Microtubule (MT) and F-actin cytoskeletal cross-talk and organization are important aspects of axon guidance mechanisms, but how associated proteins facilitate this function remains largely unknown. While the MT-associated protein, CKAP5 (XMAP215/ch-TOG), has been best characterized as a MT polymerase, we have recently highlighted a novel role for CKAP5 in facilitating interactions between MT and F-actin in vitro and in embryonic Xenopus laevis neuronal growth cones. However, the mechanism by which it does so is unclear. Here, using in vitro reconstitution assays coupled with total internal reflection fluorescence microscopy, we report that the TOG5 domain of CKAP5 is necessary for its ability to bind to and bundle actin filaments, as well as to cross-link MTs and F-actin in vitro. Additionally, we show that this novel MT/F-actin cross-linking function of CKAP5 is possible even in MT polymerase-incompetent mutants of CKAP5 in vivo. Indeed, CKAP5 requires both MT and F-actin binding, but not MT polymerization, to promote MT-F-actin alignment in growth cones and axon outgrowth. Taken together, our findings provide mechanistic insights into how MT populations penetrate the growth cone periphery through CKAP5-facilitated interaction with F-actin during axon outgrowth and guidance.

Department of Biology Boston College Chestnut Hill MA 02467

Department of Cell Biology Faculty of Science Charles University Prague 12800 Czech Republic

Department of Medicine Boston University Chobanian and Avedisian School of Medicine and Boston Medical Center Boston MA 02118

Department of Molecular and Cell Biology UC Berkeley Berkeley CA 94720

Institute of Biotechnology Czech Academy of Sciences BIOCEV Vestec Czech Republic 25240

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Al-Bassam J, Chang F (2011). Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP. Trends Cell Biol 21, 604–614. PubMed PMC

Al-Bassam J, Kim H, Brouhard G, van Oijen A, Harrison SC, Chang F (2010). CLASP promotes microtubule rescue by recruiting tubulin dimers to the microtubule. Dev Cell 19, 245–258. PubMed PMC

Al-Bassam J, Kim H, Flor-Parra I, Lal N, Velji H, Chang F (2012). Fission yeast Alp14 is a dose-dependent plus end-tracking microtubule polymerase. Mol Biol Cell 23, 2878–2890. PubMed PMC

Applegate KT, Besson S, Matov A, Bagonis MH, Jaqaman K, Danuser G (2011). plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics. J Struct Biol 176, 168–184. PubMed PMC

Ayaz P, Ye X, Huddleston P, Brautigam CA, Rice LM (2012). A TOG:αβ-tubulin complex structure reveals conformation-based mechanisms for a microtubule polymerase. Science 337, 857–860. PubMed PMC

Bearce EA, Erdogan B, Lowery LA (2015). TIPsy tour guides: How microtubule plus-end tracking proteins (+TIPs) facilitate axon guidance. Front Cell Neurosci 9, 241. PubMed PMC

Biswas S, Kalil K (2018). The microtubule-associated protein Tau mediates the organization of microtubules and their dynamic exploration of actin-rich Lamellipodia and filopodia of cortical growth cones. J Neurosci 38, 291–307. PubMed PMC

Brouhard GJ, Stear JH, Noetzel TL, Al-Bassam J, Kinoshita K, Harrison SC, Howard J, Hyman AA (2008). XMAP215 is a processive microtubule polymerase. Cell 132, 79–88. PubMed PMC

Byrnes AE, Slep KC (2017). TOG-tubulin binding specificity promotes microtubule dynamics and mitotic spindle formation. J Cell Biol 216, 1641–1657. PubMed PMC

Cammarata GM, Bearce EA, Lowery LA (2016). Cytoskeletal social networking in the growth cone: How +TIPs mediate microtubule-actin cross-linking to drive axon outgrowth and guidance. Cytoskeleton (Hoboken) 73, 461–476. PubMed PMC

Castoldi M, Popov AV (2003). Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr Purif 32, 83–88. PubMed

Challacombe JF, Snow DM, Letourneau PC (1997). Dynamic microtubule ends are required for growth cone turning to avoid an inhibitory guidance cue. J Neurosci 17, 3085–3095. PubMed PMC

Coles CH, Bradke F (2015). Coordinating neuronal actin-microtubule dynamics. Curr Biol 25, R677–691. PubMed

Engel U, Zhan Y, Long JB, Boyle SN, Ballif BA, Dorey K, Gygi SP, Koleske AJ, Vanvactor D (2014). Abelson phosphorylation of CLASP2 modulates its association with microtubules and actin. Cytoskeleton (Hoboken) 71, 195–209. PubMed PMC

Flor-Parra I, Iglesias-Romero AB, Chang F (2018). The XMAP215 ortholog Alp14 promotes microtubule nucleation in fission yeast. Curr Biol 28, 1681–1691.e4. PubMed PMC

Fox JC, Howard AE, Currie JD, Rogers SL, Slep KC (2014). The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array. Mol Biol Cell 25, 2375–2392. PubMed PMC

Gell C, Friel CT, Borgonovo B, Drechsel DN, Hyman AA, Howard J (2011). Purification of tubulin from porcine brain. Methods Mol Biol 777, 15–28. PubMed

Geraldo S, Gordon-Weeks PR (2009). Cytoskeletal dynamics in growth-cone steering. J Cell Sci 122, 3595–3604. PubMed PMC

Geraldo S, Khanzada UK, Parsons M, Chilton JK, Gordon-Weeks PR (2008). Targeting of the F-actin-binding protein drebrin by the microtubule plus-tip protein EB3 is required for neuritogenesis. Nat Cell Biol 10, 1181–1189. PubMed

Gunzelmann J, Rüthnick D, Lin TC, Zhang W, Neuner A, Jäkle U, Schiebel E (2018). The microtubule polymerase Stu2 promotes oligomerization of the γ-TuSC for cytoplasmic microtubule nucleation. Elife 7:e39932. PubMed PMC

Higaki T, Akita K, Katoh K (2020). Coefficient of variation as an image-intensity metric for cytoskeleton bundling. Sci Rep 10, 22187. PubMed PMC

Hur EM, Saijilafu, Lee BD, Kim SJ, Xu WL, Zhou FQ (2011). GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules. Genes Dev 25, 1968–1981. PubMed PMC

Kinoshita K, Noetzel TL, Pelletier L, Mechtler K, Drechsel DN, Schwager A, Lee M, Raff JW, Hyman AA (2005). Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis. J Cell Biol 170, 1047–1055. PubMed PMC

Lee AC, Suter DM (2008). Quantitative analysis of microtubule dynamics during adhesion-mediated growth cone guidance. Dev Neurobiol 68, 1363–1377. PubMed PMC

Lee H, Engel U, Rusch J, Scherrer S, Sheard K, Van Vactor D (2004). The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance. Neuron 42, 913–926. PubMed

Letourneau PC (1983). Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos. J Cell Biol 97, 963–973. PubMed PMC

Lowery LA, Faris AE, Stout A, Van Vactor D (2012). Neural explant cultures from Xenopus laevis. J Vis Exp e4232. PubMed PMC

Lowery LA, Stout A, Faris AE, Ding L, Baird MA, Davidson MW, Danuser G, Van Vactor D (2013). Growth cone-specific functions of XMAP215 in restricting microtubule dynamics and promoting axonal outgrowth. Neural Dev 8, 22. PubMed PMC

Lowery LA, Van Vactor D (2009). The trip of the tip: Understanding the growth cone machinery. Nat Rev Mol Cell Biol 10, 332–343. PubMed PMC

Marx A, Godinez WJ, Tsimashchuk V, Bankhead P, Rohr K, Engel U (2013). Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules. Mol Biol Cell 24, 1544–1558. PubMed PMC

Mortuza GB, Cavazza T, Garcia-Mayoral MF, Hermida D, Peset I, Pedrero JG, Merino N, Blanco FJ, Lyngsø J, Bruix M, et al. (2014). XTACC3-XMAP215 association reveals an asymmetric interaction promoting microtubule elongation. Nat Commun 5, 5072. PubMed PMC

Neukirchen D, Bradke F (2011). Cytoplasmic linker proteins regulate neuronal polarization through microtubule and growth cone dynamics. J Neurosci 31, 1528–1538. PubMed PMC

Nieuwkoop PD, Faber J (1994). Normal Table of Xenopus Laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg Till the End of Metamorphosis. New York: Garland Pub.

Nithianantham S, Cook BD, Beans M, Guo F, Chang F, Al-Bassam J (2018). Structural basis of tubulin recruitment and assembly by microtubule polymerases with tumor overexpressed gene (TOG) domain arrays. Elife 7:e38922. PubMed PMC

Nwagbara BU, Faris AE, Bearce EA, Erdogan B, Ebbert PT, Evans MF, Rutherford EL, Enzenbacher TB, Lowery LA (2014). TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types. Mol Biol Cell 25, 3350–3362. PubMed PMC

O'Brien LL, Albee AJ, Liu L, Tao W, Dobrzyn P, Lizarraga SB, Wiese C (2005). The Xenopus TACC homologue, maskin, functions in mitotic spindle assembly. Mol Biol Cell 16, 2836–2847. PubMed PMC

Prokop A (2013). The intricate relationship between microtubules and their associated motor proteins during axon growth and maintenance. Neural Dev 8, 17. PubMed PMC

Sabo J, Zdimalova MD, Slater PG, Dostal V, Herynek S, Libusova L, Lowery LA, Braun M, Lansky Z (2024). CKAP5 enables formation of persistent actin bundles templated by dynamically instable microtubules. Curr Biol 34, 260–272.e7. PubMed PMC

Schaefer AW, Kabir N, Forscher P (2002). Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones. J Cell Biol 158, 139–152. PubMed PMC

Sive HL, Grainger RM, Harland RM (2010). Microinjection of Xenopus oocytes. Cold Spring Harb Protoc 2010, pdb.prot5536. PubMed

Slater PG, Cammarata GM, Samuelson AG, Magee A, Hu Y, Lowery LA (2019). XMAP215 promotes microtubule–F-actin interactions to regulate growth cone microtubules during axon guidance in Xenopus laevis. J Cell Sci 132, jcs224311. PubMed PMC

Srivastava J, Barber D (2008). Actin co-sedimentation assay; for the analysis of protein binding to F-actin. J Vis Exp 28, 690. PubMed PMC

Thakur HC, Singh M, Nagel-Steger L, Kremer J, Prumbaum D, Fansa EK, Ezzahoini H, Nouri K, Gremer L, Abts A, et al. (2014). The centrosomal adaptor TACC3 and the microtubule polymerase chTOG interact via defined C-terminal subdomains in an Aurora-A kinase-independent manner. J Biol Chem 289, 74–88. PubMed PMC

Thawani A, Kadzik RS, Petry S (2018). XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex. Nat Cell Biol, 20(5), 575–585. PubMed PMC

van der Vaart B, Manatschal C, Grigoriev I, Olieric V, Gouveia SM, Bjelic S, Demmers J, Vorobjev I, Hoogenraad CC, Steinmetz MO, Akhmanova A (2011). SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase. J Cell Biol 193, 1083–1099. PubMed PMC

Van Troys M, Vandekerckhove J, Ampe C (1999). Structural modules in actin-binding proteins: Towards a new classification. Biochim Biophys Acta 1448, 323–348. PubMed

Vitriol EA, Zheng JQ (2012). Growth cone travel in space and time: The cellular ensemble of cytoskeleton, adhesion, and membrane. Neuron 73, 1068–1081. PubMed PMC

Wang HW, Nogales E (2005). Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly. Nature 435, 911–915. PubMed PMC

Widlund PO, Stear JH, Pozniakovsky A, Zanic M, Reber S, Brouhard GJ, Hyman AA, Howard J (2011). XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region. Proc Natl Acad Sci U S A 108, 2741–2746. PubMed PMC

Zanic M, Widlund PO, Hyman AA, Howard J (2013). Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels. Nat Cell Biol 15, 688–693. PubMed

Zhou FQ, Cohan CS (2004). How actin filaments and microtubules steer growth cones to their targets. J Neurobiol 58, 84–91. PubMed