FITC-DEXTRAN AS A PROBE FOR ENDOSOME FUNCTION AND LOCALIZATION IN KIDNEY
FITC-DEXTRAN AS A PROBE FOR ENDOSOME FUNCTION AND LOCALIZATION IN KIDNEY
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DOI：
10.1152/ajpcell.1990.258.2.c309
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发表时间：
1990-02-01
影响因子：
--
通讯作者：
BROWN, D
中科院分区：
文献类型：
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作者：
LENCER, WI;WEYER, P;BROWN, D
组织固定和切片后，异硫氰酸荧光素 (FITC) 标记的内体定位于肾上皮细胞中，并使用相同的探针测量分离的内吞囊泡的特定膜转运特性。给大鼠静脉注射 10 kDa FITC-葡聚糖，并用多聚甲醛赖氨酸高碘酸盐固定肾脏。在光转换反应后通过落射荧光显微镜和电子显微镜观察切除组织的半薄(1μm)冷冻切片中FITC标记的囊泡。大多数 FITC 标记的内涵体位于尿管顶部的上皮细胞中。通过免疫细胞化学，大多数 FITC 标记的囊泡中不存在抗溶酶体糖蛋白 LGP 120，尽管注意到了一些共定位。 FITC标记核内体的限制膜包含液泡质子泵（pHmin = 6.23 .+-. 0.033）和水通道（渗透水渗透系数，Pf = 0.052 .+-. 0.005 cm/s），并且对乙二醇和尿素具有高度渗透性，但对葡萄糖相对不渗透。允许将特定膜功能归因于可以在上皮细胞顶端内吞途径中可视化的囊泡的方法应该是各种系统中内吞途径研究的通用用途。
Fluroescein isothiocyanate (FITC)-labeled endosomes were localized in kidney epithelial cells after tissue fixation and sectioning, and specific membrane transport properties of isolated endocytic vesicles were measured using the same probe. Rats were infused intravenously with 10 kDa FITC-dextran, and kidneys were fixed with paraformaldehyde lysine periodate. FITC-labeled vesicles were visualized in semithin (1 .mu.m) frozen sections of excised tissue by epifluorescent microscopy and by electron microscopy after a photoconversion reaction. Most FITC-labeled endosomes were apically located in epthelial cells lining the urinary tubules. By immunocytochemistry the anti-lysosomal glycoportein LGP 120 was absent from most of the FITC-labeled vesicles, although some colocalization was noted. The limiting membrane of FITC-labeled endosomes contained a vacuolar proton pump (pHmin = 6.23 .+-. 0.033) and a water channel (osmotic water permeability coeffient, Pf = 0.052 .+-. 0.005 cm/s) and was highly permeable to ethylene glycol and urea but relatively impermeable to glucose. Methods allowing the attribution of specific membrane function to vesicles that can be visualized in the apical endocytic pathway of epithelial cells should be a general use for the study of endocytic pathways in a variety of systems.