Quantification of Genetically Encoded Lipid Biosensors

Wills, Rachel C.; Pacheco, Jonathan; Hammond, Gerald

doi:10.1007/978-1-0716-1142-5_4

Cited by 15 publications

(10 citation statements)

References 100 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For confocal image analysis, quantification of P4Mx2 signal at ER-PM or mitochondria-PM contact sites was accomplished by using the FRB-ER or mitochondria-FRB channels to generate a binary mask and measuring the normalized intensity of P4Mx2 inside this mask compared to outside. This and the auto thresholding procedure used to generate these masks has been outlined in detail in a recent protocols paper (Wills et al, 2021). Briefly, the FRB images were Gaussian filtered at lengths defined by 1 and 2 multiples of the far red-fluor airy disc size (for ER), or 1, 2, 3 and 4 times this size for mitochondria.…”

Section: Methodsmentioning

confidence: 99%

Orthogonal targeting of SAC1 to mitochondria implicates ORP2 as a major player in PM PI4P turnover

Doyle,

Rectenwald,

Timple

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Oxysterol binding protein (OSBP)-related proteins (ORPs) 5 and 8 have been shown to deplete the lipid phosphatidylinositol 4-phosphate (PI4P) at sites of membrane contact between the endoplasmic reticulum (ER) and plasma membrane (PM). This is believed to be caused by transport of PI4P from the PM to the ER, where PI4P is degraded by an ER-localized SAC1 phosphatase. This is proposed to power the anti-port of phosphatidylserine (PS) lipids from ER to PM, up their concentration gradient. Alternatively, ORPs have been proposed to sequester PI4P, dependent on the concentration of their alternative lipid ligand. Here, we aimed to distinguish these possibilities in living cells by orthogonal targeting of PI4P transfer and degradation to PM-mitochondria contact sites. Surprisingly, we found that orthogonal targeting of SAC1 to mitochondria enhanced PM PI4P turnover independent of targeting to contact sites with the PM. This turnover could be slowed by knock-down of soluble ORP2, which also has a major impact on PM PI4P levels even without SAC1 over-expression. The data reveal a role for contact site-independent modulation of PM PI4P levels and lipid antiport.

show abstract

Section: Methodsmentioning

confidence: 99%

Orthogonal targeting of SAC1 to mitochondria implicates ORP2 as a major player in PM PI4P turnover

Doyle,

Rectenwald,

Timple

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, we did not succeed in obtaining stable soluble proteins. To overcome this issue, we generated mammalian expression vectors encoding fluorescently labeled PfPX domains (EGFP-PfPX WT and EGFP-PfPX RKR ) to express them in mammalian cells, a validated alternative methodology used to determine PPI binding specificities ( 61 – 63 ). Mammalian intracellular endocytic membranes have unique and dynamic PPI compositions and the presence of PI3P on the cytoplasmic face of early endosomes is well-established ( 64 – 66 ).…”

Section: Resultsmentioning

confidence: 99%

A Phosphoinositide-Binding Protein Acts in the Trafficking Pathway of Hemoglobin in the Malaria Parasite Plasmodium falciparum

Mukherjee

Crochetière

Sergerie

et al. 2022

mBio

View full text Add to dashboard Cite

show abstract

“…The mean pixel intensity was then measured within a binary mask of the selected compartment, normalized to the mean pixel intensity outside of that mask for each cellular ROI. Masks were generated using an automated thresholding algorithm on images of a marker for that target organelle (such as the Fis1-tail targeted to mitochondria), employing wavelet decomposition as described in detail previously (Wills et al, 2021). Alternatively, when no marker construct was employed (for the experiments reported in figures 1 and 2), we used manual thresholding of P4Mx1 fluorescence at the first frame to define Golgi-associated fluorescence and generate a binary mask of that threshold for each frame.…”

Section: Methodsmentioning

confidence: 99%

OSBP is a major determinant of Golgi phosphatidylinositol 4-phosphate homeostasis

Doyle,

Timple,

Hammond

2023

Preprint

Self Cite

View full text Add to dashboard Cite

The lipid phosphatidylinositol 4-phosphate (PI4P) plays a master regulatory role at Golgi membranes, orchestrating membrane budding, non-vesicular lipid transport and membrane organization. It follows that harmonious Golgi function requires strictly maintained PI4P homeostasis. One of the most abundant PI4P effector proteins is the oxysterol binding protein (OSBP), a lipid transfer protein that exchanges trans Golgi PI4P for ER cholesterol. Although this protein consumes PI4P as part of its lipid anti-porter function, whether it actively contributes to Golgi PI4P homeostasis has been questioned. Here, we employed a series of acute and chronic genetic manipulations, together with orthogonal targeting of OSBP, to interrogate its control over Golgi PI4P abundance. Modulating OSBP levels at ER:Golgi membrane contact sites produces reciprocal changes in PI4P levels. Additionally, we observe that OSBP has a high capacity for PI4P turnover, even at orthogonal organelle membranes. However, despite also visiting the plasma membrane, endogenous OSBP makes no impact on PI4P levels in this compartment. We conclude that OSBP is a major determinant of Golgi PI4P homeostasis.

show abstract

Quantification of Genetically Encoded Lipid Biosensors

Cited by 15 publications

References 100 publications

Orthogonal targeting of SAC1 to mitochondria implicates ORP2 as a major player in PM PI4P turnover

Orthogonal targeting of SAC1 to mitochondria implicates ORP2 as a major player in PM PI4P turnover

A Phosphoinositide-Binding Protein Acts in the Trafficking Pathway of Hemoglobin in the Malaria Parasite Plasmodium falciparum

OSBP is a major determinant of Golgi phosphatidylinositol 4-phosphate homeostasis

Contact Info

Product

Resources

About