Molecular mechanisms of +TIPs in axonal extension

During development of the nervous system axons must extend to their specific target cells,such as other neurons or muscles, in order to connect into the functional networks that formthe basis of brain function. Due to the high precision required, this process is susceptible toperturbations which cause a host of neurodevelopmental disorders including forms of mentalretardation. Furthermore, many of the same mechanisms which drive axon growth arereiterated during axon regeneration following injury, stroke or degeneration.Axon extension is performed by growth cones (GCs) at the tip of the extending axons,guided to their targets through extracellular signals. Intracellularly, the cytoskeletal machinerydrives the morphogenetic processes implementing GC behaviour. Whereas F-actinpredominantly regulates the directionality of axon growth, microtubules (MTs) essentiallyimplement axon extension, and sustain the structural integrity of axons and the transportbetween soma and GCs. Therefore, an important aim of axon growth research is to understandhow MT networks are regulated. This is the overarching objective of my project.MT-binding proteins (MTBPs) interact with MTs, with one another and with F-actin,thus forming complex regulatory networks. This complexity can be tackled with efficientgenetic approaches possible in the fruit fly Drosophila, using its well established axon growthreadouts in vivo complementary to the high resolution readouts for cytoskeleton in culturedprimary neurons. I focussed my work on a specific class of MTBPs, so called +TIP, whichbind to the polymerising plus ends of MTs to regulate their dynamics, a process thatessentially contributes to MT network formation and organisation.To be able to carry out my project, I first optimised the use of the culture system. Iestablished approaches to assess MT network organisation, and MT dynamics. My initialstudies showed that fundamental properties of MT networks are highly conserved betweenvertebrates and Drosophila. I used these new approaches to contribute to ongoing work onfunctions of the actin-MT linker Shot during axon growth. I demonstrated novel roles of Shotin regulating MT polymerisation dynamics and guidance which simultaneously validated thenewly established imaging strategies. I then carried out a systematic analysis of thelocalisation patterns of different +TIPs (CLASP, CLIP, APC1, APC2, p150glued and Lis1)which I found to be highly conserved with patterns of their vertebrate homologues describedin the literature. I then used loss of function analyses identified promising axonal growthphenotypes to be used in my further study.I selected CLIP-190 as a promising candidate because of its prominent localisation inGCs and its strong mutant axon overgrowth phenotype. Well into my studies, further testsunfortunately revealed the axon growth phenotype to be due to loss of the neighbouring gene,Lrch, an actin-binding protein for which my experiments revealed a putative role in MTregulation downstream of F-actin networks. Meticulous loss of CLIP-190 analyses, includingdouble- and triple mutant combinations with all of the above +TIP members, failed to revealany neuronal phenotypes in primary neurons. Neither did CLIP-190 loss show axon growth orNMJ phenotypes in vivo. Accordingly, in neurons CLIP-190 is tethered in large patches in theGCs, via its central coiled-coil domain, and a link to myosin VI, but shows little affinity forMT plus ends, via its EB1-binding domain. Parallel studies with the close mammalianhomologue CLIP-170 showed the same localisation patterns in neurons, suggesting that CLIP-170 doesn? t play a significant role in the nervous system. Overall, my studies in Drosophilaneurons have firmly established this system for the systematic analysis of MT regulation,likely to produce mechanistic understanding that can be applied also to mammals.