Frøkjaer-Jensen J
Department of General Physiology and Biophysics, University of Copenhagen, Panum Institute, Denmark.
J Electron Microsc Tech. 1991 Nov;19(3):291-304. doi: 10.1002/jemt.1060190305.
Conventional EM sections of chemically fixed capillary endothelial cells reveal numerous apparently free smooth plasmalemmal vesicles. However, the method of ultrathin (less than 150 A) serial sectioning has shown that the smooth vesicle profiles arise merely as a result of the EM thin sectioning of two sets of complex vesicular invaginations from the luminal and abluminal cell surfaces, which end blindly in the cytoplasm. While 50-70% of the total population of vesicular profiles appear to lack connections to the cell surface in conventional (500-700 A thick) EM thin sections less than 1% truly free vesicles can be found by the ultrathin serial section analyses. In the present study it is examined whether similar conclusions apply to endothelial cells which were directly frozen by slam-freezing and subsequently freeze-substituted. The three-dimensional organization of the plasmalemmal vesicular system was analyzed in four series of 19, 18, 13, and 10 ultrathin sections (approximately 110 A thick) of capillaries from frog mesenteries quickly excised from decapitated frogs (Rana pipiens). None of 920 vesicular profiles (diameter 500-1,200 A) which appeared free in individual thin sections of the series represented free vesicles; all profiles either communicated with other vesicles, the cell surface, or in rare cases turned out to be part of cytoplasmic tubular membrane structures. It is concluded that free smooth plasmalemmal vesicles are very rare in rapidly frozen as well as in directly fixed frog capillary endothelium. The volume density of profiles (13-15%), the proportion of apparently free vesicle profiles (70%), and interconnected profiles (20%) were similar to the picture previously found in single EM sections of frog mesenteric capillaries. No transendothelial channels were found in the four series of ultrathin sections of capillaries. However, continuities between the luminal and abluminal cell surfaces were seen in the endothelium of venules. Furthermore, in the ultrathin series of the capillaries, vesicular units belonging to the two sets of invaginations and cytoplasmic tubular membrane structures were in more cases found in very close contact-as fused to share one unit membrane. If this finding is representative for the in vivo situation, it may reflect that the vesicular system represents a highly dynamic system with possibilities for mixing of membranes, cellular traffic of lipid, membrane proteins, and receptors between internal compartments and the cell surfaces, as well as occasional exchange of macromolecules between blood and tissue through rare temporary connections between the two sets of surface invaginations, without actually moving vesicles.
化学固定的毛细血管内皮细胞的传统电子显微镜切片显示出许多明显游离的光滑质膜小泡。然而，超薄（小于150埃）连续切片法表明，光滑小泡轮廓仅仅是由于对来自管腔和管腔外细胞表面的两组复杂泡状内陷进行电子显微镜超薄切片而产生的，这些内陷在细胞质中盲端终止。在传统的（500 - 700埃厚）电子显微镜超薄切片中，约50 - 70%的小泡轮廓似乎与细胞表面没有连接，但通过超薄连续切片分析发现真正游离的小泡不到1%。在本研究中，研究了类似的结论是否适用于通过快速冷冻直接冷冻并随后进行冷冻置换的内皮细胞。对从断头蛙（豹蛙）快速切除的蛙肠系膜毛细血管的四个系列的19、18、13和10个超薄切片（约110埃厚）中的质膜小泡系统的三维组织进行了分析。在该系列的单个超薄切片中看似游离的920个小泡轮廓（直径500 - 1200埃）中，没有一个代表游离小泡；所有轮廓要么与其他小泡、细胞表面相通，要么在极少数情况下被证明是细胞质管状膜结构的一部分。得出的结论是，在快速冷冻以及直接固定的蛙毛细血管内皮中，游离的光滑质膜小泡非常罕见。轮廓的体积密度（13 - 15%）、看似游离的小泡轮廓比例（70%）和相互连接的轮廓比例（20%）与先前在蛙肠系膜毛细血管的单个电子显微镜切片中发现的情况相似。在毛细血管的四个系列超薄切片中未发现跨内皮通道。然而，在小静脉内皮中可见管腔和管腔外细胞表面之间的连续性。此外，在毛细血管的超薄系列中，属于两组内陷的小泡单元和细胞质管状膜结构在更多情况下被发现紧密接触——融合以共享一个单位膜。如果这一发现代表体内情况，它可能反映出小泡系统是一个高度动态的系统，具有膜混合、脂质、膜蛋白和受体在内部隔室与细胞表面之间进行细胞运输的可能性，以及偶尔通过两组表面内陷之间罕见的临时连接在血液和组织之间交换大分子的可能性，而实际上小泡并不移动。
