Dynamics of the endoplasmic reticulum during early development of Drosophila melanogaster

Abstract

In this study, we analyze for the first time endoplasmic reticulum (ER) dynamics and organization during oogenesis and embryonic divisions of Drosophila melanogaster using a Protein Disulfide Isomerase (PDI) GFP chimera protein. An accumulation of ER material into the oocyte takes place during the early steps of oogenesis. The compact organization of ER structures undergoes a transition to an expanded reticular network at fertilization. At the syncytial stage, this network connects to the nuclear envelope as each nucleus divides. Time-lapse confocal microscopy on PDI transgenic embryos allowed us to characterize a rapid redistribution of the ER during the mitotic phases. The ER network is massively recruited to the spindle poles in prophase. During metaphase most of the ER remains concentrated at the spindle poles and shortly thereafter forms several layers of membranes along the ruptured nuclear envelope. Later, during telophase an accumulation of ER material occurs at the spindle equator. We also analyzed the subcellular organization of the ER network at the ultrastructural level, allowing us to corroborate the results from confocal microscopy studies. This dynamic redistribution of ER suggests an unexpected regulatory function for this organelle during mitosis.

Cisternal Organization of the Endoplasmic Reticulum during Mitosis

“…These include the ER markers Sec61␤ (Anderson and Hetzer, 2007; this work); ss-GFP (or RFP)-KDEL (Terasaki, 2000;Altan-Bonnet et al, 2006;this work); ␤-galactosyltransferase (Terasaki, 2000;Altan-Bonnet et al, 2006;this work); reticulon4HD (this work); ER Tracker Red (this work); SP12 (Poteryaev et al, 2005), PDI (Bobinnec et al, 2003); and the nuclear envelope markers emerin (this work), LBR (Ellenberg et al, 1997;this work), and POM121, gp210 (Anderson and Hetzer, 2007). In all cases, the pattern within a single optical section always seems as long, smooth, curvilinear tracings spanning 3 m or more.…”

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

“…In particular, it is unknown whether they undergo a semi-closed mitosis as reported for syncytial embryos (Stafstrom and Staehelin, 1984) and embryonic-derived cells in culture (Debec and Marcaillou, 1997), in which the mitotic spindles are enclosed in membranous structures that become permeable to 70-kDa molecules in prophase (Paddy et al, 1996). To further address this question, we examined dividing neuroblasts expressing GFP-protein disulfide isomerase (PDI), a component of the endoplasmic reticulum lumen (Bobinnec et al, 2003). This analysis revealed the presence of GFP-PDI-labeled membranous structures surrounding the mitotic spindle that persisted into anaphase (Supplemental Figure 6B).…”

“…Although a spindle envelope was initially identified in syncytial Drosophila embryos (Stafstrom and Staehelin, 1984;Harel et al, 1989;Bobinnec et al, 2003;Frescas et al, 2006;Wagner et al, 2006) and embryo-derived Drosophila cell lines (Debec and Marcaillou, 1997;Maiato et al, 2006), it was unclear whether all Drosophila cells had one. In the S2Rϩ cell line for example, NE remnants were observed in cells fixed shortly after NE breakdown, but not at later stages of metaphase (Maiato et al, 2006).…”

mRNA Localization and ER-based Protein Sorting Mechanisms Dictate the Use of Transitional Endoplasmic Reticulum-Golgi Units Involved in Gurken Transport inDrosophilaOocytes

“…Mutant fly stocks used are dCOG5-GFP (w;GFP-Fws/Cyo) (Farkas et al, 2003); PDI-GFP (PDI::GFP/GFP-TM3,Sb) (Bobinnec et al, 2003) (Norvell et al, 1999); cni AR55 (b cni AR55 pr cn/Cyo), cni CF5 (b cni CF5 pr cn wx bw/Cyo) and cni AA12 (b cni AA12 /Cyo) (Roth et al, 1995); grk 2B6 (grk[2B6]b cn s L bo/Cyo) and grk 2E12 (w; grk[2E12]b/Cyo) (Neuman-Silberberg and Schupbach, 1993); and fly lines expressing Gurken lacking the transmembrane and the cytoplasmic domain g⌬TC4.1 and the cytoplasmic domain g⌬C100 (Queenan et al, 1999). The stocks carrying the constructs encoding the truncated versions of Gurken proteins were crossed into the grk 2B6 background and females from this cross were mated with male grk 2E12 to yield orange-eyed black flies (g⌬TC4.1; grk 2B6 /grk 2E12 and g⌬C100; grk 2B6 /grk 2E12 ).…”

“…In Drosophila oocytes, the visualization of the ER-resident protein protein disulfide isomerase tagged with GFP (PDI-GFP) shows that the ER pervades the entire oocyte (Bobinnec et al, 2003).…”