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Asymmetric endocytosis and remodeling of β1-integrin adhesions during growth cone chemorepulsion by MAG

Nature Neuroscience volume 13, pages 829–837 (2010)Cite this article

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Abstract

Gradients of chemorepellent factors released from myelin may impair axon pathfinding and neuroregeneration after injury. We found that, analogously to the process of chemotaxis in invasive tumor cells, axonal growth cones of Xenopus spinal neurons modulate the functional distribution of integrin receptors during chemorepulsion induced by myelin-associated glycoprotein (MAG). A focal MAG gradient induced polarized endocytosis and concomitant asymmetric loss of β1-integrin and vinculin-containing adhesions on the repellent side during repulsive turning. Loss of symmetrical β1-integrin function was both necessary and sufficient for chemorepulsion, which required internalization by clathrin-mediated endocytosis. Induction of repulsive Ca2+ signals was necessary and sufficient for the stimulated rapid endocytosis of β1-integrin. Altogether, these findings identify β1-integrin as an important functional cargo during Ca2+-dependent rapid endocytosis stimulated by a diffusible guidance cue. Such dynamic redistribution allows the growth cone to rapidly adjust adhesiveness across its axis, an essential feature for initiating chemotactic turning.

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Figure 1: MAG-gradient stimulated asymmetric membrane internalization.
Figure 2: MAG stimulated endocytosis of β1-integrin.
Figure 3: Polarized redistribution of ECM adhesion proteins by a MAG gradient.
Figure 4: Polarized β1-integrin function mediates growth cone repulsion by MAG.
Figure 5: MAG- induced surface removal of β1-integrin.
Figure 6: Cytoplasmic Ca2+ regulates endocytosis of β1-integrin.
Figure 7: MAG-induced surface removal of β1-integrin and repulsion require clathrin-mediated endocytosis.

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Acknowledgements

We thank K. Yamada (US National Institutes of Health), D. DeSimone (University of Virginia) and M. McNiven (Mayo Clinic) for generous gifts of antibodies to β1-integrin, α5-integrin and dynamin (MC64 control rabbit IgG), respectively. The pFLAG-CMV2-AP180-C plasmid was from M. McNiven (Mayo Clinic) and the pCS2+RFP plasmid (Addgene) was from R. Moon (University of Washington). We also thank R. Muthu for assistance with β1 Fab purification; T. Gomez, B. Horazdovsky, C. Howe, M. McNiven, R. Pagano and members of the Henley group for critical comments; A. Windebank for sharing laboratory space at the beginning of these studies; and J. Tarara, S. Henle, E. Liang, J. Nesbitt, Z. Chen, B.-Y. Lin, V. Petrov and D. Triner for technical assistance. This work was supported by a John M. Nasseff, Sr., Career Development Award in Neurologic Surgery Research from the Mayo Clinic (J.R.H.) and career development funds from the Craig Neilsen Foundation (J.R.H.). A Robert D. and Patricia E. Kern Predoctoral Fellowship award supported J.H.H.

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  1. Mohammad Abu-Rub

    Present address: Present address: Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland.,

Authors and Affiliations

  1. Mayo Graduate School, Mayo Clinic, Rochester, Minnesota, USA

    Jacob H Hines

  2. Department of Neurologic Surgery, Mayo Clinic, Rochester, Minnesota, USA

    Mohammad Abu-Rub & John R Henley

  3. Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, USA

    John R Henley

Authors
  1. Jacob H Hines
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  2. Mohammad Abu-Rub
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Contributions

J.H.H. and J.R.H. conceived the project and designed experiments; J.H.H., M.A.-R. and J.R.H. performed experiments, analyzed data and wrote the paper; J.R.H. supervised the project.

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Correspondence to John R Henley.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 5660 kb)

Supplementary Video 1

Representative time-lapse images of FM-dye endocytosis in unstimulated growth cones. A microscopic gradient of control solution (medium + BSA vehicle) was applied to the growth cone from a micropipette positioned 90° relative to the direction of axon extension (see left arrow in frame 1). After 5 min, confocal imaging was initiated (1 Hz; t = 5:00). The growth cone surface membrane was then transiently labeled by a focal pulse of FM 5-95 (apparent in frame 2, t = 5:01) using a second micropipette positioned 100 μm in front of the leading edge of the growth cone (see top arrow in frame 1). Each video represents a 25–30 s imaging period. Time frames (min:s) are indicated at the top left. Video 1 corresponds to Fig. 1a. Scale bars, 5 μm. (MOV 2015 kb)

Supplementary Video 2

Representative time-lapse images of FM-dye endocytosis in unstimulated growth cones. A microscopic gradient of control solution (medium + BSA vehicle) was applied to the growth cone from a micropipette positioned 90° relative to the direction of axon extension (see left arrow in frame 1). After 5 min, confocal imaging was initiated (1 Hz; t = 5:00). The growth cone surface membrane was then transiently labeled by a focal pulse of FM 5-95 (apparent in frame 2, t = 5:01) using a second micropipette positioned 100 μm in front of the leading edge of the growth cone (see top arrow in frame 1). Each video represents a 25–30 s imaging period. Time frames (min:s) are indicated at the top left. Scale bars, 5 μm. (MOV 1655 kb)

Supplementary Video 3

Representative time-lapse images of fluorescent dextran internalization in unstimulated growth cones. Confocal images show examples of Xenopus spinal neuron growth cones internalizing the fluid phase endocytosis marker tetramethylrhodamine-dextran, which was focally applied as a pulse from a micropipette positioned 80 μm in front of the growth cone. Uninternalized dextran was rapidly washed away during imaging using a second micropipette containing imaging saline (see schematic and Online Methods). Bright-field images were captured before applying the dextran pulse and time (s) following the application is denoted in fluorescence images. Pseudocolor represents higher (white) and lower (blue) fluorescence intensities. Macroendocytic structures were typically internalized in the growth cone periphery and moved retrogradely to the central domain within minutes, often fusing with one another. Scale bars, 5 μm. (MOV 2829 kb)

Supplementary Video 4

Representative time-lapse images of fluorescent dextran internalization in unstimulated growth cones. Confocal images show examples of Xenopus spinal neuron growth cones internalizing the fluid phase endocytosis marker tetramethylrhodamine-dextran, which was focally applied as a pulse from a micropipette positioned 80 μm in front of the growth cone. Uninternalized dextran was rapidly washed away during imaging using a second micropipette containing imaging saline (see schematic and Online Methods). Bright-field images were captured before applying the dextran pulse and time (s) following the application is denoted in fluorescence images. Pseudocolor represents higher (white) and lower (blue) fluorescence intensities. Macroendocytic structures were typically internalized in the growth cone periphery and moved retrogradely to the central domain within minutes, often fusing with one another. Video 4 corresponds to Supplementary Fig. 1a. Scale bars, 5 μm. (MOV 3701 kb)

Supplementary Video 5

Representative time-lapse images of fluorescent dextran internalization in unstimulated growth cones. Confocal images show examples of Xenopus spinal neuron growth cones internalizing the fluid phase endocytosis marker tetramethylrhodamine-dextran, which was focally applied as a pulse from a micropipette positioned 80 μm in front of the growth cone. Uninternalized dextran was rapidly washed away during imaging using a second micropipette containing imaging saline (see schematic and Online Methods). Bright-field images were captured before applying the dextran pulse and time (s) following the application is denoted in fluorescence images. Pseudocolor represents higher (white) and lower (blue) fluorescence intensities. Macroendocytic structures were typically internalized in the growth cone periphery and moved retrogradely to the central domain within minutes, often fusing with one another. Scale bars, 5 μm. (MOV 1631 kb)

Supplementary Video 6

Representative time-lapse images of asymmetric FM-dye endocytosis in the growth cone induced by a MAG gradient. These experiments were carried out as in Videos 1 and 2 except that a continuous MAG gradient was applied to each growth cone in place of a control gradient. The membrane dye FM 5-95 was focally applied using a second micropipette positioned at the front of the growth cone. “Hot spots” of endocytosis (white arrows) were more prevalent on the side of the growth cone nearest the MAG pipette (left, see schematic in frame 1). Video 6 corresponds to Fig. 1b. Scale bars, 5 μm. (MOV 1569 kb)

Supplementary Video 7

Representative time-lapse images of asymmetric FM-dye endocytosis in the growth cone induced by a MAG gradient. These experiments were carried out as in Videos 1 and 2 except that a continuous MAG gradient was applied to each growth cone in place of a control gradient. The membrane dye FM 5-95 was focally applied using a second micropipette positioned at the front of the growth cone. “Hot spots” of endocytosis (white arrows) were more prevalent on the side of the growth cone nearest the MAG pipette (left, see schematic in frame 1). Video 7 corresponds to Fig. 1f. Scale bars, 5 μm. (MOV 1288 kb)

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Hines, J., Abu-Rub, M. & Henley, J. Asymmetric endocytosis and remodeling of β1-integrin adhesions during growth cone chemorepulsion by MAG. Nat Neurosci 13, 829–837 (2010). https://doi.org/10.1038/nn.2554

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