Research Group Phagosome Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
Int J Biochem Cell Biol. 2013 Sep;45(9):2057-65. doi: 10.1016/j.biocel.2013.07.003. Epub 2013 Jul 17.
Within a cell, the regulation of organelle positioning is considered to be critical in spatio-temporal responses. The position of late endocytic organelles (named here lysosomes for simplicity) is tightly controlled and has a functional impact on processes like endocytosis, phagocytosis and autophagocytosis. The cytoplasmic distribution profile of lysosomes can be easily determined in cells where the cytoplasm/nuclear ratio in a cross-section area is high. However, determining lysosomal position in cells with lower cytoplasm/nuclear ratio, such as macrophages is more challenging. Here, we describe a method that can be efficiently and accurately used to determine the position of organelles in macrophages using confocal microscopy in two-dimensional (2D) images. Using this approach in macrophages, we confirmed previous observations in epithelial cells that both changes in cytoplasmic pH and the levels of active Rab34 induced a re-distribution of lysosomes to the cell centre or periphery. Noteworthy is that this Rab34-dependent re-distribution of lysosomes did not significantly affect the spatial distribution profile of phagolysosomes in the cytoplasm. We conclude that although Rab34 regulates both lysosomal positioning and lysosome to phagosome fusion, the latter effect is not due to the regulation of the cytoplasmic accessibility of lysosomes to phagosomes by Rab34.
在细胞内，细胞器定位的调节被认为对时空反应至关重要。晚期内吞细胞器（这里简称为溶酶体）的位置受到严格控制，对胞吞作用、吞噬作用和自噬作用等过程有功能影响。在细胞质/核比率高的细胞中，可以很容易地确定溶酶体的细胞质分布情况。然而，在细胞质/核比率较低的细胞（如巨噬细胞）中确定溶酶体的位置则更具挑战性。在这里，我们描述了一种可以在二维（2D）图像中使用共聚焦显微镜有效地、准确地确定巨噬细胞中细胞器位置的方法。使用这种方法在巨噬细胞中，我们证实了先前在上皮细胞中观察到的结果，即细胞质 pH 的变化和活性 Rab34 的水平都会导致溶酶体重新分布到细胞中心或边缘。值得注意的是，这种 Rab34 依赖性溶酶体的重分布不会显著影响细胞质中吞噬溶酶体的空间分布情况。我们得出的结论是，尽管 Rab34 调节溶酶体的定位和溶酶体与吞噬体的融合，但后一种效应不是由于 Rab34 调节溶酶体对吞噬体的细胞质可及性。
