Falcón-Pérez Juan M, Nazarian Ramin, Sabatti Chiara, Dell'Angelica Esteban C
Department of Human Genetics, University of California, Los Angeles, CA 90095, USA.
J Cell Sci. 2005 Nov 15;118(Pt 22):5243-55. doi: 10.1242/jcs.02633. Epub 2005 Oct 25.
Late endosomes and lysosomes of mammalian cells in interphase tend to concentrate in the perinuclear region that harbors the microtubule-organizing center. We have previously reported abnormal distribution of these organelles - as judged by reduced percentages of cells displaying pronounced perinuclear accumulation - in mutant fibroblasts lacking BLOC-3 (for ;biogenesis of lysosome-related organelles complex 3'). BLOC-3 is a protein complex that contains the products of the genes mutated in Hermansky-Pudlak syndrome types 1 and 4. Here, we developed a method based on image analysis to estimate the extent of organelle clustering in the perinuclear region of cultured cells. Using this method, we corroborated that the perinuclear clustering of late endocytic organelles containing Lamp1 (for ;lysosome-associated membrane protein 1') is reduced in BLOC-3-deficient murine fibroblasts, and found that it is apparently normal in fibroblasts deficient in BLOC-1 or BLOC-2, which are another two protein complexes associated with Hermansky-Pudlak syndrome. Wild-type and mutant fibroblasts were transfected to express human LAMP1 fused at its cytoplasmic tail to green fluorescence protein (GFP). At low expression levels, LAMP1-GFP was targeted correctly to late endocytic organelles in both wild-type and mutant cells. High levels of LAMP1-GFP overexpression elicited aberrant aggregation of late endocytic organelles, a phenomenon that probably involved formation of anti-parallel dimers of LAMP1-GFP as it was not observed in cells expressing comparable levels of a non-dimerizing mutant variant, LAMP1-mGFP. To test whether BLOC-3 plays a role in the movement of late endocytic organelles, time-lapse fluorescence microscopy experiments were performed using live cells expressing low levels of LAMP1-GFP or LAMP1-mGFP. Although active movement of late endocytic organelles was observed in both wild-type and mutant fibroblasts, quantitative analyses revealed a relatively lower frequency of microtubule-dependent movement events, either towards or away from the perinuclear region, within BLOC-3-deficient cells. By contrast, neither the duration nor the speed of these microtubule-dependent events seemed to be affected by the lack of BLOC-3 function. These results suggest that BLOC-3 function is required, directly or indirectly, for optimal attachment of late endocytic organelles to microtubule-dependent motors.
处于分裂间期的哺乳动物细胞的晚期内体和溶酶体往往集中在含有微管组织中心的核周区域。我们之前报道过，在缺乏BLOC - 3（即“溶酶体相关细胞器复合物3的生物发生”）的突变成纤维细胞中，这些细胞器的分布异常——通过显示明显核周聚集的细胞百分比降低来判断。BLOC - 3是一种蛋白质复合物，包含在1型和4型Hermansky - Pudlak综合征中发生突变的基因产物。在这里，我们开发了一种基于图像分析的方法来估计培养细胞的核周区域中细胞器聚集的程度。使用这种方法，我们证实了在缺乏BLOC - 3的小鼠成纤维细胞中，含有Lamp1（即“溶酶体相关膜蛋白1”）的晚期内吞细胞器的核周聚集减少，并且发现，在缺乏BLOC - 1或BLOC - 2的成纤维细胞中，这种聚集显然正常，BLOC - 1和BLOC - 2是另外两种与Hermansky - Pudlak综合征相关的蛋白质复合物。野生型和突变型成纤维细胞被转染以表达在其细胞质尾部与绿色荧光蛋白（GFP）融合的人LAMP1。在低表达水平时，LAMP1 - GFP在野生型和突变型细胞中都能正确靶向晚期内吞细胞器。高水平的LAMP1 - GFP过表达引发晚期内吞细胞器的异常聚集，这种现象可能涉及LAMP1 - GFP反平行二聚体的形成，因为在表达相当水平的非二聚化突变变体LAMP1 - mGFP的细胞中未观察到这种现象。为了测试BLOC - 3是否在晚期内吞细胞器的移动中起作用，使用表达低水平LAMP1 - GFP或LAMP1 - mGFP的活细胞进行了延时荧光显微镜实验。虽然在野生型和突变型成纤维细胞中都观察到了晚期内吞细胞器的活跃移动，但定量分析显示，在缺乏BLOC - 3的细胞内，朝向或远离核周区域的微管依赖性移动事件的频率相对较低。相比之下，这些微管依赖性事件的持续时间和速度似乎都不受BLOC - 3功能缺失的影响。这些结果表明，BLOC - 3的功能直接或间接地是晚期内吞细胞器与微管依赖性马达最佳附着所必需的。
