Fisher K E, Lowry D S, Roberson R W
Department of Plant Biology, Box 871601, Arizona State University, Tempe, AZ 85287-1601, USA.
J Microsc. 2000 Jun;198(Pt 3):260-9. doi: 10.1046/j.1365-2818.2000.00700.x.
We have used the vital fluorescent dye, FM4-64, as a marker of membrane development during zoospore formation in living zoosporangia of Allomyces macrogynus. Membrane development was visualized and documented using standard epifluorescence and laser scanning confocal microscopy. Video-enhanced light microscopy and transmission electron microscopy, using cryopreparation methods, were also employed in this study. In the first 10-12 min after the induction of zoospore formation, only the plasma membrane labelled with FM4-64. During this time, nuclei were strictly located in the cortical cytoplasm with their associated centrosomes positioned immediately adjacent to the plasma membrane (Lowry & Roberson, 1997). Between 12 and 20 min post-induction, increased fluorescence appeared along regions of the plasma membrane adjacent to the nuclei. From these sites, membranes (i.e. cleavage elements) extended laterally within the cortex and then, in conjunction with nuclear migration, rapidly elongated into the sporangial cytoplasm. By 25-35 min post-induction, cleavage elements had ramified throughout the cytoplasm forming a complex, interconnected membranous network. Transmission electron microscopy revealed that cleavage elements were paired membrane sheets with a lumen consisting of an electron opaque, granular matrix. Cleavage elements developed into a highly ordered network by 35-40 min post-induction, which fully delimited zoospore initials into polyhedral-shaped cells. Zoospore discharge occurred between 40 and 50 min post-induction. Our results have shown that cleavage elements undergo four stages of development during zoospore formation in A. macrogynus: (i) development of membrane initials, (ii) cortical extension, (iii) cytoplasmic elongation and ramification and (iv) zoospore initial delimitation.
我们使用了活性荧光染料FM4-64，作为大孢异水霉活游动孢子囊中游动孢子形成过程中膜发育的标记物。使用标准落射荧光显微镜和激光扫描共聚焦显微镜对膜发育进行可视化和记录。本研究还采用了视频增强光学显微镜和使用冷冻制备方法的透射电子显微镜。在诱导游动孢子形成后的最初10-12分钟内，只有质膜被FM4-64标记。在此期间，细胞核严格位于皮层细胞质中，其相关的中心体紧邻质膜（Lowry和Roberson，1997）。诱导后12至20分钟之间，质膜上靠近细胞核的区域出现荧光增强。从这些部位开始，膜（即分裂元件）在皮层内横向延伸，然后与细胞核迁移一起迅速延伸到孢子囊细胞质中。诱导后25-35分钟时，分裂元件在整个细胞质中分支，形成一个复杂的、相互连接的膜网络。透射电子显微镜显示，分裂元件是成对的膜片，其腔由电子不透明的颗粒状基质组成。诱导后35-40分钟时，分裂元件发展成高度有序的网络，将游动孢子原基完全分隔成多面体形状的细胞。游动孢子在诱导后40至50分钟之间释放。我们的结果表明，在大孢异水霉游动孢子形成过程中，分裂元件经历四个发育阶段：（i）膜原基的发育，（ii）皮层延伸，（iii）细胞质伸长和分支，以及（iv）游动孢子原基的分隔。
