# Microenvironmental stiffness directs microtubule perturbation in chondrocyte mitosis via ILK-refilinB/Smad3 axis **Authors:** Mengmeng Duan, Chenchen Zhou, Guanyue Su, Chunhe Zhang, Jie Ren, Qingjia Chi, Xiaojing Liu, Li Yang, Haiqing Bai, Yang Claire Zeng, Seongmin Kim, Yunhao Zhai, Crystal Yuri Oh, Adam Yongxin Ye, Yuting Chen, Longlong Si, Xiaoheng Liu, Jing Xie PMC · DOI: 10.1038/s41413-025-00491-4 · 2026-01-30 ## TL;DR This study shows how the stiffness of the environment affects microtubule behavior in chondrocytes during cell division, through a specific signaling pathway. ## Contribution The study identifies refilin B and the ILK-p-Smad3 axis as key regulators of microtubule dynamics in chondrocytes in response to mechanical stiffness. ## Key findings - Mechanical stiffness influences microtubule dynamics during chondrocyte mitosis. - Refilin B regulates microtubule assembly via the p-Smad3 signaling pathway. - Integrin-linked kinase (ILK) mediates microtubule changes in response to stiffness. ## Abstract Cells actively sense and transduce microenvironmental mechanical inputs into chemical signals via cytoskeletal rearrangements. During these mechanosensation and mechanotransduction processes, the role of the actin cytoskeleton is well-understood, whereas the role of the tubulin cytoskeleton remains largely elusive. Here, we report the dynamic changes in microtubules in response to microenvironmental stiffness during chondrocyte mitosis. Mechanical stiffness was found to be coupled with microtubule generation, directing microtubule dynamics in mitotic chondrocytes. Refilin B was found to be a key regulator of microtubule assembly in chondrocytes in response to mechanical stiffness. It was found to play its role in microtubule formation via the p-Smad3 signaling pathway. Additionally, integrin-linked kinase (ILK), triggered by mechanical stiffness, was found to play an indispensable role in the process of microtubule dynamics mediated by refilin B. Our data emphasizes stiffness-mediated dynamic changes in the microtubules of chondrocytes in a quiescent state (G0) and at anaphase, which improves our understanding of the mechanical regulation of microtubule assembly during the chondrocyte cell cycle and provides insights into microenvironment mechanics during tissue maintenance, wound healing, and disease occurrence. ## Linked entities - **Genes:** Rflnb (refilin B) [NCBI Gene 76566], ILK (integrin linked kinase) [NCBI Gene 3611] ## Full-text entities - **Genes:** Gapdh (glyceraldehyde-3-phosphate dehydrogenase) [NCBI Gene 14433] {aka Gapd}, Ccnb1 (cyclin B1) [NCBI Gene 268697] {aka Ccnb1-rs1, Ccnb1-rs13, CycB1, Cycb-4, Cycb-5, Cycb1-rs1}, Vcl (vinculin) [NCBI Gene 22330] {aka 9430097D22}, Rflnb (refilin B) [NCBI Gene 76566] {aka 1500005K14Rik, Fam101b, RefilinB, cfm}, Itgb5 (integrin beta 5) [NCBI Gene 16419] {aka ESTM23, [b]-5, [b]5, [b]5A, [b]5B, beta-5}, Zyx (zyxin) [NCBI Gene 22793] {aka 9530098H06Rik}, Tubb5 (tubulin, beta 5 class I) [NCBI Gene 22154] {aka B130022C14Rik, M(beta)5}, Tubb2a (tubulin, beta 2A class IIA) [NCBI Gene 22151] {aka M(beta)2, Tubb2}, Itga5 (integrin alpha 5 (fibronectin receptor alpha)) [NCBI Gene 16402] {aka Cd49e, Fnra, VLA5}, Itgb2 (integrin beta 2) [NCBI Gene 16414] {aka 2E6, Cd18, LAD, LCAMB, Lfa1, MF17}, Alb (albumin) [NCBI Gene 11657] {aka Alb-1, Alb1, BCL001, BCL002, BPL001}, Tuba1a (tubulin, alpha 1A) [NCBI Gene 22142] {aka Tuba-1, Tuba1}, Samd3 (sterile alpha motif domain containing 3) [NCBI Gene 268288] {aka Gm623}, Rflna (refilin A) [NCBI Gene 73121] {aka 3110032G18Rik, Fam101a, cfm, cfm2}, Smad4 (SMAD family member 4) [NCBI Gene 17128] {aka D18Wsu70e, DPC4, Madh4}, Tuba1b (tubulin, alpha 1B) [NCBI Gene 22143] {aka Tuba2}, Flnb (filamin, beta) [NCBI Gene 286940] {aka Fln-b}, Rhoa (ras homolog family member A) [NCBI Gene 11848] {aka Arha, Arha1, Arha2}, Smad2 (SMAD family member 2) [NCBI Gene 17126] {aka 7120426M23Rik, Madh2, Madr2, Smad-2, mMad2}, Smad3 (SMAD family member 3) [NCBI Gene 17127] {aka Madh3}, Akt1 (Akt serine/threonine kinase 1) [NCBI Gene 11651] {aka Akt, LTR-akt, PKB, PKB/Akt, PKBalpha, Rac}, Vim (vimentin) [NCBI Gene 22352], Itga4 (integrin alpha 4) [NCBI Gene 16401] {aka CD49D, Itga4B}, Syngr1 (synaptogyrin 1) [NCBI Gene 20972] {aka Syngr1b, p29}, Actb (actin, beta) [NCBI Gene 11461] {aka Actx, E430023M04Rik, beta-actin}, Pxn (paxillin) [NCBI Gene 19303] {aka Pax}, Ccnb2 (cyclin B2) [NCBI Gene 12442] {aka CycB2}, Cdk1 (cyclin dependent kinase 1) [NCBI Gene 12534] {aka Cdc2, Cdc2a, p34}, RFLNB (refilin B) [NCBI Gene 359845] {aka CFM1, FAM101B}, Acan (aggrecan) [NCBI Gene 11595] {aka Agc, Agc1, CSPCP, Cspg1, b2b183Clo, cmd}, SMAD3 (SMAD family member 3) [NCBI Gene 4088] {aka HSPC193, HsT17436, JV15-2, LDS1C, LDS3, MADH3}, Tubb6 (tubulin, beta 6 class V) [NCBI Gene 67951] {aka 2310057H16Rik}, Ilk (integrin linked kinase) [NCBI Gene 16202] {aka ESTM24, ILK-1, ILK-2, p59ILK}, Pik3r1 (phosphoinositide-3-kinase regulatory subunit 1) [NCBI Gene 18708] {aka PI3K, p50alpha, p55alpha, p85alpha}, ILK (integrin linked kinase) [NCBI Gene 3611] {aka HEL-S-28, ILK-1, ILK-2, P59, p59ILK}, Itga6 (integrin alpha 6) [NCBI Gene 16403] {aka 5033401O05Rik, Cd49f, VLA-6}, Tuba1c (tubulin, alpha 1C) [NCBI Gene 22146] {aka M[a]6, Tuba6}, Itgam (integrin alpha M) [NCBI Gene 16409] {aka CD11b/CD18, CR3, CR3A, Cd11b, F730045J24Rik, Ly-40}, Itgav (integrin alpha V) [NCBI Gene 16410] {aka 1110004F14Rik, 2610028E01Rik, CD51, D430040G12Rik}, Tgfb1 (transforming growth factor, beta 1) [NCBI Gene 21803] {aka TGF-beta1, TGFbeta1, Tgfb, Tgfb-1}, Tubb4b (tubulin, beta 4B class IVB) [NCBI Gene 227613] {aka 4930542G03Rik, Tubb2c, Tubb2c1}, Itgal (integrin alpha L) [NCBI Gene 16408] {aka (p180), Cd11a, LFA-1, LFA-1A, Ly-15, Ly-21} - **Diseases:** OA (MESH:D010003), fibrosis (MESH:D005355), FA (MESH:C565561), trauma (MESH:D014947), joint diseases (MESH:D007592), skeletal malformation (MESH:C535850), hypertrophy (MESH:D006984) - **Chemicals:** glutaraldehyde (MESH:D005976), SDS (MESH:D012967), gold (MESH:D006046), trichloroacetic acid (MESH:D014238), nocodazole (MESH:D015739), Alexa Fluor 488 (MESH:C000711379), ammonium bicarbonate (MESH:C027043), blebbistatin (MESH:C472645), amino acids (MESH:D000596), 4, 6-diamidino-2-phenylindole (MESH:C007293), bicinchoninic acid (MESH:C047117), dopamine (MESH:D004298), 3'v3 (-), L-glutamine (MESH:D005973), peptide (MESH:D010455), Alexa Fluor 647 (MESH:C569686), streptomycin (MESH:D013307), EDTA (MESH:D004492), acetone (MESH:D000096), chondroitin sulfate (MESH:D002809), DMSO (MESH:D004121), Triton X-100 (MESH:D017830), NP-40 (MESH:C010615), PDMS (MESH:C013830), PFA (MESH:C003043), silicon (MESH:D012825), penicillin (MESH:D010406), PVDF (MESH:C024865), DPBS (MESH:C012939), orthovanadate (MESH:D014638), Lipofectamine (MESH:C086724), PBS (MESH:D007854), CO2 (MESH:D002245), hyaluronic acid (MESH:D006820), alcohol (MESH:D000438) - **Species:** Rattus norvegicus (brown rat, species) [taxon 10116], Mus musculus (house mouse, species) [taxon 10090], Homo sapiens (human, species) [taxon 9606] - **Mutations:** C-25 C, P0023A ## Figures 8 figures with captions in the complete paper: https://tomesphere.com/paper/PMC12858872/full.md --- Source: https://tomesphere.com/paper/PMC12858872