Abstract
BackgroundS2 cells are one of the most widely used Drosophila melanogaster cell lines. A series of studies has shown that they are particularly suitable for RNAi-based screens aimed at the dissection of cellular pathways, including those controlling cell shape and motility, cell metabolism, and host–pathogen interactions. In addition, RNAi in S2 cells has been successfully used to identify many new mitotic genes that are conserved in the higher eukaryotes, and for the analysis of several aspects of the mitotic process. However, no detailed and complete description of S2 cell mitosis at the ultrastructural level has been done. Here, we provide a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM).ResultsWe analyzed by TEM a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behaviors. This unbiased approach provided a comprehensive ultrastructural view of the dividing cells, and allowed us to discover that S2 cells exhibit a previously uncharacterized behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase.ConclusionsWe discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells.
Highlights
S2 cells are one of the most widely used Drosophila melanogaster cell lines
Our observations suggest that the inner double membrane component of the Quadruple nuclear membrane (QNM) corresponds to the vertebrate nuclear membrane, which disintegrates at the beginning of prometaphase concomitantly with its associated lamin meshwork (NEBD)
We do not know whether it is different from the structure of Drosophila embryonic or neuroblast spindles, which are currently incompletely characterized at the ultrastructural level
Summary
S2 cells are one of the most widely used Drosophila melanogaster cell lines. A series of studies has shown that they are suitable for RNAi-based screens aimed at the dissection of cellular pathways, including those controlling cell shape and motility, cell metabolism, and host–pathogen interactions. Extensive work carried out in the past 15 years has shown that Drosophila S2 cells are an excellent system for the molecular dissection of mitosis using RNA interference (RNAi). There are multiple advantages for using S2 cells in RNAi-based studies of mitosis. They have a very favorable cytology for spindle and chromosome visualization in both fixed and living cells [3, 4]. Simultaneous RNAi against multiple genes can be performed, allowing pathway dissection. The Drosophila genome is fully sequenced and well annotated and mitotic genes are highly evolutionarily conserved among metazoans [7, 8], allowing RNAi data on S2 cells to be extrapolated to human cells
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