a significant enrichment of MAP65 -1 on the seeds (Fig. S7c -e), indicating a 211 \npreference for the GMPCPP lattice. 212 \nTo determine whether MAP65 -1 specifically recognizes the expanded lattice state, we 213 \ntreated microtubules with taxol, which also induces lattice expansion29. However, taxol-214 \ntreated microtubules showed significantly reduced MAP65-1 accumulation (Fig. S7f and 215 \ng), suggesting that lattice expansion alone does not account for MAP65-1 binding. 216 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nSince some MAPs that recognize specific lattice states can also induce lattice expansion 217 \nor compaction upon binding30, we examined microtubules incubated with GFP-MAP65-1 218 \nusing microfluidics (Fig. S7h and i). Even at a high concentration (500 nM), MAP65 -1 did 219 \nnot induce measurable changes in lattice length. Thus, we concluded that MAP65-1 does 220 \nnot recognize microtubule curvature through lattice expansion or compaction. 221 \n 222 \nMAP65-1 and PRC1 recognize microtubules with structural irregularities 223 \nIn our experiments, we define  lattice defects as irregularities that arise  during 224 \npolymerization (for example, a change in protofilament number), whereas damage refers 225 \nto alterations that occur after polymerization, such as local tubulin loss or breaks in the 226 \nlattice. Bent microtubules are  particularly prone to lattice damage, as reflected by 227 \nincreased tubulin turnover along their shafts31. 228 \nGiven MAP65 -1’s ability to recognize bent microtubules , we  thus hypothesized that 229 \nMAP65-1/PRC1 might detect structural irregularities in the microtubule lattice, such as 230 \ndefects and damage . To test this, we manipulated the  frequency of microtubule 231 \nirregularities using two approaches: (1) by reducing the tubulin concentration for 232 \npolymerization to half of that used in fast -growth conditions, generating microtubules 233 \nwith fewer defects 32 (referred to as slow growth conditions); and (2), by treating 234 \nmicrotubules with taxol to induce lattice damage 33, followed by thorough washing to 235 \nremove the drug (Fig. 5a-g). 236 \nIn taxol pre -treated microtubules, we occasionally observed regions with noticeably 237 \nreduced lattice fluorescence (Fig. 5a-d, white arrowhead), which likely represent damage 238 \nsites. This observation is consistent with previous reports showing that treatment with 239 \ntaxol overnight at room temperature causes considerable damage to the microtubule 240 \nlattice33. Notably, MAP65 -1-mediated microtubule nucleation events often originated 241 \nfrom these damage sites. The median distance between nucleation events was 242 \nsignificantly reduced in taxol -pre-treated microtubules (10.06 µm) compared to fast -243 \ngrowth conditions (16.88 µm; P = 0.015, Fig. 5c). In contrast, under slow -growth 244 \nconditions, the median distance between nucleation events increased to 63.00 µm (P = 245 \n0.0016). This corresponds to a spatial frequency nearly four times lower than that 246 \nobserved in fast-growth conditions. 247 \nWe validated these findings in an alternative approach, in which GMPCPP -stabilized 248 \nmicrotubules were polymerized under high - and low -defect conditions 34, followed by 249 \nincubation with MAP65 -1 and free tubulin (see Methods, Fig. S8a). Under low -defect 250 \nconditions, the median distance between nucleation events significantly increased from 251 \n3.44 µm to 6.74 µm, nearly a twofold di^erence, compared to the high-defect regime (Fig. 252 \nS8b and c). These results indicate that the spatial frequency of MAP65 -1-mediated 253 \nmicrotubule nucleation is promoted by the presence of structural irregularities in the 254 \nlattice. 255 \nTo determine whether this function is conserved, we tested whether PRC1 also promotes 256 \nnucleation in a defect- and damage-dependent manner. We found that PRC1-mediated 257 \nnucleation occurred approximately three times more often on taxol -pre-treated 258 \nmicrotubules and about two times less often on microtubules grown under slow-growth 259 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nconditions (Fig. 5e-g). We concluded that both MAP65-1 and PRC1 preferentially promote 260 \nmicrotubule nucleation on lattices with increased structural irregularity. 261 \n 262 \nMAP65-1 mediated microtubule nucleation often co-localizes with annealing sites 263 \nA remaining question was whether MAP65 -1/PRC1 promote microtubule nucleation 264 \ndirectly at defect or damage sites. To test this, we generated microtubules with defined 265 \nannealing sites, which often form structural defects due to the imperfect alignment of 266 \nfrayed microtubule ends. We polymerized two populations of GMPCPP -stabilized 267 \nmicrotubules, mixed them in equal amounts and allowed them to anneal overnight (Fig. 268 \n5h). This approach generated a mixed population of annealed and non -annealed 269 \nmicrotubules. We then incubated the mixture with MAP65-1 and free tubulin, followed by 270 \nwashing and stabilization with taxol. 271 \nStrikingly, approximately 45% of the nucleation events co-localized with annealing sites 272 \n(25 out of 55 events; Fig. 5i and j). This was significantly higher than expected from 273 \nrandomly distributed nucleation sites: among 55 randomly selected microtubule 274 \npatches, only 6 contained nucleation events (Fisher’s exact test, P < 0.0001). 275 \nFurthermore, the median distance between nucleation events significantly decreased in 276 \nannealed microtubules (5.11 µm) compared to non -annealed ones (14.86 µm; P = 277 \n0.0099, Fig. 5k), almost a three -fold di^erence. Thus, we concluded that nucleation 278 \noccurs at annealing sites more frequently than expected by chance, supporting the 279 \nhypothesis that MAP65 -1/PRC1 can recognize structural defects, where they promote 280 \nmicrotubule nucleation. 281 \n 282 \nBundled microtubules are more resistant to breakage after free tubulin removal 283 \nConsidering that MAP65 -1 preferentially promotes nucleation – and thus bundling – on 284 \nmicrotubules with structural defects and damage, we wondered whether microtubules 285 \nbundled by MAP65-1 are more resistant to breakage. To test this, we removed free tubulin 286 \nfrom our in vitro assays, a condition that causes microtubules to gradually lose tubulin 287 \ndimers from their lattices, soften, and eventually break. After polymerizing and capping 288 \nmicrotubules, we simultaneously removed free tubulin and added 100 nM MAP65-1 and 289 \nimaged microtubules over time (Fig. 6a and b). Remarkably, bundled microtubules 290 \nsurvived almost 3 times longer compared to single ones (Fig. 6b), showing that MAP65-1 291 \nprotects microtubule bundles from breakage. 292 \nMechanical stress in the form of tension has been shown to accelerate microtubule 293 \npolymerization in vitro 35. Based on our findings that MAP65 -1 promotes nucleation on 294 \nmicrotubules with  more lattice defects  and damage, we propose a model in which 295 \nmechanical stress enhances microtubule polymerization speed  in plant cells. Under 296 \nhigher polymerization speeds, microtubules accumulate structural irregularities, for 297 \ninstance in the form of lattice defects, which are recognized by MAP65 -1. Microtubules 298 \naligning with mechanical stress in plant cells also likely accumulate lattice damage 299 \ncaused by bending. MAP65-1 thus preferentially stabilizes, softens, nucleates on, and 300 \nbundles these microtubules. As a consequence, MAP65-1 activity promotes microtubule 301 \narray alignment along mechanical stress patterns (Fig. 6c). 302 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nDiscussion 303 \nIn summary, we showed that MAP65 -1 preferentially binds to and promotes nucleation 304 \non bent microtubules, supporting a model in which MAP65-1 acts as a mechanosensitive 305 \nregulator of cytoskeletal organization. This is particularly relevant in plant cells, where 306 \ncortical microtubules typically align with tensile stress patterns rather than local 307 \ngeometric cues. For instance, in many plant tissues, adjacent cells with di^erent 308 \ngeometries exhibit consistent supracellular microtubule alignment 11,36. Our finding 309 \nprovides a scenario for microtubules to be di^erentially sensitive to geometrical and 310 \nmechanical cues. In turgid cells, tensile stress  would promote  fast microtubule 311 \npolymerization and the formation of defects in the lattice; the subsequent recruitment of 312 \nMAP65-1 would  secure the viability of defective microtubules through microtubule 313 \naddition (nucleation and bundling) . As a consequence  of MAP65 recruitment , MAP65 -314 \ndecorated microtubules would also become softer. They would then be less inclined to 315 \nalign with the flattest part of the cell cortex (compared to when they were sti^) and 316 \ninstead align more in the direction of tensile stress at the cell cortex,  i.e., under high 317 \ncurvature, in a feedback loop . Importantly, we do not expect single microtubules to 318 \nreorient and align with the direction of highest tensile stress or curvature; instead, a few 319 \nmicrotubules stochastically growing in t hat direction are expected to be su^icient to 320 \nsupport the proposed feedback loop. 321 \nThe co-localization of nucleation events with microtubule annealing sites supports the 322 \nidea that MAP65 -1 recognizes structurally vulnerable regions and reinforces them 323 \nthrough nucleation and bundling. Importantly, we demonstrate that microtubules 324 \nbundled by MAP65 -1 are more resistant to breakage following tubulin depletion, 325 \nsuggesting a stabilizing role for MAP65-1-mediated bundling under stress conditions (Fig. 326 \n6c). Interestingly, an alternative scenario where tensile stress would promote the 327 \nexpanded lattice conformation of microtubules and further recruitment of MAP65 was 328 \nnot supported by our experiments. MAP65-1-dependent stress perception would thus be 329 \nmediated through the accumulation of defects and lattice damage. In this scenario, 330 \nMAP65-1 recruitment to damaged microtubules would serve a dual purpose: it would 331 \nacutely reinforce microtubules in response to mechanical stress, and, because the 332 \nresulting bundles are particularly stable, it would also leave a lasting record of past stress 333 \nevents within the cell. This would, in turn, make the microtubule array align better with  334 \nstress.  335 \nThe exact molecular mechanism by which MAP65 -1/PRC1 promote microtubule 336 \nnucleation remains to be elucidated. Since the tubulin concentration on the lattice was 337 \nnot high enough to explain the spatial frequency of nucleation activity, it is likely that 338 \nMAP65-1/PRC1 stabilize nucleation intermediates by enhancing lateral or longitudinal 339 \na^inity between tubulin dimers 17. Accordingly, the yeast homolog Ase1 has been 340 \nproposed to reduce the detachment of terminal tubulin subunits at depolymerizing 341 \nmicrotubule tips 37, which shows that MAP65 -1/PRC1 could influence the tubulin 342 \ndissociation rate from the microtubule lattice. 343 \nNote that further synergies may be envisioned here. In particular, higher osmolarity does 344 \nnot only reduce cortical tension, but also leads to reduced microtubule growth 38. Thus, 345 \nin non -pressurized cells, slow -growing microtubules might experience less defects in 346 \ntheir lattice, leading to reduced recruitment of MAP65 -1. The resulting sti^er 347 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nmicrotubules would be more sensitive to cell geometry and align with the flat part of the 348 \ncell cortex, by default. 349 \nThe mechanism by which MAP65 -1 recognizes bent microtubules seems to di^er from 350 \nother MAPs that have been shown to bind bent microtubules through a preference for the 351 \nexpanded or compacted lattice states 27,28,39. Although we did not observe preferential 352 \nbinding to the taxol -expanded lattice, MAP65-1 exhibited a preference for the GMPCPP 353 \nlattice. This suggests that MAP65-1 may recognize bent microtubules through alternative 354 \nfeatures of the microtubule lattice that are not explained solely by expansion or 355 \ncompaction.28,37,38 Furthermore, one interesting possibility is that MAP65 -1 detects 356 \nmicrotubules under tension, a hypothesis that remains to be tested. 357 \nBecause MAP65-1/PRC1 have not been described to preferentially bind GTP-tubulin, the 358 \nrecognition of lattice defects also seems di^erent from other MAPs that recognize defect 359 \nsites through the recognition of GTP-tubulin incorporation, like CLASP , CLIP-170, and the 360 \nEBs40–43. Moreover, MAP65-1 may recognize bent microtubules through their increased 361 \nlattice damage, as bent microtubules show much higher levels of repair (with an 362 \nincreased incorporation of tubulin from solution) along their lattice than straight ones31. 363 \nFinally, the interplay of MAP65 -1/PRC1 with other MAPs that are responsive to 364 \nmechanical stress is likely complex in cells. For example, PRC1 and Ase1 interact with 365 \nand recruit CLASP44,45, and microtubule bundles formed by MAP65-1 are protected from 366 \nsevering by KATANIN 46. Recent simulations of plant cortical microtubule arrays also 367 \nsupport a key role for bundling in helping microtubule self -organization together with 368 \nKATANIN’s severing function 47. Therefore, MAP65 -1 might further contribute to the 369 \nmicrotubule response to mechanical stress in cells by recruiting CLASP and counter -370 \nacting the severing by KATANIN. Overall, it will be interesting to test how depletion of 371 \nMAP65 in plant cells can a^ect their response to changes in mechanical stress, although 372 \nthis is a challenging endeavor because the MAP65 family has nine members in A. 373 \nthaliana48. Future work could address whether other MAP65 members also localize to 374 \nbent microtubules or promote nucleation, clarifying whether they have a conserved or 375 \nspecialized role. 376 \nTogether, our findings position MAP65 -1 and PRC1 as key players in the dynamic 377 \nregulation of the microtubule cytoskeleton, contributing to a form of cytoskeletal 378 \n“memory” , where mechanical stress-induced microtubule patterns are reinforced and 379 \nlikely maintained over time. Since both MAP65-1 and PRC1 have conserved functions in 380 \ncell division, it also remains open whether the nucleation mechanism described here 381 \ncontributes to spindle or phragmoplast formation and has further implications in cell 382 \nproliferation that were previously unknown. Thus, both proteins have the potential to 383 \nactively contribute to tissue morphogenesis across the eukaryotic kingdom – particularly 384 \nin plants, where growth anisotropy mainly relies on the cortical microtubule -cellulose 385 \ndeposition nexus.  386 \n  387 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nMethods 388 \nMAP65-1, PRC1 and KIF11 purification 389 \nPlasmids encoding His -MAP65-1-His (referred to as MAP65 -1) and GFP -MAP65-1-His 390 \n(referred to as GFP -MAP65-1) with MAP65-1 sequences from Arabidopsis thaliana were 391 \npreviously generated24. The recombinant proteins were purified from Rosetta 2(DE3) E. 392 \ncoli cells. Bacteria were grown at 37 °C to an OD of 0.5 followed by transfer to 20 °C for 393 \none hour. Protein expression continued at 20 °C overnight with 0.5 mM IPTG. The next day, 394 \ncells were collected by centrifugation and frozen. Cell pellets were resuspended in lysis 395 \nbu^er containing 50 mM Sodium Phosphate Bu^er, pH 7.9, 200 mM NaCl, 20 mM 396 \nimidazole, 0.5% Triton X -100, 0.5 mM DTT and a protease inhibitor cocktail (cOmplete, 397 \nEDTA-free). Samples were then sonicated in a beaker on ice (Bandelin Sonopuls, 9X 20 398 \nseconds on/o^, 40% duty cycle). The lysate was then centrifuged for 30 min at 16,000 399 \nrpm at 4 °C and applied to a column containing Ni Sepharose High Performance beads 400 \n(Cytiva Life Sciences). The column was washed with a bu^er containing 50 mM Sodium 401 \nPhosphate Bu^er, pH 7.9, 100 mM NaCl, 30 mM imidazole and 0.5 mM DTT. The proteins 402 \nwere eluted with 500 mM imidazole and dialyzed overnight in a bu^er containing 50 mM 403 \nSodium Phosphate Bu^er, pH 7.9, 100 mM NaCl and 0.5 mM DTT. Further purification 404 \nfollowed by loading the protein on a gel filtration column (HiLoad 16/600 Superdex 200 405 \npg, Cytiva Life Sciences) connected to the NGC Chromatography system (Bio-Rad) in 50 406 \nmM Sodium Phosphate Bu^er, pH 7.9, 100 mM NaCl and 0.5 mM DTT. Proteins were 407 \nconcentrated using Amicon Ultra-15 Centrifugal Filters (Merck). 408 \nHuman PRC1 was expressed for 96 h in SF9 cells from a pOCC7 plasmid encoding PRC1 409 \nlabeled with a His6 tag and a 3C precision cleavage site. For purification, cell pellets were 410 \nthawed on ice and resuspended in purification bu^er (50 mM NaH2PO4, 500 mM NaCl, 411 \n2 mM MgCl2, 1 mM DTT, 0.1% Tween20, pH 7.8) with protease inhibitor. The lysate was 412 \ncleared with an ultracentrifuge spin with 40,000 rpm for 1 h at 4 °C. The supernatant was 413 \nfiltered through a 0.45 μm filter and loaded on a 1 mL HiTrap column with a superloop. 414 \nThe column was washed with IMAC wash bu^er (purification bu^er with 20 mM 415 \nimidazole) and the protein was eluted with IMAC elution bu^er (purification bu^er with 416 \n300 mM imidazole) with an elution gradient. Protein -containing fractions were pooled 417 \nand concentrated with Amicon filters (cuto^ 100 kDa). 3C protease was added (1:150, 418 \nv/v) and the His6 tag was cleaved overnight at 4°C. The protein solution was diluted 6-fold 419 \nto reduce the imidazole concentration and passed over the HiTrap column again. The 420 \nprotease remained bound to the column with its His6 tag. The flow through was 421 \nconcentrated to 0.5 mL, cleared at 17,000 g for 10 min and gel-filtered over a Superose6 422 \ncolumn with purification bu^er. 10% glycerol was added, and the protein was flash frozen 423 \nin liquid nitrogen and stored at -80°C. 424 \nHuman KIF11 was expressed and purified as previously described49. 425 \n 426 \nTubulin purification and labeling 427 \nFresh bovine brains were used as the source of brain tubulin, which was purified by three 428 \ncycles of temperature-dependent polymerization and depolymerization in Brinkley Bu^er 429 \n80 (BRB80 bu^er; composed of 80 mM PIPES, pH 6.8, 1 mM EGTA and 1 mM MgCl 2 430 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nsupplemented with 1 mM GTP). We obtained MAP -free tubulin by using low (32.6% 431 \nglycerol, 1.5 mM ATP , 0.5 mM GTP , 3 mM MgCl2) and high salt bu^ers (High Molarity PIPES 432 \nbu^er; 1 M PIPES, pH 6.9, adjusted with KOH, 10 mM MgCl 2, 20 mM EGTA) and cation -433 \nexchange chromatography (EMD SO, 650 M, Merck) in 50 mM PIPES, pH 6.8, 1 mM MgCl2 434 \nand 1 mM EGTA. 435 \nFor the labeling (with ATTO-488, ATTO-565, Alexa-Fluor-647 or biotin), microtubules were 436 \npolymerized with purified brain tubulin at 37 °C for 30 min and layered onto cushions of 437 \n0.1 M NaHEPES, pH 8.6, 1 mM MgCl 2, 1 mM EGTA, 60% glycerol, followed by 438 \nsedimentation by ultracentrifugation at 37 °C. Microtubules were then resuspended in 439 \n0.1 M NaHEPES, pH 8.6, 1 mM MgCl 2, 1 mM EGTA, 40% glycerol and labeled by adding 440 \n1:10 volume 100 mM NHS-ATTO (ATTO Tec) or NHS-LC-LC-Biotin (EZ-link, Thermo) for 10 441 \nmin at 37 °C. Two volumes of 2X BRB80 with 100 mM potassium glutamate and 40% 442 \nglycerol were used to stop the labeling reaction, followed by microtubule sedimentation 443 \nonto cushions of BRB80 supplemented with 60% glycerol. Finally, microtubules were 444 \nresuspended in BRB80, and a last cycle of polymerization and depolymerization was 445 \nperformed before storage. 446 \n 447 \nCoverslip treatment 448 \nCoverslips were cleaned by successive treatment with the following solutions: 30 min 449 \nacetone and 15 min 96% ethanol followed by two washes with ultrapure water, then 2 450 \nhours in Hellmanex III (2% in water) followed by two washes with ultrapure water. 451 \nCoverslips were then airdried and treated with UV for 25 min. Next, coverslips were 452 \nincubated for 3 days in a solution containing a 1:9 mix of triethoxysilane -PEG-biotin and 453 \ntriethoxysilane-PEG (30 kDa, Creative PEGWorks) at 1 mg/ml in 96% ethanol and 0.1% 454 \nHCl with gentle agitation at room temperature. Coverslips were then rinsed once in 455 \nabsolute ethanol and twice in ultrapure water, airdried and stored at 4 °C. 456 \n 457 \nMicrotubule growth, capping and nucleation dynamics 458 \nMicrotubules seeds were polymerized in a total volume of 100 μl with 6 μM tubulin 459 \n(labeled with 30% ATTO-565 or Alexa Fluor 647 and 70% biotinylated tubulin) in 1X BRB80 460 \nsupplemented with 0.5 mM GMPCPP at 37 °C for 1 hour. 2 μl of 50 μM taxol (Merck) were 461 \nthen added followed by incubation at room temperature for 30 min and 462 \nultracentrifugation at 156,000 xg at 25 °C for 10 min. Seeds were then resuspended in 1X 463 \nBRB80 supplemented with 0.5 mM GMPCPP and 1 μM taxol. 464 \nA flow cell chamber with a volume of approximately 20 μl was built using double -sided 465 \nadhesive tape and a glass coverslip functionalized and passivated as mentioned above. 466 \nThe top and bottom pieces were cut into the desired sizes using a diamond engraving 467 \npen. Flow chambers were first perfused with 50 μg/ml Neutravidin (Fisher Scientific) in 468 \n1X BRB80 for 1 min, followed by passivation with 0.1 mg/ml PLL -g-PEG (PII 20 K -G35-469 \nPEG2K, Jenkam Technology) in 10 mM Na-HEPES, pH 7.4, for 1 min and washed with 1X 470 \nBRB80. Microtubules seeds were then flushed into the chamber. Non -attached seeds 471 \nwere washed out by using 1X BRB80 supplemented with 1 mg/ml casein (BRB80/casein). 472 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nMicrotubule polymerization with seeds as template was achieved with a mix containing 473 \n11 μM tubulin (10 to 20% labeled with ATTO -565 or Alexa Fluor 647) in 0.7X BRB80 and 474 \n0.38X MAP bu^er (500 mM Phosphate bu^er, 1 mM KCl, 10 mM DTT, pH 7.9) 475 \nsupplemented with 1 mM GTP , an oxygen scavenger cocktail (22 mM DTT, 1.2 mg/ml 476 \nglucose, 8 μg/ml catalase and 40 μg/ml glucose oxidase), 1 mg/ml casein and 0.033% 477 \nmethyl cellulose (1,500 cP , Sigma) at 37 °C. Microtubules were capped by substituting 478 \nGTP with 0.5 mM GMPCPP (Jena Bioscience) and using 3 µM 100% labeled tubulin (with 479 \nATTO-565 or Alexa Fluor 647) at 37 °C. Capping microtubules extends their lifetime for 480 \nlong-term observation (for instance up to 30 min) and overcomes dynamic instability in 481 \nvitro. To observe microtubule nucleation in the presence of PRC1 or MAP65 -1, the same 482 \nbu^er as in microtubule polymerization was used, supplemented with 4 µM tubulin 100% 483 \nlabeled with ATTO-488 and the corresponding protein at the desired concentration (the 484 \nprotein stock was diluted in 1X BRB80). Microtubule nucleation dynamics in the presence 485 \nof PRC1/MAP65-1 was observed for 30 minutes, unless stated otherwise. 486 \nFor the experiments varying the amount of microtubule defects: for fast growth 487 \nconditions, 11 µM tubulin was used for polymerization; for slow growth conditions, 5.5 488 \nµM tubulin was used. 489 \nTo observe stabilized microtubules nucleated by varying MAP65 -1 concentrations, 490 \nmicrotubule incubation with 100% ATTO-488-labeled tubulin and MAP65-1 (in the same 491 \nbu^er as for microtubule polymerization) proceeded for 20 min, followed by a wash 492 \nbu^er (with the same composition as the microtubule polymerization bu^er, but without 493 \nfree tubulin and with 20 µM taxol). 494 \n 495 \nNucleated microtubule transport by KIF11 496 \nMicrotubules were polymerized in microcentrifuge tubes as described below with 10% 497 \nlabeled ATTO-565 and with biotin on their caps and seeds. After washing away taxol,  498 \nmicrotubule nucleation proceeded for 30 min in the presence of 100 nM MAP65 -1 and 4 499 \nµM 100% ATTO-488-labeled tubulin followed by flushing of a kinesin bu^er containing 10 500 \nnM KIF11, 2 µM of tubulin 100% labeled with Alexa Fluor 647 and 0.7X BRB80 501 \nsupplemented with 1 mM GTP , 1 mM ATP , an oxygen scavanger cocktail (22 mM DTT, 1.2 502 \nmg/ml glucose, 8 μg/ml catalase and 40 μg/ml glucose oxidase), 1 mg/ml casein and 503 \n0.033% methyl cellulose (1,500 cP , Sigma). Microtubules were observed for 20 min. 504 \n 505 \nMicrotubule polymerization for bending and taxol treatment 506 \nMicrotubules were first polymerized in microcentrifuge tubes by using 11 µM tubulin 507 \n(labeled with 10% ATTO -565 or Alexa Fluor 647) in 200 µl of a bu^er containing 1.2X 508 \nBRB80, 0.6X MAP bu^er, 0.5 mM GTP and previously polymerized seeds for 40 min at 37 509 \n°C. 5 µl of 30 µM taxol were then added, following by centrifugation for 30 min at 15,000 510 \nrpm at room temperature. Microtubules were then resuspended in capping mix 511 \ncontaining 0.5 µM tubulin (labeled with 60% biotin and 40% ATTO-565 or Alexa Fluor 647) 512 \nin 1.2X BRB80, 0.6X MAP bu^er, 0.5 mM GMPCPP and 10 µM taxol. Stepwise capping of 513 \nmicrotubules was achieved by adding 0.5 µM tubulin at a time followed by incubation for 514 \n15 min at 37 °C for a total of ten times. Microtubules were diluted 1:200 in BRB80/taxol 515 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n(1X BRB80, 10 µM taxol) until usage. The same conditions were used for microtubules 516 \nreferred to as taxol pre-treated, which were incubated overnight at room temperature in 517 \nBRB80/taxol. 518 \nMicrotubules were flushed into passivated and functionalized chambers as described 519 \nabove, followed by successive addition of 100 µl BRB80/taxol from both ends of the flow 520 \nchamber in an alternate fashion to cause microtubule bending due to fluid flow, since 521 \nmicrotubules could attach via the seed and the cap to the coverslips. Taxol was washed 522 \naway by using BRB80/casein. To observe GFP -MAP65-1 binding to bent microtubules, 523 \nMAP65-1 was added with 15% GFP -MAP65-1 and 85% MAP65 -1 in the presence of 2 µM 524 \nnon-fluorescent tubulin. To observe microtubule nucleation on bent microtubules, 525 \nMAP65-1-mediated nucleation proceeded for 20 min followed by stabilization with a 526 \nwash bu^er with no tubulin and 10 µM taxol. 527 \n 528 \nMicrotubule nucleation using annealed microtubule population 529 \nMicrotubules were polymerized in a bu^er containing 1X BRB80, 1.25 mM GMPCPP , 1.25 530 \nmM MgCl2 and 2.5 µM tubulin (labeled with 20% ATTO -565 or Alexa Fluor 647 and 20% 531 \nbiotin) for 5 hours at 28 °C, followed by addition of 120 µl 1X BRB80 and centrifugation at 532 \n13,000 rpm for 15 min. Microtubules were then resuspended in 150 µl of 1X BRB80 and 533 \nmixed in equal amounts, followed by incubation at 30 °C overnight to allow for annealing 534 \nto happen. Microtubules were then flushed into flow chambers and MAP65 -1-mediated 535 \nnucleation proceeded by 20 min followed by stabilization with a wash bu^er with no 536 \ntubulin and 10 µM taxol. 537 \n 538 \nMicrotubule nucleation using a population of microtubules with high and low defect 539 \nregime 540 \nFor the high defect regime, microtubules were polymerized in 10 µl of a bu^er containing 541 \n1X BRB80, 2 mM GMPCPP , 0.1 mM MgCl and 20 µM tubulin (labeled with 20% Alexa Fluor 542 \n647 and 20% biotin) for 30 min at 37 °C, followed by addition of 190 µl of 1X BRB80 and 543 \ncentrifugation at 156,000 xg for 15 min. Microtubules were then resuspended in 150 µl of 544 \n1X BRB80. For the low defect regime, microtubules were polymerized in 80 µl of a bu^er 545 \ncontaining 1X BRB80, 1.25 mM GMPCPP , 1.25 mM MgCl2 and 2.5 µM tubulin (labeled with 546 \n20% ATTO-565 and 20% biotin) for 5 hours at 28 °C, followed by addition of 120 µl of 1X 547 \nBRB80 and centrifugation at 13,000 rpm for 15 min. Microtubules were then resuspended 548 \nin 150 µl of 1X BRB80. Microtubules were then flushed in sequentially and MAP65 -1-549 \nmediated nucleation proceeded by 20 min followed by stabilization with a wash bu^er 550 \nwith no tubulin and 10 µM taxol. 551 \n 552 \nPlant growth conditions 553 \nArabidopsis thaliana seeds were surface -sterilized by treatment with a solution 554 \ncontaining 2% bleach and 0.05% Triton X -100 for 5 min followed by three washes with 555 \nsterile distilled water and resuspension in 0.05% agarose. Seeds were sown on ½ MS 556 \nmedium (basal salt mixture, Duchefa Biochemie) supplemented with 0.5% sucrose and 557 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n0.8% plant agar (Duchefa Biochemie). Plates containing seeds were stratified at 4 °C for 558 \n2 to 3 days in the dark. Plants were grown in a 16-hour/21 °C light and 8-hour/18 °C dark 559 \nregime with 60% humidity. 560 \n 561 \nProtoplast isolation and observation 562 \nRoots from seedlings grown for eleven days were dissected and inserted in an enzyme 563 \nsolution composed of solution A (2 mM CaCl2, 2 mM MgCl2,10 mM MES, pH 5.5 adjusted 564 \nwith KOH, and 600 mOsmol/l Mannitol) and the following enzymes: 17 mg/ml Cellulysine 565 \n(Calbiochem), 17 mg/ml Cellulase RS (Duchefa Biochemie) and 0.4 mg/ml Pectyolase 566 \nfrom Aspergillus japonicus (Sigma-Aldrich). Roots were digested for two to four hours at 567 \nroom temperature on a rotating stage at 15 rpm. Next, the solution was filtered through a 568 \n70 µm filter and the filter was washed with 1 ml of solution A. The filtered protoplasts 569 \nwere then centrifuged for 4 min at 1,000 rpm. The supernatant was removed and 1 ml of 570 \nsolution A was added, followed by gentle flicking. The protoplasts were centrifuged again 571 \nfor 4 min at 1,000 rpm, followed by supernatant removal and resuspension in 200 µl of 572 \nsolution A. The protoplasts were then applied on microwells and allowed to sediment for 573 \n10 min. 3 ml of solution B (the same as solution A, but with 280 mOsmol/l Mannitol) were 574 \nthen added to promote protoplast pressurization. Microscopic observation started 2 575 \nhours after treatment with solution B and continued for another 2 hours. The 12 X 40 µm 576 \nmicrowells were produced as previously described 25. We selected protoplasts that had 577 \nan aspect ratio of at least 1.1 (major axis divided by minor axis) to make sure that only 578 \nenclosed protoplasts were taken into account for our analysis. We included protoplasts 579 \nwith an aspect ratio range of 1.28 to 1.12. 580 \n 581 \nImaging conditions and image analysis 582 \nTo observe plant cells, a point scanning confocal microscope (Zeiss LSM 900) with 583 \nAiryscan 2 and Axiocam 705 camera was used. In vitro microtubules were either 584 \nvisualized with the Zeiss LSM 900 with a stage that was kept at 37 °C through a cage 585 \nincubator (PECON), or an objective -based orbital TIRF microscope (Nikon Eclipse Ti2, 586 \nmodified by Visitron Systems) and EMCCD camera (Andor iXon Life) at minimal laser 587 \nintensity with a stage kept at 37 °C through a warm stage controller (OkoLabs). For the 588 \nZeiss LSM 900 microscope, the ZenBlue software version 3.2 was used. For the TIRF 589 \nmicroscope, the VisiView software version 6.0 was used. 590 \nTo observe microtubule nucleation dynamics in the presence of PRC1/MAP65-1, images 591 \nwere taken every 4 s for a total of 30 minutes. Nucleation dynamics were observed 592 \nthrough kymographs. The distance between nucleation events was measured manually 593 \nusing the segmented line on Fiji. 594 \nTo distinguish between incorporation and nucleation events in the case of stabilized 595 \nmicrotubules, typically 50 images were taken per field of view (FOV) with an interval of 596 \n0.5 to 1 s. Images were processed for background subtraction and smoothing. Line scans 597 \nof the green fluorescence intensity were drawn along maximum intensity projections of 598 \nindividual microtubules. The elongation of microtubule ends was used as a reference for 599 \na full microtubule for every FOV – an event that surpassed the intensity found at the 600 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\npolymerized microtubule ends was scored as a nucleation event if it co -localized with 601 \nthe microtubule lattice. 602 \nTo quantify the recruitment of free tubulin on the microtubule lattice by MAP65-1, a 1-µm 603 \nsection in length spanning the whole microtubule width was drawn using the polygon tool 604 \nin Fiji. This section was moved along the microtubule from the beginning to the end. For 605 \neach individual microtubule, the integrated density measured from each microtubule 606 \nsection was divided by the integrated density in a background region with the same area 607 \nright next to the microtubule. 608 \nTo assess whether microtubule nucleation sites overlapped with microtubule annealing 609 \nsites, we measured the distance between the peak maximum of the nucleation 610 \nfluorescence signal and the nearest annealing site. If this distance was less than 500 nm, 611 \nthe nucleation site was classified as overlapping with the annealing site. As a control, we 612 \nselected 55 random 500 nm -long patches along annealed microtubules. A patch was 613 \nscored as containing a nucleation event if the peak maximum of a nucleation 614 \nfluorescence signal fell within the 500 nm interval. 615 \n 616 \nFRAP assay 617 \nThe GFP-MAP65-1 and free tubulin signals were bleached by using 5 cycles of the 405 618 \nlaser line at 100% at a speed of 50 ms per pixel. Images were acquired every 0.9 s for 5 619 \nmin. Bleaching events that resulted in less than 30% of the initial fluorescence intensity 620 \nwere used for the analysis. The fluorescence intensity was normalized to the initial 621 \nmaximum value and plotted over time by using the Stowers Plugins Collection 622 \n(https://research.stowers.org/imagejplugins) after background subtraction (rolling ball 623 \n50 pixels). 624 \n 625 \nMicrotubule curvature and orientation analysis 626 \nMicrotubules were tracked with the JFilament Fiji plugin 627 \n(https://imagej.net/plugins/jfilament). Using custom-written python code, the curves of 628 \nthe tracked filaments were first smoothed by parametric Spline interpolation. Then, the 629 \nmenger curvature was computed and the orientation was calculated via finite di^erence. 630 \nGFP-MBD and MAP65 -1-RFP fluorescence values were normalized for every protoplast 631 \nagainst the mean fluorescence intensity (for each corresponding channel) in a circle with 632 \na diameter of 2 µm drawn in a region of the cytoplasm with no microtubules present. 633 \n 634 \nStatistical methods 635 \nAll statistical analyses were performed with GraphPad Prism. For the spatial frequency 636 \nquantifications, microtubules for each experimental condition were concatenated in a 637 \nrandom order and the distance between two adjacent nucleation events was measured 638 \n(similarly to what was done previously for the quantification of spatial incorporation 639 \nfrequency32).  640 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nData sets were tested for their normality. All of the tested datasets had at least one group 641 \nthat was non -normally distributed, thus non -parametric tests were employed to test if 642 \ndi^erences were statistically significant (Mann-Whitney and Kruskal-Wallis). 643 \nTo test if the microtubule nucleation frequency di^ered in regions containing microtubule 644 \nannealing sites compared to randomly selected patches of the annealed microtubule 645 \npopulation, a Fisher’s exact test was used. 646 \n 647 \nData availability 648 \nThe datasets generated and analyzed in this study are available from the corresponding 649 \nauthors upon request. 650 \n 651 \nReferences 652 \n1. Mitchison, T. & Kirschner, M. Dynamic instability of microtubule growth. Nature 653 \n312, 237–242 (1984). 654 \n2. Burbank, K. S. & Mitchison, T. J. Microtubule dynamic instability. Current Biology 655 \n16, R516–R517 (2006). 656 \n3. Kuo, Y.-W. & Howard, J. Cutting, Amplifying, and Aligning Microtubules with 657 \nSevering Enzymes. Trends Cell Biol 31, 50–61 (2021). 658 \n4. Walczak, C. E. & Shaw, S. L. A MAP for Bundling Microtubules. Cell 142, 364–367 659 \n(2010). 660 \n5. Tovey, C. A. & Conduit, P . T. Microtubule nucleation by γ-tubulin complexes and 661 \nbeyond. Essays Biochem 62, 765–780 (2018). 662 \n6. Hamant, O., Inoue, D., Bouchez, D., Dumais, J. & Mjolsness, E. Are microtubules 663 \ntension sensors? Nat Commun 10, 2360 (2019). 664 \n7. Matis, M. The Mechanical Role of Microtubules in Tissue Remodeling. BioEssays 665 \n42, 1900244 (2020). 666 \n8. Ju, R. J. et al. Compression-dependent microtubule reinforcement enables cells 667 \nto navigate confined environments. Nat Cell Biol 26, 1520–1534 (2024). 668 \n9. Lechler, T. & Mapelli, M. Spindle positioning and its impact on vertebrate tissue 669 \narchitecture and cell fate. Nat Rev Mol Cell Biol 22, 691–708 (2021). 670 \n10. Seetharaman, S. et al. Microtubules tune mechanosensitive cell responses. Nat 671 \nMater 21, 366–377 (2022). 672 \n11. Hamant, O. et al. Developmental Patterning by Mechanical Signals in 673 \nArabidopsis. Science (1979) 322, 1650–1655 (2008). 674 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n12. Yan, Y ., Sun, Z., Yan, P ., Wang, T. & Zhang, Y . Mechanical regulation of cortical 675 \nmicrotubules in plant cells. New Phytologist 239, 1609–1621 (2023). 676 \n13. Bringmann, M. et al. Cracking the elusive alignment hypothesis: the microtubule–677 \ncellulose synthase nexus unraveled. Trends Plant Sci 17, 666–674 (2012). 678 \n14. Lee, Y .-R. J. & Liu, B. Microtubule nucleation for the assembly of acentrosomal 679 \nmicrotubule arrays in plant cells. New Phytologist 222, 1705–1718 (2019). 680 \n15. Vinopal, S. & Bradke, F . Centrosomal and acentrosomal microtubule nucleation 681 \nduring neuronal development. Curr Opin Neurobiol 92, 103016 (2025). 682 \n16. Sallee, M. D. & Feldman, J. L. Microtubule organization across cell types and 683 \nstates. Current Biology 31, R506–R511 (2021). 684 \n17. Roostalu, J. & Surrey, T. Microtubule nucleation: beyond the template. Nat Rev 685 \nMol Cell Biol 18, 702–710 (2017). 686 \n18. Wadsworth, P . The multifunctional spindle midzone in vertebrate cells at a glance. 687 \nJ Cell Sci 134, jcs250001 (2021). 688 \n19. Ehrhardt, D. W. Straighten up and fly right—microtubule dynamics and 689 \norganization of non-centrosomal arrays in higher plants. Curr Opin Cell Biol 20, 690 \n107–116 (2008). 691 \n20. Chan, J., Jensen, C. G., Jensen, L. C. W., Bush, M. & Lloyd, C. W. The 65-kDa carrot 692 \nmicrotubule-associated protein forms regularly arranged filamentous cross-693 \nbridges between microtubules. Proceedings of the National Academy of Sciences 694 \n96, 14931–14936 (1999). 695 \n21. Mollinari, C. et al. PRC1 is a microtubule binding and bundling protein essential to 696 \nmaintain the mitotic spindle midzone. Journal of Cell Biology 157, 1175–1186 697 \n(2002). 698 \n22. Portran, D. et al. MAP65/Ase1 promote microtubule flexibility. Mol Biol Cell 24, 699 \n1964–1973 (2013). 700 \n23. Basnet, N. et al. Direct induction of microtubule branching by microtubule 701 \nnucleation factor SSNA1. Nat Cell Biol 20, 1172–1180 (2018). 702 \n24. Gaillard, J. et al. Two Microtubule-associated Proteins of Arabidopsis MAP65s 703 \nPromote Antiparallel Microtubule Bundling. Mol Biol Cell 19, 4534–4544 (2008). 704 \n25. Colin, L. et al. Cortical tension overrides geometrical cues to orient microtubules 705 \nin confined protoplasts. Proceedings of the National Academy of Sciences 117, 706 \n32731–32738 (2020). 707 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n26. Lindeboom, J. J. et al. A Mechanism for Reorientation of Cortical Microtubule 708 \nArrays Driven by Microtubule Severing. Science (1979) 342, 1245533 (2013). 709 \n27. Paquette, A. L. et al. Competition for microtubule lattice spacing between a 710 \nmicrotubule expander and compactor. bioRxiv 2025.02.28.640185 (2025) 711 \ndoi:10.1101/2025.02.28.640185. 712 \n28. Bechstedt, S., Lu, K. & Brouhard, G. J. Doublecortin Recognizes the Longitudinal 713 \nCurvature of the Microtubule End and Lattice. Current Biology 24, 2366–2375 714 \n(2014). 715 \n29. Alushin, G. M. et al. High-Resolution Microtubule Structures Reveal the Structural 716 \nTransitions in αβ-Tubulin upon GTP Hydrolysis. Cell 157, 1117–1129 (2014). 717 \n30. Liu, H. & Shima, T. Preference of CAMSAP3 for expanded microtubule lattice 718 \ncontributes to stabilization of the minus end. Life Sci Alliance 6, e202201714 719 \n(2023). 720 \n31. Nandakumar, S. et al. Kinesin-induced buckling reveals the limits of microtubule 721 \nself-repair. bioRxiv 2025.09.08.672697 (2025) doi:10.1101/2025.09.08.672697. 722 \n32. Schaedel, L. et al. Lattice defects induce microtubule self-renewal. Nat Phys 15, 723 \n830–838 (2019). 724 \n33. Reid, T. A., Coombes, C. & Gardner, M. K. Manipulation and quantification of 725 \nmicrotubule lattice integrity. Biol Open 6, 1245–1256 (2017). 726 \n34. Reuther, C., Santos-Otte, P ., Grover, R., Korten, T. & Diez, S. Microtubule lattice 727 \ndefects facilitate spastin-mediated severing. bioRxiv 2025.08.29.673059 (2025) 728 \ndoi:10.1101/2025.08.29.673059. 729 \n35. Trushko, A., Schä^er, E. & Howard, J. The growth speed of microtubules with 730 \nXMAP215-coated beads coupled to their ends is increased by tensile force. 731 \nProceedings of the National Academy of Sciences 110, 14670–14675 (2013). 732 \n36. Hervieux, N. et al. A Mechanical Feedback Restricts Sepal Growth and Shape in 733 \nArabidopsis. Current Biology 26, 1019–1028 (2016). 734 \n37. Krattenmacher, J. et al. Ase1 selectively increases the lifetime of antiparallel 735 \nmicrotubule overlaps. Current Biology 34, 4071-4080.e6 (2024). 736 \n38. Molines, A. T. et al. Physical properties of the cytoplasm modulate the rates of 737 \nmicrotubule polymerization and depolymerization. Dev Cell 57, 466-479.e6 738 \n(2022). 739 \n39. Peet, D. R., Burroughs, N. J. & Cross, R. A. Kinesin expands and stabilizes the 740 \nGDP-microtubule lattice. Nat Nanotechnol 13, 386–391 (2018). 741 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n40. Aher, A. et al. CLASP Mediates Microtubule Repair by Restricting Lattice Damage 742 \nand Regulating Tubulin Incorporation. Current Biology 30, 2175-2183.e6 (2020). 743 \n41. Li, Y . et al. Compressive forces stabilize microtubules in living cells. Nat Mater 22, 744 \n913–924 (2023). 745 \n42. de Forges, H. et al. Localized Mechanical Stress Promotes Microtubule Rescue. 746 \nCurrent Biology 26, 3399–3406 (2016). 747 \n43. Aumeier, C. et al. Self-repair promotes microtubule rescue. Nat Cell Biol 18, 748 \n1054–1064 (2016). 749 \n44. Liu, J. et al. PRC1 Cooperates with CLASP1 to Organize Central Spindle Plasticity 750 \nin Mitosis. Journal of Biological Chemistry 284, 23059–23071 (2009). 751 \n45. Bratman, S. V & Chang, F . Stabilization of Overlapping Microtubules by Fission 752 \nYeast CLASP. Dev Cell 13, 812–827 (2007). 753 \n46. Burkart, G. M. & Dixit, R. Microtubule bundling by MAP65-1 protects against 754 \nsevering by inhibiting the binding of katanin. Mol Biol Cell 30, 1587–1597 (2019). 755 \n47. Deinum, E. E., Tindemans, S. H., Lindeboom, J. J. & Mulder, B. M. How selective 756 \nsevering by katanin promotes order in the plant cortical microtubule array. 757 \nProceedings of the National Academy of Sciences 114, 6942–6947 (2017). 758 \n48. Smertenko, A. P . et al. The C-Terminal Variable Region Specifies the Dynamic 759 \nProperties of Arabidopsis Microtubule-Associated Protein MAP65 Isotypes. Plant 760 \nCell 20, 3346–3358 (2008). 761 \n49. Meißner, L., Niese, L., Schüring, I., Mitra, A. & Diez, S. Human kinesin-5 KIF11 762 \ndrives the helical motion of anti-parallel and parallel microtubules around each 763 \nother. EMBO J 43, 1244-1256–1256 (2024). 764 \n  765 \nAcknowledgments 766 \nMRM and OH were supported by a grant from the European Research Council (ERC, grant 767 \nagreement number 101019515, “Musix” , awarded to OH). MRM was also supported by a 768 \ngrant from the German Research Foundation (DFG, project number 545084341, awarded 769 \nto MRM). LS was supported by the DFG grant SFB 1027 and the ERC grant StG 101115795 770 \n“CROSSTALK”. 771 \n 772 \nAuthor contributions 773 \nMRM, LS, OH and SD conceived and guided the project. MRM, LS, OH and SD designed 774 \nthe experiments. MRM and CG performed the experiments. MG and BK purified the 775 \ntubulin. LM, LN and SD provided KIF11 and PRC1 and related expertise. MRM and CG 776 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nanalyzed the experiments. MRM, LS and OH wrote the manuscript with input from all 777 \nauthors. 778 \n 779 \nCompeting interest declaration 780 \nThe authors declare no competing interests. 781 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. 1: MAP65-1/PRC1 promote microtubule nucleation on existing lattices\na, Schematic representation of the experimental setup used to study the function of MAP65-1 and \nPRC1 in lattice-templated microtubule appearance. Microtubules were grown with ATTO-565-labeled \ntubulin at a concentration of 11 μM (step I) before they were capped with GMPCPP (step II) and \nexposed to ATTO-488-labeled free tubulin at a concentration of 4 μM (step III). Step III was observed \nlive for a total of 30 minutes. b, Schematic representation of the flow chambers used as an \nexperimental setup throughout the manuscript. 20-μl flow chambers were built from passivated \ncoverslips cut with a diamond pen and attached by double-sided tape. Solutions were sequentially \nflushed in through one side of the flow chamber. After step III shown in a, flow chambers were sealed \nto prevent evaporation and observed for 30 min. c, Microtubule dynamics imaged by TIRFM in the \npresence of ATTO-488-labeled free tubulin (green) and ATTO-565 microtubules (magenta). In the \ncontrol (top), only growth from the free microtubule ends (elongation) was observed. In the presence \nof both 100 nM MAP65-1 (middle) and 300 pM PRC1 (bottom), microtubules appeared and grew in \nparallel to the existing lattices (microtubule appearance events are indicated with a white star). Scale \nbars, 5 µm. d, Schematic representation of lattice-templated microtubule appearance. Microtubule \nappearance and growth could be clearly distinguished from elongation at the free ends because it \noccurred overlapping with the original microtubule lattice “template”. e, Kymograph of the \ncorresponding stills in c in the presence of MAP65-1. Appearance events are indicated with a white \nstar. Scale bars, 5 µm (horizontal) and 1 min (vertical). f, Experimental setup used to test if \nmicrotubules that appear in the presence of MAP65-1 are independent from the “template” \nmicrotubule lattice. 10 nM KIF11 was added in the presence of 2 μM free tubulin, and some of the \nnew microtubules were effectively used as cargo. g, KIF11-mediated microtubule sliding observed by \nTIRFM after incubation of microtubules with 100 nM MAP65-1 and 4 μM ATTO-488-labeled tubulin. \nScale bar, 5 μm. h, Kymograph of the corresponding microtubule shown in g. Scale bars, 5 μm \n(horizontal) and 1 min (vertical). i, Quantification of nucleated microtubule behavior (mobile or static) \nupon addition of KIF11 (n = 24 nucleated microtubules, N = 3). j, Schematic representation of the \nexperimental setup used to study PRC1-mediated microtubule nucleation in k. The same steps were \ntaken as in a, except microtubule seeds and caps were polymerized with Alexa-Fluor-647-labeled \ntubulin (blue) instead of ATTO 565. k, Microtubule dynamics in the absence (control) and in the \npresence of 1 nM PRC1 observed by TIRFM. Scale bar, 50 μm.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. 2: MAP65-1 concentrates tubulin on the microtubule lattice\na, Schematic representation of the experimental setup used to study free tubulin recruitment by \nMAP65-1. Microtubules were grown with Alexa-Fluor-647-labeled tubulin at a concentration of 11 μM \n(step I) before they were capped with GMPCPP (step II) and exposed to ATTO-565-labeled free \ntubulin at a concentration of 4 μM and 100 nM or 200 nM GFP-MAP65-1 (step III). b, Recruitment of \nfree tubulin in the absence of GTP to the microtubule lattice by 100 and 200 nM GFP-MAP65-1 (cyan) \nobserved by CLSM. Microtubules in magenta and free tubulin in green. Scale bar, 5 μm. c, Schematic \nrepresentation of the setup used to analyze free tubulin recruitment by MAP65-1 to the microtubule \nlattice. ROIs with a length of 1 µm were drawn to measure fluorescence intensity across the full width \nof the microtubule, which was then divided to the fluorescence intensity in the background to estimate \ntubulin concentration. d, Graphs represent line scans along the microtubule shown in b in the \npresence of 200 nM GFP-MAP65-1. e, Line scan of free tubulin with the intensity normalized to the \nbackground free tubulin level. The graph corresponds to the microtubule shown in b with 200 nM \nGFP-MAP65-1 and the corresponding graph in d. f, Quantification of tubulin recruitment to the \nmicrotubule lattice (normalized to the background level) in the presence of 100 nM (n = 150 \nmicrotubule segments of 1 µm) and 200 nM (n = 113 microtubule segments of 1 µm) MAP65-1. Bars \nrepresent the median values. Statistics: Mann-Whitney test, **** P < 0.0001. g, FRAP assay observed \nby TIRFM. The dashed white rectangle indicates the bleached region where fluorescence intensity \nwas measured over time. Scale bar, 5 μm. h, Graph with icons representing the average normalized \nfluorescence intensity recovery over time for both GFP-MAP65-1 (cyan) and free tubulin (green; n = \n17 microtubules). Shaded region above and below the icons corresponds to the standard deviation for \neach time point.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. 3: MAP65-1 preferentially localizes to bent microtubules in cells\na, Average intensity Z-projection of CLSM of epidermal hypocotyl cells from 5-day-old plants co-\nexpressing p35S::GFP-MBD and pMAP65-1::MAP65-1-mCherry. Scale bar, 5 μm. b, Arrowheads \nindicate examples of straight and bent microtubules extracted from the image shown in a. Scale bar, 5 \nμm. c, Quantification of mean MAP65-1 intensity on straight (maximum curvature < 0.1 μm-1, n = 27) \nand bent (maximum curvature > 0.1 μm-1, n = 31) microtubules, N = 5 cells. Bars indicate the median \nvalues. Statistics: Mann-Whitney test, **** P < 0.0001. d, Quantification of mean MBD intensity per \nmicrotubule against microtubule maximum curvature. The pink line indicates a simple linear \nregression (Spearman r = 0.67, P < 0.0001, n = 58 microtubules). e, Quantification of mean MAP65-1 \nintensity per microtubule against microtubule maximum curvature. The pink line indicates a simple \nlinear regression (Spearman r = 0.64, P < 0.0001, n = 58 microtubules). f, Schematic representation \nof the protoplast experiment. Protoplasts are first extracted in a solution with 600 mOsmol/L mannitol, \nthen allowed to sediment into rectangular microwells followed by an exchange to a solution with 280 \nmOsmol/L mannitol that causes the protoplasts to inflate. g, Average intensity Z-projection of CLSM of \nenclosed protoplasts extracted from roots of plants co-expressing p35S::GFP-MBD and pMAP65-\n1::MAP65-1-mCherry. Scale bar, 5 μm. h, Quantification of normalized mean MAP65-1 intensity \nagainst mean microtubule orientation. The pink line indicates a simple linear regression (Spearman r \n= -0.25, P = 0.0028, n = 135 microtubules).\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. 4: MAP65-1 promotes microtubule nucleation on bent microtubules in vitro\na, Microtubules attached to the surface at both ends, straight or forced to statically bend by fluid flow, \nobserved by TIRFM. 100 nM MAP65-1 was added at a ratio of 15% GFP-MAP65-1 to 85% non-\nfluorescent MAP65-1. Scale bar, 5 μm.  b, Quantification of mean MAP65-1 intensity on straight \n(maximum curvature < 0.1 μm-1, n = 24) and bent (maximum curvature > 0.1 μm-1, n = 29) \nmicrotubules, N = 3. Bars indicate the median values. Statistics: Mann-Whitney test, **** P < 0.0001. \nc, Quantification of mean MAP65-1 intensity per microtubule against microtubule maximum curvature. \nThe pink line indicates a simple linear regression (Spearman r = 0.52, P < 0.0001, n = 53). d, \nMicrotubule nucleation mediated by 100 nM MAP65-1 on straight and bent microtubules observed by \nTIRFM. Nucleation events are indicated with a white star. Scale bar, 5 μm. e, Graphs represent line \nscans along the microtubules shown in d (original microtubule in magenta, nucleated/elongated \nmicrotubules in green). f, Quantification of the distance between nucleation events on straight \n(maximum curvature < 0.1 μm-1, n = 11 events) and bent (maximum curvature > 0.1 μm-1, n = 40 \nevents) microtubules. Bars indicate the median values. T otal microtubule lengths of 723.52 μm \n(straight) and 819.45 μm (bent) were analyzed, N = 3. Statistics: Mann-Whitney test, ** P = 0.0012. g, \nSpatial nucleation frequency (number of events per µm) according to microtubule maximum curvature \n(n = 14 events for 0–0.1 µm-1, 17 events for 0.1–0.3 µm-1, 10 events for 0.3–0.5 µm-1, 8 events for \n0.5–0.7 µm-1 and 6 events for 0.7–0.9 µm-1). T otal microtubule lengths = 723.52 µm (0–0.1 µm-1), \n461.18 µm (0.1–0.3 µm-1), 143.07 µm (0.3–0.5 µm-1), 94.91 µm (0.5–0.7 µm-1) and 72.87 µm (0.7–0.9 \nµm-1).\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. 5: MAP65-mediated microtubule nucleation depends on the amount of microtubule defects\na, Microtubule nucleation on taxol pre-treated microtubule mediated by 100 nM MAP65-1 imaged by \nTIRFM. Free tubulin (green) labeled with ATTO 488 and microtubules (magenta) with ATTO 565. \nWhite arrowhead indicates presumed microtubule damage site from where nucleation (white star) \nhappens. Scale bar, 5 µm. b, Kymograph of the corresponding microtubule shown in a. Scale bars, 5 \nµm (horizontal) and 1 min (vertical). c, Quantification of the distance between nucleation events on \nfast grown, taxol pre-treated and slowly grown microtubules. A total of 16 (fast growth), 26 (taxol pre-\ntreated) and 6 (slow growth) nucleation events were observed. Bars indicate the median values. T otal \nmicrotubule lengths of 430.04 µm, 398.67 µm and 453.21 µm were analyzed respectively, N  3. \nStatistics: Mann-Whitney test, Bonferroni-corrected, * P = 0.015 and ** P = 0.0016. d, Graph \nrepresents line scan along the microtubule shown in a, showing fluorescence intensity at presumed \ndamage site (white arrowhead). e, Microtubule nucleation on taxol pre-treated microtubule mediated \nby 300 pM PRC1 imaged by TIRFM. Free tubulin (green) labeled with ATTO 488 and microtubules \n(magenta) with ATTO 565. Nucleation events are indicated with white stars. Scale bar, 5 µm. f, \nKymograph of corresponding microtubule shown in e. Scale bars, 5 µm (horizontal) and 1 min \n(vertical). g, Quantification of the distance between nucleation events on fast grown, taxol pre-treated \nand slowly grown microtubules. A total of 26 (fast growth), 46 (taxol pre-treated) and 13 (slow growth) \nnucleation events were observed. Bars indicate the median values. T otal microtubule lengths of \n536.38 µm, 254.24 µm and 446.95 µm were analyzed respectively, N  3. Statistics: Mann-Whitney \ntest, Bonferroni-corrected, ** P = 0.0024 and *** P = 0.0002.  h, Schematic illustration of the \nexperimental setup to generate annealed microtubules. Microtubules were slowly polymerized with \nGMPCPP and in the presence of ATTO-565-labeled or Alexa-Fluor-647-labeled tubulin (step I). The \ntwo microtubule populations were then mixed and incubated overnight at 30°C (step II). The next day, \nthe mix of non-annealed and annealed microtubules was used for the experiments (step III). i, \nMicrotubule nucleation mediated by 100 nM MAP65-1 observed on annealed microtubules by TIRFM. \nNucleation event is indicated with white star. Scale bar, 5 µm. j, Graph represents line scan along the \nmicrotubule shown in i (microtubule 1 in magenta, microtubule 2 in blue, nucleated microtubule in \ngreen). k, Quantification of the distance between nucleation events on non-annealed and annealed \nmicrotubules. A total of 9 (non-annealed) and 54 (annealed) nucleation events were observed. Bars \nindicate the median values. T otal microtubule lengths of 244.35 µm and 406.52 µm were analyzed \nrespectively, N = 2. Statistics: Mann-Whitney test, ** P = 0.0099.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. 6: Bundled microtubules survive longer in the absence of free tubulin\na, Microtubule dynamics upon removal of free tubulin and incubation with 100 nM MAP65-1 imaged \nby TIRFM (microtubules labeled with ATTO-565 in magenta, seeds and caps in blue). White \narrowheads indicate microtubule breakage. Scale bar, 5 µm. b, Quantification of microtubule survival \nover time upon removal of free tubulin in the presence of 100 nM MAP65-1 for single (N = 8, n = 33 \nmicrotubules) and bundled (N = 5, n = 69 microtubules) microtubules. Statistics: Logrank (Mantel-\nCox) test, **** P < 0.0001. c, Schematic representation of the hypothetical function of MAP65-1 in \nreinforcing microtubule alignment with mechanical stress. Microtubules polymerize faster under \ntensile stress (lower right part of the panel); this induces more defects in the lattice (along with more \ndamage sites due to the high curvature of these microtubules); these defects and damage sites recruit \nMAP65, which then stabilizes, nucleates on and bundles microtubules. MAP65 binding also leads to \nmicrotubule softening. The alignment of microtubule arrays is the emerging property of their stiffness: \nstiff microtubules tend to align along the flattest direction of the cell cortex. For soft microtubules, this \nemerging property is disrupted, and microtubules instead align to a greater extent along the direction \nof maximal tensile stress, i.e., the surface experiencing the highest curvature for a pressurized cell. \nThese mechanisms bring forth a positive feedback loop, where then microtubules under stress again \npolymerize faster, have more defects (and damage due to bending) and recruit MAP65. MAP65 thus \nprotects microtubule under stress and promotes its own recruitment. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. S1, related to Fig. 1: MAP65-1 is a well-known microtubule bundler\na, Confocal laser scanning microscopy (CLSM) of epidermal hypocotyl cells from 5-day-old plants co-\nexpressing p35S::GFP-MBD and pMAP65-1::MAP65-1-mCherry. White arrowhead indicates a \nmicrotubule that grows and gets bundled in the last time point (marked with a dashed white square). \nScale bar, 5 µm. b, Close-up of dashed white square indicated in panel a. c, In vitro microtubule \ndynamics imaged by total internal reflection fluorescence microscopy (TIRFM) in the presence of 8 \nμM ATTO-565-labeled free tubulin (magenta) and 50 nM GFP-MAP65-1 (cyan). Stable GMPCPP \nseeds labeled with Alexa Fluor 647 appear in blue. White arrowhead indicates a microtubule that \ngrows and gets bundled in the last time point. Scale bar, 5 µm.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. S2, related to Fig. 1: Nucleation does not happen in the control and is MAP65-1 \nconcentration-dependent\na, Kymograph of the corresponding control microtubule shown in Fig. 1c (top panel), exhibiting growth \nfrom the free ends in the absence of MAP65-1/PRC1. Free tubulin labeled with ATTO 488 (green) and \nmicrotubule labeled with ATTO 565 (magenta). Scale bars, 5 µm (horizontal) and 1 min (vertical). b, \nTIRFM images showing microtubule nucleation by MAP65-1 with different concentrations. White stars \nindicate nucleation events. Scale bar, 5 µm. c, Quantification of the distance between nucleation \nevents using different MAP65-1 concentrations. A total of 11 (100 nM), 34 (150 nM) and 32 (200 nM) \nnucleation events were observed. Bars indicate the median values. Total microtubule lengths of \n1009.49 µm, 705.76 µm and 587.54 µm were analyzed respectively, N = 3. Statistics: Kruskal-Wallis \ntest, *** P = 0.0001 and **** P < 0.0001.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. S3, related to Fig. 1: Microtubule nucleation thresholds in solution for MAP65-1 and PRC1\n4 µM ATTO-488-labeled free tubulin was incubated either in the presence of PRC1 or MAP65-1 with \nthe indicated concentrations. For the condition of 500 nM MAP65-1, no seeds are visible. White stars \nindicate nucleation events in solution. Scale bars, 25 µm.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. S4, related to Fig. 1: 1 nM PRC1 promotes extensive nucleation\na, Close-up of microtubule from Fig. 1k showing nucleation events (white stars) along the microtubule \nlattice in the presence of 4 µM free ATTO-488-labeled tubulin (green) and 1 nM PRC1. Scale bar, 5 \nµm. b, Kymograph of corresponding microtubule shown in a. White stars indicate nucleation events. \nScale bars, 5 µm (horizontal) and 1 min (vertical).\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. S5, related to Fig. 2: In the absence of GTP, no growth happens from microtubule ends\na, Microtubule incubated with 100 nM GFP-MAP65-1 and 4 µM free ATTO-565-labeled tubulin in the \nabsence of GTP imaged by TIRFM. Scale bar, 5 µm. b, Kymograph of the corresponding microtubule \nshown in a. Scale bars, 5 µm (horizontal) and 1 min (vertical).\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. S6, related to Fig. 4: Nucleation events do not always happen at the highest curvature \nregions\nExamples of site of microtubule nucleation on bent microtubules, showing the stochastic nature of this \nevent. The nucleation does not always happen at the site of highest microtubule curvature. Scale bar, \n5 µm.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. S7, related to Fig. 4: MAP65-1 does not specifically recognize an expanded lattice\na, Quantification of the frequency of the nucleation origin site in microtubules grown with 11 µM of \n10% ATTO-565-labeled tubulin and incubated with 100 nM MAP65-1 and 4 µM ATTO-488-labeled \ntubulin as shown in Fig. 1a. n = 46 nucleation events, N = 4. b, Average composition of the \nmicrotubule lattice in length from the microtubules that were used for the quantification in a. Total \nmicrotubule length analyzed (GMPCPP seed, GMPCPP cap, and GDP lattice) = 578.94 µm. c, \nMicrotubule incubated with 100 nM GFP-MAP65-1 and 3 µM black tubulin and observed by TIRFM. \nScale bar, 5 µm. d, Graph represents line scans along microtubule shown in c, with MAP65-1 in cyan \nand the microtubule in magenta. The peak corresponds to a GMPCPP region of the microtubule, the \nseed. e, Quantification of GFP-MAP65-1 intensity on the seed (GMPCPP) and lattice (GDP) portions \nof the microtubule, n = 17 microtubules. Bars indicate the median values. Statistics: Mann-Whitney \ntest, *** P = 0.0001. f, Microtubules incubated with 100 nM GFP-MAP65-1 and 3 µM black tubulin or \nwith 20 µM taxol observed by TIRFM. Scale bar, 5 µm. g, Quantification of GFP-MAP65-1 intensity on \nthe GDP lattice with and without 20 µM taxol, n = 15-17 microtubules. Bars indicate the median \nvalues. Statistics: Mann-Whitney test, **** P < 0.0001. h, Stills of a microtubule before and after \naddition of 500 nM GFP-MAP65-1 and 4 µm black tubulin imaged by TIRFM in a microfluidics setup. \nScale bar, 5 µm. i, Kymograph of the corresponding microtubule shown in h, showing no obvious \nchange in microtubule length upon binding of MAP65-1. White arrow indicates moment in which \nMAP65-1 is flushed in. Scale bars, 5 µm (horizontal) and 1 min (vertical).\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint \n\nFig. S8, related to Fig. 5: Microtubule nucleation frequency depends on the amount of \nmicrotubule defects\na, Schematic representation of the experimental setup to obtain microtubules with high and low \namounts of defects. Microtubules are either polymerized in a high or low defect regime (step I), \nlabeled respectively with 20% Alexa Fluor 647 and 20% ATTO 565. Next, 1X BRB80 is added and \nmicrotubules are centrifuged and resuspended in fresh 1X BRB80 (step II). Microtubules are then \nsequentially flushed in the flow chamber for incubation with 100 nM MAP65-1 and free tubulin (step \nIII). b, Microtubule nucleation mediated by 100 nM MAP65-1 on microtubules polymerized under a \nhigh or low defect regime imaged by TIRFM. Scale bar, 5 µm. c, Quantification of the distance \nbetween nucleation events on microtubules polymerized under a high or low defect regime. A total of \n95 (high defect) and 67 (low defect) nucleation events were observed. Bars indicate the median \nvalues. Total microtubule lengths of 545.41 µm and 626.598 µm were analyzed respectively, N = 3. \nStatistics: Mann-Whitney test, * P = 0.0433.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted September 15, 2025. ; https://doi.org/10.1101/2025.09.14.676082doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}