Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to α-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.

Interactions between dynamic microtubules and actin filaments (F-actin) underlie a range of cellular processes including cell polarity and motility. In growth cones, dynamic microtubules are continually extending into selected filopodia, aligning alongside the proximal ends of the F-actin bundles. This interaction is essential for neuritogenesis and growth-cone pathfinding. However, the molecular components mediating the interaction between microtubules and filopodial F-actin have yet to be determined. Here we show that drebrin, an F-actin-associated protein, binds directly to the microtubule-binding protein EB3. In growth cones, this interaction occurs specifically when drebrin is located on F-actin in the proximal region of filopodia and when EB3 is located at the tips of microtubules invading filopodia. When this interaction is disrupted, the formation of growth cones and the extension of neurites are impaired. We conclude that drebrin targets EB3 to coordinate F-actin-microtubule interactions that underlie neuritogenesis.

Interactions between dynamic microtubules and actin filaments are essential to a wide range of cell biological processes including cell division, motility and morphogenesis. In neuronal growth cones, interactions between microtubules and actin filaments in filopodia are necessary for growth cones to make a turn. Growth-cone turning is a fundamental behaviour during axon guidance, as correct navigation of the growth cone through the embryo is required for it to locate an appropriate synaptic partner. Microtubule-actin filament interactions also occur in the transition zone and central domain of the growth cone, where actin arcs exert compressive forces to corral microtubules into the core of the growth cone and thereby facilitate microtubule bundling, a requirement for axon formation. We now have a fairly comprehensive understanding of the dynamic behaviour of the cytoskeleton in growth cones, and the stage is set for discovering the molecular machinery that enables microtubule-actin filament coupling in growth cones, as well as the intracellular signalling pathways that regulate these interactions. Furthermore, recent experiments suggest that microtubule-actin filament interactions might also be important for the formation of dendritic spines from filopodia in mature neurons. Therefore, the mechanisms coupling microtubules to actin filaments in growth-cone turning and dendritic-spine maturation might be conserved.

Recent experiments show that the microtubule-associated protein (MAP) 1B is a major phosphorylation substrate for the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β) in differentiating neurons. GSK-3β phosphorylation of MAP1B appears to act as a molecular switch regulating the control that MAP1B exerts on microtubule dynamics in growing axons and growth cones. Maintaining a population of dynamically unstable microtubules in growth cones is important for axon growth and growth cone pathfinding. We have mapped two GSK-3β phosphorylation sites on mouse MAP1B to Ser1260 and Thr1265 using site-directed point mutagenesis of recombinant MAP1B proteins, in vitro kinase assays and phospho-specific antibodies. We raised phospho-specific polyclonal antibodies to these two sites and used them to show that MAP1B is phosphorylated by GSK-3β at Ser1260 and Thr1265 in vivo. We also showed that in the developing nervous system of rat embryos, the expression of GSK-3β phosphorylated MAP1B is spatially restricted to growing axons, in a gradient that is highest distally, despite the expression of MAP1B and GSK-3β throughout the entire neuron. This suggests that there is a mechanism that spatially regulates the GSK-3β phosphorylation of MAP1B in differentiating neurons. Heterologous cell transfection experiments with full-length MAP1B, in which either phosphorylation site was separately mutated to a valine or, in a double mutant, in which both sites were mutated, showed that these GSK-3β phosphorylation sites contribute to the regulation of microtubule dynamics by MAP1B.