Peroxisome elongation and constriction but not fission can occur independently of dynamin-like protein 1
Peroxisome elongation and constriction but not fission can occur independently of dynamin-like protein 1
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DOI：
10.1242/jcs.01268
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发表时间：
2004-08-01
影响因子：
4
通讯作者：
Schrader, M
中科院分区：
文献类型：
--
作者：
Koch, A;Schneider, G;Schrader, M
哺乳动物动力样蛋白 DLP1 属于大型 GTP 酶的动力家族，该家族与细胞膜的管状和裂变事件有关。我们之前已经证明，GTP 水解缺陷的显性失活 DLP1 突变体 (K38A) 的表达会抑制哺乳动物细胞中的过氧化物酶体分裂。在本研究中，我们进行了 RNA 干扰实验，以“敲低”稳定表达带有 C 端过氧化物酶体靶向信号 1 的 GFP 构建体的 COS-7 细胞中 DLP1 的表达。DLP1 沉默的细胞中的过氧化物酶体高度伸长，具有分段形态。超微结构和定量研究证实，DLP1 沉默诱导的管状过氧化物酶体保留了收缩膜的能力，但无法分裂成球形细胞器。 DLP1 siRNA 与 Pex11pbeta（一种参与过氧化物酶体增殖的过氧化物酶体膜蛋白）共转染，诱导过氧化物酶体区室的进一步延伸和网络形成。对 DLP1 沉默的活细胞进行延时显微镜观察发现，细长的过氧化物酶体以微管依赖性方式移动并发出管状突起。 COS-7 细胞中的 DLP1 沉默还导致线粒体显着伸长，以及更分散、伸长的高尔基体结构，而没有检测到 rER、溶酶体和细胞骨架的形态变化。这些观察结果清楚地表明 DLP1 作用于多个膜细胞器。他们进一步表明，过氧化物酶体的伸长、收缩和裂变需要不同的蛋白质组，并且动力蛋白样蛋白质 DLP1 主要在后一个过程中发挥作用。
The mammalian dynamin-like protein DLP1 belongs to the dynamin family of large GTPases, which have been implicated in tubulation and fission events of cellular membranes. We have previously shown that the expression of a dominant-negative DLP1 mutant deficient in GTP hydrolysis (K38A) inhibited peroxisomal division in mammalian cells. In this study, we conducted RNA interference experiments to 'knock down' the expression of DLP1 in COS-7 cells stably expressing a GFP construct bearing the C-terminal peroxisomal targeting signal 1. The peroxisomes in DLP1-silenced cells were highly elongated with a segmented morphology. Ultrastructural and quantitative studies confirmed that the tubular peroxisomes induced by DLP1-silencing retained the ability to constrict their membranes but were not able to divide into spherical organelles. Co-transfection of DLP1 siRNA with Pex11pbeta, a peroxisomal membrane protein involved in peroxisome proliferation, induced further elongation and network formation of the peroxisomal compartment. Time-lapse microscopy of living cells silenced for DLP1 revealed that the elongated peroxisomes moved in a microtubule-dependent manner and emanated tubular projections. DLP1-silencing in COS-7 cells also resulted in a pronounced elongation of mitochondria, and in more dispersed, elongated Golgi structures, whereas morphological changes of the rER, lysosomes and the cytoskeleton were not detected. These observations clearly demonstrate that DLP1 acts on multiple membranous organelles. They further indicate that peroxisomal elongation, constriction and fission require distinct sets of proteins, and that the dynamin-like protein DLP1 functions primarily in the latter process.