Dataset Information

Quantitative analysis of microtubule dynamics during adhesion-mediated growth cone guidance.

ABSTRACT: During adhesion-mediated neuronal growth cone guidance microtubules undergo major rearrangements. However, it is unknown whether microtubules extend to adhesion sites because of changes in plus-end polymerization and/or translocation dynamics, because of changes in actin-microtubule interactions, or because they follow the reorganization of the actin cytoskeleton. Here, we used fluorescent speckle microscopy to directly quantify microtubule and actin dynamics in Aplysia growth cones as they turn towards beads coated with the cell adhesion molecule apCAM. During the initial phase of adhesion formation, dynamic microtubules in the peripheral domain preferentially explore apCAM-beads prior to changes in growth cone morphology and retrograde actin flow. Interestingly, these early microtubules have unchanged polymerization rates but spend less time in retrograde translocation due to uncoupling from actin flow. Furthermore, microtubules exploring the adhesion site spend less time in depolymerization. During the later phase of traction force generation, the central domain advances and more microtubules in the peripheral domain extend because of attenuation of actin flow and clearance of F-actin structures. Microtubules in the transition zone and central domain, however, translocate towards the adhesion site in concert with actin arcs and bundles, respectively. We conclude that adhesion molecules guide neuronal growth cones and underlying microtubule rearrangements largely by differentially regulating microtubule-actin coupling and actin movements according to growth cone region and not by controlling plus-end polymerization rates.

SUBMITTER: Lee AC

PROVIDER: S-EPMC2745986 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Quantitative analysis of microtubule dynamics during adhesion-mediated growth cone guidance.

Lee Aih Cheun AC Suter Daniel M DM

Developmental neurobiology 20081001 12

During adhesion-mediated neuronal growth cone guidance microtubules undergo major rearrangements. However, it is unknown whether microtubules extend to adhesion sites because of changes in plus-end polymerization and/or translocation dynamics, because of changes in actin-microtubule interactions, or because they follow the reorganization of the actin cytoskeleton. Here, we used fluorescent speckle microscopy to directly quantify microtubule and actin dynamics in Aplysia growth cones as they turn ...[more]

PMID: 18698606

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Project description: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.

Project description:Guidance of axons to their proper synaptic target sites requires spatially and temporally precise modulation of biochemical signals within growth cones. Ionic calcium (Ca2+) is an essential signal for axon guidance that mediates opposing effects on growth cone motility. The diverse effects of Ca2+ arise from the precise localization of Ca2+ signals into microdomains containing specific Ca2+ effectors. For example, differences in the mechanical and chemical composition of the underlying substrata elicit local Ca2+ signals within growth cone filopodia that regulate axon guidance through activation of the protease calpain. However, how calpain regulates growth cone motility remains unclear. Here, we identify the adhesion proteins talin and focal adhesion kinase (FAK) as proteolytic targets of calpain in Xenopus laevis spinal cord neurons both in vivo and in vitro Inhibition of calpain increases the localization of endogenous adhesion signaling to growth cone filopodia. Using live cell microscopy and specific calpain-resistant point-mutants of talin (L432G) and FAK (V744G), we find that calpain inhibits paxillin-based adhesion assembly through cleavage of talin and FAK, and adhesion disassembly through cleavage of FAK. Blocking calpain cleavage of talin and FAK inhibits repulsive turning from focal uncaging of Ca2+ within filopodia. In addition, blocking calpain cleavage of talin and FAK in vivo promotes Rohon-Beard peripheral axon extension into the skin. These data demonstrate that filopodial Ca2+ signals regulate axon outgrowth and guidance through calpain regulation of adhesion dynamics through specific cleavage of talin and FAK.SIGNIFICANCE STATEMENT The proper formation of neuronal networks requires accurate guidance of axons and dendrites during development by motile structures known as growth cones. Understanding the intracellular signaling mechanisms that govern growth cone motility will clarify how the nervous system develops and regenerates, and may identify areas of therapeutic intervention in disease or injury. One important signal that controls growth cones is that of local Ca2+ transients, which control the rate and direction of axon outgrowth. We demonstrate here that Ca2+-dependent inhibition axon outgrowth and guidance is mediated by calpain proteolysis of the adhesion proteins talin and focal adhesion kinase. Our findings provide mechanistic insight into Ca2+/calpain regulation of growth cone motility and axon guidance during neuronal development.

Dataset Information

Quantitative analysis of microtubule dynamics during adhesion-mediated growth cone guidance.

Publications

Quantitative analysis of microtubule dynamics during adhesion-mediated growth cone guidance.

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