The role of ER exit sites in maintaining P-body organization and transmitting ER stress response during Drosophila melanogaster oogenesis

Background

Organisation of the cell cytoplasm was thought to be through lipid bilayers compartmentalising the cell into membrane bound organelles. However, in recent years the formation of membraneless compartments which concentrate cellular components into condensates through in a liquid-liquid phase separation like manner has been of increasing interest. This field of biocondensate formation has focused on “bottom up” assembly based on weak interactions of intrinsically disordered domains favouring the formation of condensates (Banani et al., 2017). Whether other factors may aid the formation of biocondensates in a physiological setting is an open question.

Processing bodies (P bodies) are cytoplasmic granules composed of translationally repressed mRNAs and proteins related to mRNA degradation. The ribonucleoprotein complexes with intrinsically disordered regions found in P bodies favour liquid-liquid phase separation and biocondensate formation (Banani et al., 2017; Luo et al., 2018). The fruit fly Drosophila melanogaster is a good model system for studying P bodies and the formation of biocondensates in vivo (Wilby and Weil 2023). In the ovary egg chambers of these flies, P bodies are used for translation repression of transcripts as they are spatio-temporally localised from nurse cells to the oocyte (Lin et al., 2008; Weil et al., 2012). Core P body proteins, in particular Trailer Hitch (Tral), colocalise with the endoplasmic reticulum (ER) membrane and are important for the spatio-temporal regulation of ER exit sites (Wilhelm et al., 2005; Kugler et al., 2009). More recently, the ER has also been shown to regulate P bodies where it facilitates fission (Lee et al., 2020). In this preprint, Milano and colleagues investigate how ER exit sites may affect the formation and composition of P bodies.

Key findings

In this preprint, the authors investigate the interaction of P bodies with ER exit sites. Notably they find that this interaction is important for P body maturation and composition and if it is prevented then P body function is impaired. They also show ER stress can be communicated to P bodies at these sites and hypothesise that essential mRNAs may be protected in these P bodies during ER stress.

P bodies associate with ER exit sites – these are larger, less mobile, more mature and less dynamic compared to cytoplasmic ones.

Using GFP tagged ME31B as a marker for P bodies, the authors performed confocal fluorescence microscopy to measure the volume and sphericity of P bodies. They also used STED super-resolution imaging to assess the “roughness” of their surface. Larger and less spherical P bodies indicate they are more mature. They found that P bodies associated with the ER and ER exit sites (ERES) were larger and more mature than cytoplasmic P bodies.

Following this, the authors noted that in live imaging experiments ERES-associated P bodies did not travel far compared to cytoplasmic ones. They showed that cytoplasmic P bodies are tracking along microtubules by knocking down BicD which is part of a complex linking P bodies to microtubules. Furthermore, ERES-associated P bodies underwent fission where transport along microtubules was able to pull off new cytoplasmic P bodies from the ERES-associated ones.

ER exit sites may have a role in organising P bodies and removing them compromises the integrity/stability of P bodies leading to a reduction in the repression of maternal oskar mRNA in the ovary.

Next, the authors assessed the importance of COPII vesicle formation at ERES for P body composition. By knocking down the COPII vesicle scaffold protein Sec16, they showed smaller and less mature condensates of ME31B and Tral. When they knocked down the coat proteins, the condensates were much larger but failed to mature.

The authors then prevented the initiation of ERES by knocking down Sar1. They observed that fewer P bodies formed and that P body proteins and maternal oskar mRNA were mislocalised in the cytoplasm and not present in P bodies. Knockdown of Sar1 reduced translational repression and increased degradation of oskar mRNA. The authors did not observe reduced access of translation repression proteins such as Me31B and Cup which associate with the mRNA, suggesting this was due to compromised P body function.

ER stress is communicated to P bodies via ER exit sites

Using DTT to stress the ER, the authors could show that ME31B granules get larger and less mature, more reminiscent of stress granules. Additionally, these granules require ERES as they did not form in a Sar1 knockdown. Under ER stress, transcripts of ATP synthase and me31B – which do not normally associate with P bodies – started to accumulate with Me31B granules. As the levels of transcripts stayed the same or increased, the authors speculated that the Me31B granules (P bodies) are protecting essential transcripts during ER stress.

The proposed mechanism of P body formation at ER exit sites (ERES) in which the ERES act as a hub for P-body formation and maintenance (figure 7 from Milano et al.). Mature P bodies dock at the ERES with mobile liquid like P bodies being pulled away by the microtubule network.

What I like about the preprint/why I think this new work is important

I came across this preprint whilst reading about ER exit sites. I am fascinated by the growing research field looking at membrane contact sites and so was intrigued by this contact with a membraneless cell entity. This research also draws on ideas around intrinsically disordered domains and how they contribute to biocondensate formation. The combination of these three research areas in one paper drew me in. I think that the increasing interest in intracellular organelle communication, and in the weak interactions of intrinsically disordered domains in proteins, make this paper one that a lot of people will want to read.

Future directions and questions for the authors

• How do you propose the P bodies associating with ERES are being held there? And how close is this association?
• You show that ERES-associated P bodies are more mature than cytoplasmic ones and that they undergo fission events to form cytoplasmic P bodies. Is the composition of the cytoplasmic P body that is pulled off different to the parental, ERES-associated P body? If so, then how do you propose this can be achieved?
• P bodies function to degrade mRNA as well as store and translationally repress it (Luo et al., 2018). How do you resolve the degradation function with your hypothesis that Me31B-GFP granules are protecting essential mRNAs during ER stress? Could these be a specific subset of P bodies without the degradation machinery? And are these P bodies associated with ERES?

References

• Banani, S. F., Lee, H. O., Hyman, A. A., & Rosen, M. K. (2017). Biomolecular condensates: Organizers of cellular biochemistry. Nature Reviews Molecular Cell Biology, 18(5), 285–298. https://doi.org/10.1038/nrm.2017.7
• Kugler, J. M., Chicoine, J., & Lasko, P. (2009). Bicaudal-C associates with a Trailer Hitch/Me31B complex and is required for efficient Gurken secretion. Developmental Biology, 328(1), 160–172. https://doi.org/10.1016/j.ydbio.2009.01.024
• Lee, J. E., Cathey, P. I., Wu, H., Parker, R., & Voeltz, G. K. (2020). Endoplasmic reticulum contact sites regulate the dynamics of membraneless organelles. Science, 367(6477). https://doi.org/10.1126/science.aay7108
• Lin, M. Der, Jiao, X., Grima, D., Newbury, S. F., Kiledjian, M., & Chou, T. Bin. (2008). Drosophila processing bodies in oogenesis. Developmental Biology, 322(2), 276–288. https://doi.org/10.1016/j.ydbio.2008.07.033
• Luo, Y., Na, Z., & Slavoff, S. A. (2018). P-Bodies: Composition, Properties, and Functions. Biochemistry, 57(17), 2424–2431. https://doi.org/10.1021/acs.biochem.7b01162
• Milano, S. N., Bayer, L. V., Ko, J. J., Casella, C. E., & Bratu, D. P. (2024). The role of ER exit sites in maintaining P-body organization and transmitting ER stress response during. BioRxiv. https://doi.org/10.1101/2024.07.03.601952
• Weil, T. T., Parton, R. M., Herpers, B., Soetaert, J., Veenendaal, T., Xanthakis, D., Dobbie, I. M., Halstead, J. M., Hayashi, R., Rabouille, C., & Davis, I. (2012). Drosophila patterning is established by differential association of mRNAs with P bodies. Nature Cell Biology, 14(12), 1305–1313. https://doi.org/10.1038/ncb2627
• Wilby, E. L., & Weil, T. T. (2023). Relating the Biogenesis and Function of P Bodies in Drosophila to Human Disease. Genes, 14(9). https://doi.org/10.3390/genes14091675
• Wilhelm, J. E., Buszczak, M., & Sayles, S. (2005). Efficient protein trafficking requires trailer hitch, a component of a ribonucleoprotein complex localized to the ER in Drosophila. Developmental Cell, 9(5), 675–685. https://doi.org/10.1016/j.devcel.2005.09.015

Tags: biocondensate, drosophila, endoplasmic reticulum, eres, p body

doi: https://doi.org/10.1242/prelights.38295

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