EBs clip CLIPs to growing microtubule ends

Proteins that track growing microtubule (MT) ends are important for many aspects of intracellular MT function, but the mechanism by which these +TIPs accumulate at MT ends has been the subject of a long-standing controversy. In this issue, Bieling et al. (Bieling, P., S. Kandels-Lewis, I.A. Telley, J. van Dijk, C. Janke, and T. Surrey. 2008. J. Cell Biol. 183:1223–1233) reconstitute plus end tracking of EB1 and CLIP-170 in vitro, which demonstrates that CLIP-170 plus end tracking is EB1-dependent and that both +TIPs rapidly exchange between a soluble and a plus end–associated pool. This strongly supports the hypothesis that plus end tracking depends on a biochemical property of growing MT ends, and that the characteristic +TIP comets result from the generation of new +TIP binding sites through MT polymerization in combination with the exponential decay of these binding sites

EBs clip CLIPs to growing microtubule ends

Proteins that track growing microtubule (MT) ends are important for many aspects of intracellular MT function, but the mechanism by which these +TIPs accumulate at MT ends has been the subject of a long-standing controversy. In this issue, Bieling et al. (Bieling, P., S. Kandels-Lewis, I.A. Telley, J. van Dijk, C. Janke, and T. Surrey. 2008. J. Cell Biol. 183:1223–1233) reconstitute plus end tracking of EB1 and CLIP-170 in vitro, which demonstrates that CLIP-170 plus end tracking is EB1-dependent and that both +TIPs rapidly exchange between a soluble and a plus end–associated pool. This strongly supports the hypothesis that plus end tracking depends on a biochemical property of growing MT ends, and that the characteristic +TIP comets result from the generation of new +TIP binding sites through MT polymerization in combination with the exponential decay of these binding sites

Spatial regulation of CLASP affinity for microtubules by Rac1 and GSK3β in migrating epithelial cells

Proteins that in cells specifically bind to growing microtubule plus ends (+TIPs) are thought to play important roles in polarization of the cytoskeleton. However, most +TIPs do not show a bias of their microtubule-binding behavior toward different subcellular regions. Here, we examine the dynamics of the +TIP CLASP in migrating PtK1 epithelial cells. We find that, although CLASPs track microtubule plus ends in the cell body, they dynamically decorate the entire microtubule lattice in the leading edge lamella and lamellipodium. Microtubule lattice binding is mediated by the COOH-terminal region of the CLASP microtubule-binding domain and is regulated downstream of Rac1. Phosphorylation of sites in the NH2-terminal part of the microtubule-binding domain by glycogen synthase kinase 3β likely regulates the affinity of CLASPs for microtubule lattices. These results demonstrate the striking difference of the microtubule cytoskeleton in the lamella as compared with the cell body and provide the first direct observation of subcellular regulation of a microtubule-associated protein in migrating cells

Regulation of leading edge microtubule and actin dynamics downstream of Rac1

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, “pioneer” MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells

Localization of the Kinesin-like Protein Xklp2 to Spindle Poles Requires a Leucine Zipper, a Microtubule-associated Protein, and Dynein

Xklp2 is a plus end–directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49–59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein–dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends

Neuronal deletion of GSK3beta increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2

BACKGROUND: In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity. RESULTS: Following a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3β (GSK3β) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3β regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3β activity in neurons. Following spinal cord injury, reduced GSK3β levels or complete neuronal deletion of GSK3β led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3β substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3β-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3β-CRMP-2 relevance during axon regeneration. CONCLUSIONS: Our work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3β-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth