Steinberg T H, Swanson J A, Silverstein S C
Rover Laboratory of Physiology, Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032.
J Cell Biol. 1988 Sep;107(3):887-96. doi: 10.1083/jcb.107.3.887.
After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.
将膜不透性染料鲁米诺黄引入J774细胞的细胞质基质后，该染料被隔离在细胞质液泡中并分泌到细胞外培养基中。在本研究中，我们研究了鲁米诺黄在J774巨噬细胞中的细胞内运输以及该染料被隔离其中的细胞质液泡的性质。当用德克萨斯红卵清蛋白偶联物对J774细胞的溶酶体系统进行预标记，然后通过ATP介导的质膜通透化将鲁米诺黄加载到细胞的细胞质中时，30分钟后隔离鲁米诺黄的液泡与德克萨斯红染色的溶酶体不同。再过30分钟后，鲁米诺黄和德克萨斯红共定位于相同的膜结合区室，表明鲁米诺黄已被递送至溶酶体。接下来，在将鲁米诺黄加载到细胞中之前，我们用抗巨噬细胞抗体和德克萨斯红蛋白A对J774细胞的质膜进行预标记。随后隔离鲁米诺黄的透明液泡也用德克萨斯红染色，表明它们是内吞途径的一部分。在将鲁米诺黄引入细胞的细胞质基质后15分钟或60分钟，在Percoll密度梯度上对J774细胞进行分级分离。在15分钟后分级分离的细胞中，鲁米诺黄存在于含有质膜和内体的低密度浮力级分中；该染料随后出现在含有溶酶体的较高密度的小泡中。有机阴离子转运阻滞剂丙磺舒可抑制鲁米诺黄从J774细胞的细胞质基质中分泌。我们发现丙磺舒也可逆地抑制染料的隔离，表明细胞质液泡中染料的隔离也是由有机阴离子转运体介导的。这些研究表明，从J774细胞的细胞质基质中隔离鲁米诺黄的液泡具有内体的特性。因此，除了在分选膜结合和可溶性物质方面发挥作用外，巨噬细胞内体可能在可溶性细胞质中驻留分子的积累和运输中发挥作用。
