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.

译文

细胞质的动力蛋白部分是通过推动肌动蛋白的细胞骨架,将短的微管沿轴突向下运输。最近的研究表明,类似的动力蛋白驱动力可能会作用于轴突内更长的微管上。在这里,我们检查了这些力在小干扰RNA导致部分消除了动力蛋白重链(DHC)的神经元中对轴突回缩和生长锥转向的潜在作用。尽管DHC耗尽的轴突以正常速度生长,但它们对一氧化氮供体的反应比对对照轴突的撤回作用要强得多,并且它们的生长锥无法响应底物边界而有效地转向。动态微管尖端的活细胞成像显示,DHC耗尽的生长锥中的微管主要局限在中心区域,很少延伸到丝状伪足。即使在微管动力学受到抑制的条件下,DHC的消耗也会削弱微管进入生长锥外围区域的能力,这表明动力蛋白驱动力对微管的分布起着直接作用。这些作用都通过抑制肌球蛋白II力而逆转,肌球蛋白II力是轴突收缩过程中肌动蛋白在生长锥中逆行流动和皮质肌动蛋白收缩力的基础。我们的结果与动力蛋白驱动力使微管克服轴突轴和生长锥内肌球蛋白II驱动力的模型相符。这些由达因蛋白驱动的力与轴突收缩的趋势相反,并允许微管前进到生长锥的外围区域,因此它们可以侵入丝状伪足。

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