Probing the Subcellular Distribution of Phosphatidylinositol Reveals a Surprising Lack at the Plasma Membrane
Posted on: 25 July 2019
Preprint posted on 19 June 2019
Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.201906127
Defining the Subcellular Distribution and Metabolic Channeling of Phosphatidylinositol
Posted on:
Preprint posted on 20 June 2019
Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.201906130
It’s time to rethink how some lipids are distributed inside cells. Brand new molecular probes made by two labs show that phosphatidylinositol gets around more than we realise.
Selected by Sruthi S Balakrishnan
Categories: cell biology, molecular biology
Background
The lipid phosphatidylinositol (PI) performs both structural and functional roles in cells. Structurally, it anchors proteins in the plasma membrane (PM) and maintains membrane integrity. During infections, bacteria can break this link between PI and the proteins, creating avenues into the host cell. Apart from this role at outer leaflet of the PM, PI is a source of lipid intermediates required for cellular signalling and conferring identity to compartments like the Golgi apparatus and endosomes [1].
In cells, PI is thought to be present mainly at the endoplasmic reticulum (ER), with a minor presence on other compartments like the Golgi apparatus and PM [2]. Most of the available information regarding PI abundance and localisation comes from subcellular fractionation assays, which have limited resolution and potential of cross-contamination between organelles. While lipid-binding probes have been used to great effect, there have been none developed for PI till date [3]. Accurate data about PI subcellular distribution is required to understand processes like signalling, which often have high turnover rates of PI-derived lipid intermediates.
The two preprints discussed here have independently developed methods for live-tracking PI at specific membranes, offering new insights into its localisation and abundance.
Key Findings
The first study, from Gerald Hammond’s group at the University of Pittsburgh, used a bacterial PI- specific phospholipase C (PI-PLC) to develop an activatable probe for PI. PI-PLC hydrolyses PI to produce another lipid known as DAG, for which there exists a high-specificity lipid probe. The levels of DAG now provide an indirect readout of PI. To control PI-PLC activation, the group split PI-PLC from Listeria monocytogenes into its N-terminus, fused to a protein domain called FKBP, and C-terminus, fused to a domain called FRB. FKBP and FRB dimerise in the presence of the drug rapamycin, allowing temporal control over PI-PLC activity. The FKBP domain can be targeted to different subcellular compartments, allowing spatial control of the probe.
Using different versions of the split PI-PLC constructs, they found that PI was most abundant on the Golgi apparatus and peroxisomes, followed by the mitochondria and endosomes. Contrary to expectations, the probe did not show any signal from the PM or the ER.
They then made a second set of probes, this time using a recruitable variant of an enzyme (PI4K) that converts PI to a lipid called PI4P. Levels of PI4P now serve as proxy readouts of PI. While initially there was no PI detected on the ER using this probe, the lack of signal was attributed to the presence of a PI4P-degrading enzyme known as Sac1.When Sac1 activity was blocked, PI was again detected at the ER, confirming its presence at the compartment.
The second study, from Tamas Balla’s group at NIH, used PI-PLC probes derived from Bacillus cereus. A thorough study of protein structure enabled them to reduce the catalytic activity of PI-PLC since the native form was toxic to the cell. This altered construct was sent to specific compartments using the FKBP-FRB system. After rapamycin-induced dimerisation, they measured the DAG levels as an indirect readout of PI, using a probe similar to the one used by the first group.
Using PI-PLC constructs targetted to different compartments, they found that PI is present at the ER, mitochondria, Golgi apparatus, peroxisomes and endosomes. There was no signal for PI seen at the PM, consistent with the observations made in the first study.
The relevance of PI at the ER was confirmed using a second set of experiments. PI4P, which is a signalling lipid derived from PI, has a constitutive presence at the PM. When PI was selectively removed from the ER, PI4P levels at the PM dropped significantly. Removing PI from the PM, however, had no effect on the PI4P levels.
Both of the studies highlighted two important aspects of PI distribution in cells. The first being that PI has a higher presence in non-ER compartments than previously considered. The second is that free PI is most likely never present at the PM, but rather transferred to the PM only upon requirement. Additionally, both studies have added a whole new repertoire of tools to track PI dynamics in live, intact cells – something that has been lacking in the field until now.
What I like about these preprints
Firstly, I was excited to see that two independent research groups had made similar findings. It is always a win for science when results are corroborated by different scientists. In this case, both the studies beautifully complement each other as the same findings were made using varied approaches.
Secondly, the studies have generated tools to study PI in live cells with greater resolution than previously available.
Finally, they challenged the important assumption that most of the PI in cells is concentrated at the ER. The presence of PI in high abundance at other compartments prompts a reassessment of the standard membrane composition models.
Future Directions
The derivatives of PI present at the PM are not only required for compartment identity, but also undergo fast turnover when signalling is turned on [4].The marked absence of PI from the PM, therefore, points to its supply as a potential checkpoint. This also begs the question of why PI is not allowed to remain on the PM for any length of time.
The observations of substantial PI presence at nearly all subcellular compartments also warrants a rethinking of lipid metabolism dynamics.The detection of PI at the mitochondria, in particular, is remarkable since there are very few reports of PI derivatives at this compartment.The mitochondrial membrane is known to form contact sites with other compartments like the endoplasmic reticulum. It is also a reported to be a source for autophagosomal membranes [5]. Both of these processes often involve lipid derivatives of PI and further investigations into this aspect could unearth new mechanistic details.
Questions for the Authors
- The mitochondrial outer membrane interacts with other cellular compartments, often via PI- derived lipids. Does selectively depleting PI on the mitochondria change these interactions?
- Removing PI from the ER reduces the PI4P levels at the PM. Does it also affect the numbers/ clustering of the PI-anchored proteins?
- In both studies, PI was detected at the ER at lower levels than anticipated.The readout was either of low intensity (preprint 1) or transient (preprint 2). Does this solely reflect dynamics of the proxy lipid or actual fluctuations in the levels of PI at the ER?
Bibliography
- Sunshine, & Iruela-Arispe, M. L. Membrane lipids and cell signaling: Current Opinion in Lipidology 28, 408–413 (2017).
- Yang,Y., Lee, & Fairn, G. D. Phospholipid subcellular localization and dynamics. Journal of Biological Chemistry 293, 6230–6240 (2018).
- Wills, C., Goulden, B. D. & Hammond, G. R.V. Genetically encoded lipid biosensors. MBoC 29, 1526–1532 (2018).
- Rhee, G. Regulation of Phosphoinositide Specific PLC.pdf. Annu. Rev. Biochem. 70, 281– 312 (2001).
- Hailey, W. et al. Mitochondria Supply Membranes for Autophagosome Biogenesis during Starvation. Cell 141, 656–667 (2010).
doi: https://doi.org/10.1242/prelights.12350
(3 votes) Sign up to customise the site to your preferences and to receive alerts
Register hereAlso in the cell biology category:
Motor Clustering Enhances Kinesin-driven Vesicle Transport
Sharvari Pitke
Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis
Ankita Walvekar
Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water
Safieh Shah, Benjamin Dominik Maier
Also in the molecular biology category:
Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor
Jonathan Townson
HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos
Anchel De Jaime Soguero
The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites
Felipe Del Valle Batalla
preLists in the cell biology category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
| List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
| List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
| List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
| List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
| List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
| List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
| List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
| List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
| List by | Dey Lab |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
| List by | Joseph Jose Thottacherry |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
| List by | Dey Lab, Amanda Haage |
Also in the molecular biology category:
2024 Hypothalamus GRC
This 2024 Hypothalamus GRC (Gordon Research Conference) preList offers an overview of cutting-edge research focused on the hypothalamus, a critical brain region involved in regulating homeostasis, behavior, and neuroendocrine functions. The studies included cover a range of topics, including neural circuits, molecular mechanisms, and the role of the hypothalamus in health and disease. This collection highlights some of the latest advances in understanding hypothalamic function, with potential implications for treating disorders such as obesity, stress, and metabolic diseases.
| List by | Nathalie Krauth |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |