Gilbert T, Le Bivic A, Quaroni A, Rodriguez-Boulan E
Department of Cell Biology and Anatomy, Cornell University Medical College, New York 10021.
J Cell Biol. 1991 Apr;113(2):275-88. doi: 10.1083/jcb.113.2.275.
We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.
我们通过激光扫描共聚焦显微镜对人肠上皮细胞系Caco-2中微管的三维结构进行了表征。微管在顶端极形成了一个与细胞表面平行的、约4微米厚的致密网络，在基底极形成了一个1微米厚的松散网络。在顶端和基底束之间，微管平行于细胞的主轴线排列，集中在侧膜附近。用秋水仙碱处理4小时使99.4%的微管微管蛋白解聚。采用代谢脉冲追踪结合区域选择性生物素化、免疫沉淀和链霉亲和素沉淀法，研究微管在四种顶端和一种基底外侧标记物的分选和靶向中的作用。最近研究表明，顶端蛋白通过直接和转胞吞（经基底外侧膜）途径到达Caco-2细胞的顶端表面。秋水仙碱处理减缓了顶端和基底外侧蛋白向细胞表面的转运，但对顶端蛋白的影响更为显著，且同时影响了直接和间接途径。微管破坏对顶端蛋白分布的最终影响取决于对照细胞中各个标记物的稳态极化程度。氨肽酶N（APN）和蔗糖酶-异麦芽糖酶（SI）通常具有高度极化分布（顶端比基底外侧高110倍和75倍），秋水仙碱处理后仍相对极化（9倍）。APN和SI极性的降低主要是由于基底外侧残余表达增加（占对照总表面表达的10%），因为在没有微管的情况下，80%新合成的APN仍能转运到顶端表面，尽管转运速率较慢。碱性磷酸酶和二肽基肽酶IV通常仅达到较低水平的顶端极性（20小时追踪后分别为4倍和6倍，稳态时为9倍和8倍），在秋水仙碱存在下根本不发生极化，这是因为向顶端表面的递送较慢且在基底外侧表面的停留时间增加。秋水仙碱处理的细胞在基底外侧表面出现微绒毛或其他顶端标记物的异位定位以及大的细胞内空泡。向顶端和基底外侧培养基的极化分泌也受到微管破坏的影响。因此，完整的微管网络通过直接和间接途径促进顶端蛋白向Caco-2细胞表面的转运；这一作用对于一些顶端质膜蛋白的最终极性似乎至关重要，而对另一些蛋白只是一个增强因子。
