Abstract
BackgroundMicrotubule (MT) regulators play essential roles in multiple aspects of neural development. In vitro reconstitution assays have established that the XMAP215/Dis1/TOG family of MT regulators function as MT ‘plus-end-tracking proteins’ (+TIPs) that act as processive polymerases to drive MT growth in all eukaryotes, but few studies have examined their functions in vivo. In this study, we use quantitative analysis of high-resolution live imaging to examine the function of XMAP215 in embryonic Xenopus laevis neurons.ResultsHere, we show that XMAP215 is required for persistent axon outgrowth in vivo and ex vivo by preventing actomyosin-mediated axon retraction. Moreover, we discover that the effect of XMAP215 function on MT behavior depends on cell type and context. While partial knockdown leads to slower MT plus-end velocities in most cell types, it results in a surprising increase in MT plus-end velocities selective to growth cones. We investigate this further by using MT speckle microscopy to determine that differences in overall MT translocation are a major contributor of the velocity change within the growth cone. We also find that growth cone MT trajectories in the XMAP215 knockdown (KD) lack the constrained co-linearity that normally results from MT-F-actin interactions.ConclusionsCollectively, our findings reveal unexpected functions for XMAP215 in axon outgrowth and growth cone MT dynamics. Not only does XMAP215 balance actomyosin-mediated axon retraction, but it also affects growth cone MT translocation rates and MT trajectory colinearity, all of which depend on regulated linkages to F-actin. Thus, our analysis suggests that XMAP215 functions as more than a simple MT polymerase, and that in both axon and growth cone, XMAP215 contributes to the coupling between MTs and F-actin. This indicates that the function and regulation of XMAP215 may be significantly more complicated than previously appreciated, and points to the importance of future investigations of XMAP215 function during MT and F-actin interactions.
Highlights
Microtubule (MT) regulators play essential roles in multiple aspects of neural development
XMAP215 prevents spontaneous actomyosin-mediated axon retraction To investigate the function of XMAP215 during vertebrate nervous system development, we inhibited its translation in Xenopus laevis embryos by utilizing an antisense morpholino oligonucleotide (MO) (Figure 1A)
While it was previously reported that axon outgrowth requires the mammalian ortholog, ch-TOG [12], here we provide unanticipated insights into XMAP215 function by using quantitative analysis of high-resolution timelapse imaging of Xenopus laevis embryonic neurons
Summary
Microtubule (MT) regulators play essential roles in multiple aspects of neural development. We use quantitative analysis of high-resolution live imaging to examine the function of XMAP215 in embryonic Xenopus laevis neurons. MTs, in particular, play a significant role in the neuronal growth cone during axon outgrowth [2]. They are necessary for axon elongation, axonal transport, and accurate steering of the growth cone. Despite their importance, only a few studies have single-molecule imaging combined with structurefunction analyses have provided useful insights into the mechanism by which XMAP215 catalyzes MT polymerization in vitro [10,11]. Our data suggests that XMAP215 functions as more than a simple MT polymerase and is likely involved in the coupling of MT-F-actin linkages
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