RESUMEN
Here we studied doublecortin (DCX) in cultured hippocampal and sympathetic neurons during axonal development. In both types of neurons, DCX is abundant in the growth cone, in which it primarily localizes with microtubules. Its abundance is lowest on microtubules in the neck region of the growth cone and highest on microtubules extending into the actin-rich lamellar regions. Interestingly, the microtubule polymer richest in DCX is also deficient in tau. In hippocampal neurons but not sympathetic neurons, discrete focal patches of microtubules rich in DCX and deficient in tau are present along the axonal shaft. Invariably, these patches have actin-rich protrusions resembling those of growth cones. Many of the DCX/actin filament patches exhibit vigorous protrusive activity and also undergo a proximal-to-distal redistribution within the axon at average rates approximately 2 microm/min and thus closely resemble the growth-cone-like waves described by previous authors. Depletion of DCX using small interfering RNA had little effect on the appearance of the growth cone or on axonal growth in either type of neuron. However, DCX depletion significantly delayed collateral branching in hippocampal neurons and also significantly lowered the frequency of actin-rich patches along hippocampal axons. Branching by sympathetic neurons, which occurs by growth cone splitting, was not impaired by DCX depletion. These findings reveal a functional relationship between the DCX/actin filament patches and collateral branching. Based on the striking resemblance of these patches to growth cones, we discuss the possibility that they reflect a mechanism for locally boosting morphogenetic activity to facilitate axonal growth and collateral branching.
Asunto(s)
Actinas/metabolismo , Axones/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Actinas/fisiología , Animales , Axones/química , Axones/fisiología , Células Cultivadas , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/química , Microtúbulos/fisiología , Neuropéptidos/fisiología , RatasRESUMEN
Cytoplasmic dynein transports short microtubules down the axon in part by pushing against the actin cytoskeleton. Recent studies have suggested that comparable dynein-driven forces may impinge upon the longer microtubules within the axon. Here, we examined a potential role for these forces on axonal retraction and growth cone turning in neurons partially depleted of dynein heavy chain (DHC) by small interfering RNA. While DHC-depleted axons grew at normal rates, they retracted far more robustly in response to donors of nitric oxide than control axons, and their growth cones failed to efficiently turn in response to substrate borders. Live cell imaging of dynamic microtubule tips showed that microtubules in DHC-depleted growth cones were largely confined to the central zone, with very few extending into filopodia. Even under conditions of suppressed microtubule dynamics, DHC depletion impaired the capacity of microtubules to advance into the peripheral zone of the growth cone, indicating a direct role for dynein-driven forces on the distribution of the microtubules. These effects were all reversed by inhibition of myosin-II forces, which are known to underlie the retrograde flow of actin in the growth cone and the contractility of the cortical actin during axonal retraction. Our results are consistent with a model whereby dynein-driven forces enable microtubules to overcome myosin-II-driven forces, both in the axonal shaft and within the growth cone. These dynein-driven forces oppose the tendency of the axon to retract and permit microtubules to advance into the peripheral zone of the growth cone so that they can invade filopodia.
Asunto(s)
Axones/fisiología , Citoplasma/metabolismo , Dineínas/metabolismo , Conos de Crecimiento/metabolismo , Miosina Tipo II/metabolismo , Actinas/metabolismo , Animales , Axones/ultraestructura , Movimiento Celular/fisiología , Forma de la Célula , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Conos de Crecimiento/efectos de los fármacos , Laminina/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas/citología , Óxido Nítrico/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Moduladores de Tubulina/farmacología , Vinblastina/farmacologíaRESUMEN
Microtubule-associated-protein 1b (MAP1b) is abundant in neurons actively extending axons. MAP1b is present on microtubules throughout growing axons, but is preferentially concentrated on microtubule polymer in the distal axon and growth cone. Although MAP1b has been implicated in axon growth and pathfinding, its specific functions are not well understood. Biochemical and transfection studies suggest that MAP1b has microtubule-stabilizing activity, but recent studies with neurons genetically deficient in MAP1b have not confirmed this. We have explored MAP1b functions in growing sympathetic neurons using an acute inactivation approach. Neurons without axons were injected with polyclonal MAP1b antibodies and then stimulated to extend axons. Injected cells were compared to controls in terms of axon growth behavior and several properties of axonal microtubules. The injected antibodies rapidly and quantitatively sequestered MAP1b in the cell body, making it unavailable to perform its normal functions. This immunodepletion of MAP1b had no statistically significant effect on axon growth, the amount of microtubule polymer in the axon, and the relative tyrosinated tubulin content of this polymer, and this was true in sympathetic neurons from rat, wild type mice, and tau knockout mice. Thus, robust axon growth can occur in the absence of MAP1b alone or both MAP1b and tau. However, immunodepletion of MAP1b significantly increased the sensitivity of microtubules in the distal axon and growth cone to nocodazole-induced depolymerization. These results indicate that MAP1b has microtubule-stabilizing activity in growing axons. This stabilizing activity may be required for some axonal functions, but it is not necessary for axon growth.