Beaven, Robin and Dzhindzhev, Nikola S. and Qu, Yue and Hahn, Ines and Dajas-Bailador, Federico and Ohkura, Hiroyuki and Prokop, Anderas (2015) Drosophila CLIP-190 and mammalian CLIP-170 display

Axons act like cables, electrically wiring the nervous system. Polar bundles of microtubules (MTs) form their backbones and drive their growth. Plus end–tracking proteins (+TIPs) regulate MT growth dynamics and directionality at their plus ends. However, current knowledge about +TIP functions, mostly derived from work in vitro and in nonneuronal cells, may not necessarily apply to the very different context of axonal MTs. For example, the CLIP family of +TIPs are known MT polymerization promoters in nonneuronal cells. However, we show here that neither Drosophila CLIP-190 nor mammalian CLIP-170 is a prominent MT plus end tracker in neurons, which we propose is due to low plus end affinity of the CAP-Gly domain–containing N-terminus and intramolecular inhibition through the C-terminus. Instead, both CLIP-190 and CLIP-170 form F-actin–dependent patches in growth cones, mediated by binding of the coiled-coil domain to myosin-VI. Because our loss-of-function analyses in vivo and in culture failed to reveal axonal roles for CLIP-190, even in double-mutant combinations with four other +TIPs, we propose that CLIP-190 and -170 are not essential axon extension regulators. Our findings demonstrate that +TIP functions known from nonneuronal cells do not necessarily apply to the regulation of the very distinct MT networks in axons. INTRODUCTION Axons are the cable-like neuronal protrusions that electrically wire the nervous system. The structural backbones of axons are polar bundles of microtubules (MTs) oriented with their plus ends toward the distal axon tips. MTs grow and shrink at their plus ends, and their positive net polymerization is believed to drive MT bundle extension and hence axon growth (Conde and Caceres, 2009; Dent et al., 2011; Prokop, 2013; Prokop et al., 2013). End-binding proteins (EBs) directly bind to MT plus ends and recruit other plus end–tracking proteins (+TIPs) to this position (Akhmanova and Steinmetz, 2008, 2010; Etienne-Manneville, 2010). These various +TIPs are proposed to determine MT growth, shrinkage, targeting, and stability. However, experimental support for such roles of +TIPs during axon growth remains patchy, not least due to the fact that different +TIP members display functional redundancies. The +TIPs are evolutionarily well conserved, and the fruit fly Drosophila provides powerful means for their study, enormously facilitated by low genetic redundancy and high amenability to genetic manipulations (Sánchez-Soriano et al., 2007; Prokop et al., 2013). Roles for some fly +TIPs during axon growth have been demonstrated. For example, Drosophila cytoplasmic linker-associated protein (dCLASP) acts in axonal midline guidance (Lee et al., 2004; Lowery et al., 2010), and fly EB1 promotes axon growth in part through evolutionarily conserved MT-organizing functions of Monitoring Editor Erika Holzbaur University of Pennsylvania Received: Jun 12, 2014 Revised: Jan 5, 2015 Accepted: Feb 10, 2015 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E14-06-1083) on February 18, 2015. Present addresses: *Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom; †Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom; ‡School of Biomedical Sciences, University of Nottingham, Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom. The authors declare no competing financial interests. Address correspondence to: Andreas Prokop (Andreas.Prokop@manchester .ac.uk). © 2015 Beaven et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: CLIP, cytoplasmic linker protein; EB, end-binding protein; GC, growth cone; MT, microtubule; +TIP, microtubule plus end–tracking protein. http://www.molbiolcell.org/content/suppl/2015/02/16/mbc.E14-06-1083v1.DC1. html Supplemental Material can be found at: