khushi singh.in

Research · Completed · 2024–2025

When the cell’s folding factory changes shape

IISER Pune · Prof. Richa Rikhy

Organism: Drosophila melanogaster

The question

What happens to protein quality control and tissue shape when an ER-shaping protein is reduced in fly embryos and ovaries?

The endoplasmic reticulum (ER) is a network of tubes and sheets where many proteins are made and folded, and its shape is set by dedicated ER-shaping proteins. I used RNA interference to reduce an ER-shaping protein and asked how cells cope, measuring global autophagy (cellular recycling) and protein ubiquitination, and looking for morphological defects in embryos and ovaries.

The basics: making proteins

Why ask this?

Every cell must make proteins, fold them correctly, and dispose of the ones that fail. The endoplasmic reticulum (ER) is a huge folding factory, a network of sheets and tubes whose shape is maintained by specialised proteins. Cells also run a recycling service, autophagy, which packages damaged parts for breakdown, and they tag unwanted proteins with ubiquitin. Established

Rapidly developing tissues, such as an early embryo or a growing egg, put these systems under particular strain, which makes them an ideal place to see what organelle shape is for.

How the ER gets its shape, and how autophagy works

The ER’s shape is built by proteins. Reticulons and REEPs curve the membrane into tubules (Voeltz et al. 2006), and the GTPase atlastin fuses tubules into a network of three-way junctions (Hu et al. 2009; Orso et al. 2009). Established Autophagy begins at the ER: a cup-shaped membrane grows around cargo, closes into an autophagosome and delivers it to the lysosome for digestion (Mizushima & Komatsu 2011). Established Autophagy can also remove pieces of the ER itself (ER-phagy) through receptors such as FAM134B, linking ER shape and turnover (Khaminets et al. 2015). Strong evidence

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Autophagy. The Atg1/ULK1 complex starts autophagy. The lipid kinase Vps34 makes PI3P at the ER, where a cup-shaped membrane (phagophore) forms. Atg8a (LC3 in mammals) coats it as it grows around cargo, such as ubiquitinated proteins or pieces of ER (ER-phagy, through receptors such as FAM134B; Khaminets et al. 2015), and it closes into a double-membrane autophagosome (Mizushima & Komatsu 2011).

Autophagy and the ER in development

Autophagy is not only a stress response. Embryos and eggs use it to clear maternal material, to control growth by nutrients, and to remove cells on schedule.

  • Egg to embryo (mouse)

    Fertilisation switches autophagy on, and it helps clear maternal proteins. Embryos lacking Atg5 from both egg and sperm stop at the 4–8-cell stage. (Tsukamoto et al. 2008) Strong evidence

  • Egg chamber (fly)

    Starvation switches on autophagy in germline and follicle cells, under insulin/TOR control (Barth et al. 2011). Late in oogenesis, autophagy degrades the apoptosis inhibitor dBruce so that nurse cells die on schedule. (Nezis et al. 2010) Strong evidence

  • Fat body: nutrients and growth (fly)

    TOR signalling keeps autophagy off in the larval fat body while food is plentiful; starvation releases it to recycle nutrients. (Scott et al. 2004) Established

  • The ER in the early embryo (fly)

    Before cellularisation, each syncytial nucleus gathers its own ER–Golgi unit (Frescas et al. 2006). Without the fusion GTPase atlastin, the fly ER fragments. (Orso et al. 2009) Strong evidence

Autophagy and the ER in development. Purple dots mark autophagosomes; blue rings mark ER. Autophagy renovates cells and tissues throughout development (Mizushima & Komatsu 2011). Schematics drawn for this site after the cited papers.

What I did

  • I reduced an ER-shaping protein with RNAi.
  • I measured autophagy and ubiquitination across the whole cell.
  • I immunostained embryos and ovaries to look for changes in tissue structure.
  • I presented the work as a poster at IISER Pune: Exploring autophagy and endoplasmic reticulum morphogenesis during embryogenesis and oogenesis in Drosophila melanogaster.

What I took away

An organelle’s shape is not decoration. It is tied to how well the cell keeps its proteins healthy, and in a developing tissue that shows up as form and function.

Why it matters

Protein quality control and autophagy are central to ageing and to many diseases, from neurodegeneration to metabolic disorders. Autophagy research was recognised with the 2016 Nobel Prize. Established

Methods used

  • RNA interference (RNAi)
  • Immunostaining of embryos and ovaries
  • Western blotting
  • PCR
  • Confocal microscopy

What these methods are →

References

  1. Nobel Prize in Physiology or Medicine 2016: Yoshinori Ohsumi, for discoveries of mechanisms for autophagy. nobelprize.org link
  2. English AR, Voeltz GK (2013) Endoplasmic reticulum structure and interconnections with other organelles. Cold Spring Harbor Perspectives in Biology 5:a013227. doi:10.1101/cshperspect.a013227
  3. Voeltz GK, Prinz WA, Shibata Y, Rist JM, Rapoport TA (2006) A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124:573–586. doi:10.1016/j.cell.2005.11.047
  4. Hu J, Shibata Y, Zhu PP, et al., Blackstone C (2009) A class of dynamin-like GTPases involved in the generation of the tubular ER network. Cell 138:549–561. doi:10.1016/j.cell.2009.05.025
  5. Orso G, Pendin D, Liu S, et al., Daga A (2009) Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin. Nature 460:978–983. doi:10.1038/nature08280
  6. Frescas D, Mavrakis M, Lorenz H, DeLotto R, Lippincott-Schwartz J (2006) The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei. Journal of Cell Biology 173:219–230. doi:10.1083/jcb.200601156
  7. Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y (1992) Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. Journal of Cell Biology 119:301–311. doi:10.1083/jcb.119.2.301
  8. Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741. doi:10.1016/j.cell.2011.10.026
  9. Khaminets A, Heinrich T, Mari M, et al., Dikic I (2015) Regulation of endoplasmic reticulum turnover by selective autophagy. Nature 522:354–358. doi:10.1038/nature14498
  10. Tsukamoto S, Kuma A, Murakami M, Kishi C, Yamamoto A, Mizushima N (2008) Autophagy is essential for preimplantation development of mouse embryos. Science 321:117–120. doi:10.1126/science.1154822
  11. Scott RC, Schuldiner O, Neufeld TP (2004) Role and regulation of starvation-induced autophagy in the Drosophila fat body. Developmental Cell 7:167–178. doi:10.1016/j.devcel.2004.07.009
  12. Nezis IP, Shravage BV, Sagona AP, et al., Stenmark H (2010) Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. Journal of Cell Biology 190:523–531. doi:10.1083/jcb.201002035
  13. Barth JMI, Szabad J, Hafen E, Köhler K (2011) Autophagy in Drosophila ovaries is induced by starvation and is required for oogenesis. Cell Death & Differentiation 18:915–924. doi:10.1038/cdd.2010.157