Rejman Joanna, Oberle Volker, Zuhorn Inge S, Hoekstra Dick
Department of Membrane Cell Biology, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Biochem J. 2004 Jan 1;377(Pt 1):159-69. doi: 10.1042/BJ20031253.
Non-phagocytic eukaryotic cells can internalize particles <1 microm in size, encompassing pathogens, liposomes for drug delivery or lipoplexes applied in gene delivery. In the present study, we have investigated the effect of particle size on the pathway of entry and subsequent intracellular fate in non-phagocytic B16 cells, using a range of fluorescent latex beads of defined sizes (50-1000 nm). Our data reveal that particles as large as 500 nm were internalized by cells via an energy-dependent process. With an increase in size (50-500 nm), cholesterol depletion increased the efficiency of inhibition of uptake. The processing of the smaller particles was significantly perturbed upon microtubule disruption, while displaying a negligible effect on that of the 500 nm beads. Inhibitor and co-localization studies revealed that the mechanism by which the beads were internalized, and their subsequent intracellular routing, was strongly dependent on particle size. Internalization of microspheres with a diameter <200 nm involved clathrin-coated pits. With increasing size, a shift to a mechanism that relied on caveolae-mediated internalization became apparent, which became the predominant pathway of entry for particles of 500 nm in size. At these conditions, delivery to the lysosomes was no longer apparent. The data indicate that the size itself of (ligand-devoid) particles can determine the pathway of entry. The clathrin-mediated pathway of endocytosis shows an upper size limit for internalization of approx. 200 nm, and kinetic parameters may determine the almost exclusive internalization of such particles along this pathway rather than via caveolae.
非吞噬性真核细胞能够内化尺寸小于1微米的颗粒，包括病原体、用于药物递送的脂质体或用于基因递送的脂质复合物。在本研究中，我们使用一系列尺寸确定的荧光乳胶珠（50 - 1000纳米），研究了颗粒大小对非吞噬性B16细胞的内化途径及随后细胞内命运的影响。我们的数据显示，直径达500纳米的颗粒可通过能量依赖过程被细胞内化。随着颗粒尺寸增大（50 - 500纳米），胆固醇耗竭会提高摄取抑制效率。较小颗粒的处理在微管破坏时受到显著干扰，而对500纳米珠子的处理影响可忽略不计。抑制剂和共定位研究表明，珠子内化的机制及其随后的细胞内转运强烈依赖于颗粒大小。直径小于200纳米的微球内化涉及网格蛋白包被小窝。随着尺寸增加，明显转向一种依赖小窝介导内化的机制，这成为500纳米大小颗粒的主要内化途径。在这些条件下，向溶酶体的递送不再明显。数据表明（无配体）颗粒自身的大小可决定内化途径。网格蛋白介导的内吞途径显示内化的尺寸上限约为200纳米，动力学参数可能决定此类颗粒几乎完全沿此途径而非通过小窝进行内化。
