Sheeman Brina, Carvalho Pedro, Sagot Isabelle, Geiser John, Kho David, Hoyt M Andrew, Pellman David
Department of Pediatric Oncology, The Dana Farber Cancer Institute, The Children's Hospital, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA.
Curr Biol. 2003 Mar 4;13(5):364-72. doi: 10.1016/s0960-9822(03)00013-7.
During anaphase in budding yeast, dynein inserts the mitotic spindle across the neck between mother and daughter cells. The mechanism of dynein-dependent spindle positioning is thought to involve recruitment of dynein to the cell cortex followed by capture of astral microtubules (aMTs).
We report the native-level localization of the dynein heavy chain and characterize the effects of mutations in dynein regulators on its intracellular distribution. Budding yeast dynein displays discontinuous localization along aMTs, with enrichment at the spindle pole body and aMT plus ends. Loss of Bik1p (CLIP-170), the cargo binding domain of Bik1p, or Pac1p (LIS1) resulted in diminished targeting of dynein to aMTs. By contrast, loss of dynactin or a mutation in the second P loop domain of dynein resulted in an accumulation of dynein on the plus ends of aMTs. Unexpectedly, loss of Num1p, a proposed dynein cortical anchor, also resulted in selective accumulation of dynein on the plus ends of anaphase aMTs.
We propose that, rather than first being recruited to the cell cortex, dynein is delivered to the cortex on the plus ends of polymerizing aMTs. Dynein may then undergo Num1p-dependent activation and transfer to the region of cortical contact. Based on the similar effects of loss of Num1p and loss of dynactin on dynein localization, we suggest that Num1p might also enhance dynein motor activity or processivity, perhaps by clustering dynein motors.
在出芽酵母的后期，动力蛋白将有丝分裂纺锤体插入母细胞和子细胞之间的颈部。动力蛋白依赖的纺锤体定位机制被认为涉及动力蛋白被招募到细胞皮层，随后捕获星体微管（aMTs）。
我们报道了动力蛋白重链的天然水平定位，并表征了动力蛋白调节因子中的突变对其细胞内分布的影响。出芽酵母动力蛋白沿aMTs呈现不连续定位，在纺锤极体和aMT正端富集。Bik1p（CLIP - 170）、Bik1p的货物结合结构域或Pac1p（LIS1）的缺失导致动力蛋白对aMTs的靶向减少。相比之下，动力蛋白激活蛋白复合体的缺失或动力蛋白第二个P环结构域中的突变导致动力蛋白在aMT正端积累。出乎意料的是，Num1p（一种假定的动力蛋白皮层锚定蛋白）的缺失也导致后期aMT正端动力蛋白的选择性积累。
我们提出，动力蛋白不是首先被招募到细胞皮层，而是在聚合的aMT正端被递送到皮层。然后动力蛋白可能经历Num1p依赖的激活并转移到皮层接触区域。基于Num1p缺失和动力蛋白激活蛋白复合体缺失对动力蛋白定位的相似影响，我们认为Num1p可能还会增强动力蛋白的运动活性或持续运动能力，也许是通过聚集动力蛋白分子。
