Rab11 and Its Effector Rab Coupling Protein Contribute to the Trafficking of 1 Integrins during Axon Growth in Adult Dorsal Root Ganglion Neurons and PC12 Cells

Cambridge Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 09/2010; 30(35):11654-69. DOI: 10.1523/JNEUROSCI.2425-10.2010
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Integrins play an important part in axon growth, but integrin traffic in neurons is poorly understood. Expression of the tenascin-C-binding integrin alpha9 promotes axon regeneration. We have therefore studied the mechanism by which alpha9 integrin and its partner beta1 are trafficked along axons and at the growth cone using adult DRG neurons and PC12 cells. We have focused on the small GTPase Rab11 and its effector Rab coupling protein (RCP), as they are involved in the long-range trafficking of beta1 integrins in other cells. Rab11 colocalizes with alpha9 and other alpha integrins and with beta1 integrin in growth cones and axons, and immunopurified Rab11 vesicles contain alpha9 and beta1. Endocytosed beta1 integrins traffic via Rab11. However, Rab11 vesicles in axons are generally static, and alpha9 integrins undergo bouts of movement during which they leave the Rab11 compartment. In growth cones, alpha9 and beta1 overlap with RCP, particularly at the growth cone periphery. We show that beta1 integrin trafficking during neurite outgrowth involves Rab11 and RCP, and that manipulation of these molecules alters surface integrin levels and axon growth, and can be used to enhance alpha9 integrin-dependent neurite outgrowth. Our data suggest that manipulation of trafficking via Rab11 and RCP could be a useful strategy for promoting integrin-dependent axonal regeneration.

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Available from: Richard Eva, Oct 05, 2015
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    • "Stuermer). formation and cell migration (Arjonen et al., 2012; Bridgewater et al., 2012; Eva et al., 2010). Integrin and E-cadherin trafficking are also influenced by Src and FAK (Canel et al., 2013). "
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    ABSTRACT: Reggies/flotillins are implicated in trafficking of membrane proteins to their target sites and in the regulation of the Rab11a-dependent targeted recycling of E-cadherin to adherens junctions (AJs). Here we demonstrate a function of reggies in focal adhesion (FA) formation and α5- and β1-integrin recycling to FAs. Downregulation of reggie-1 in HeLa and A431 cells by siRNA and shRNA increased the number of FAs, impaired their distribution and modified FA turnover. This was coupled to enhanced focal adhesion kinase (FAK) and Rac1 signaling and gain in plasma membrane motility. Wild type and constitutively-active (CA) Rab11a rescued the phenotype (normal number of FAs) whereas dominant-negative (DN) Rab11a mimicked the loss-of-reggie phenotype in control cells. That reggie-1 affects integrin trafficking emerged from the faster loss of internalized antibody-labeled β1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and α5- or β1-integrin, trafficking close to the substrate-near membrane and making kiss-and-run contacts with FAs. Thus, reggie-1 in interaction with Rab11a controls Rac1 and FAK activation and coordinates the targeted recycling of α5- and β1-integrins to FAs to regulate FA formation and membrane dynamics. Copyright © 2015 Elsevier GmbH. All rights reserved.
    European Journal of Cell Biology 07/2015; DOI:10.1016/j.ejcb.2015.07.003 · 3.83 Impact Factor
    • "Rab11 plays an important role in connecting the membrane with actin filaments of the growth cone peripheral domain, as actin nucleator proteins are among its effectors (Schuh, 2011). Rab11 and Arf6 are necessary for local recycling of a9 and b1 integrins (Eva et al., 2010, 2012). Rab6 and Rab10 are localised on trans-Golgi membranes, and interact , respectively, with Kif1C, the kinesin motor protein, and myosin Vb, both of which are necessary for axonal outgrowth (Schlager et al., 2010; Liu et al., 2013). "
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    ABSTRACT: Peripheral axon regeneration requires surface-expanding membrane addition. The continuous incorporation of new membranes into the axolemma allows the pushing force of elongating microtubules to drive axonal growth cones forward. Hence, a constant supply of membranes and cytoskeletal building blocks is required, often for many weeks. In human peripheral nerves, axon tips may be more than one metre away from the neuronal cell body. Therefore, in the initial phase of regeneration, membranes are derived from pre-existing vesicles or synthesised locally. Only later stages of axon regeneration are supported by membranes and proteins synthesised in neuronal cell bodies, considering the fastest anterograde transport mechanisms deliver cargo at 20 cm/day. While endo- and exocytosis of membrane vesicles are balanced in intact axons, membrane incorporation exceeds membrane retrieval during regeneration to compensate for the loss of membranes distal to the lesion site. Physiological membrane turnover rates will not be established before the completion of target re-innervation. In this review, the current knowledge on membrane traffic in axon outgrowth is summarised with a focus on endosomal vesicles as the provider of membranes and carrier of growth factor receptors required for initiating signalling pathways to promote the elongation and branching of regenerating axons in lesioned peripheral nerves. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    European Journal of Neuroscience 07/2015; DOI:10.1111/ejn.13025 · 3.18 Impact Factor
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    • "However, the construct could be transfected into PC12 cells, and we therefore performed assays using these cells. We have previously performed various integrin-related neurite outgrowth experiments with the same interventions in DRG neurons and PC12 cells, and have obtained very comparable results between the two types of cells (Eva et al., 2010, 2012b). In addition to wild-type talin, we also tested the effect of two constitutively active forms of talin, talin-1 (T1767E) and talin-1 (E1770A), in which point mutations T1767E and E1770A have been introduced to abolish the self-inhibitory folding between the head and rod domains as seen in wild-type talin (Critchley, 2009; Goult et al., 2009). "
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    ABSTRACT: Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulphate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by expression of wild-type talin and more so by two 'activated' forms of talin produced by point mutation (on laminin and aggrecan-laminin substrata). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract. Copyright © 2015. Published by Elsevier Inc.
    Molecular and Cellular Neuroscience 03/2015; 68. DOI:10.1016/j.mcn.2015.03.011 · 3.84 Impact Factor
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