Phosphatidylinositol 5-phosphate 4-kinase regulates early endosomal dynamics during clathrin-mediated endocytosis

Kamalesh, Kumari; Trivedi, Deepti; Toscano, Sarah; Sharma, Sanjeev; Kolay, Sourav; Raghu, Padinjat

doi:10.1242/jcs.202259

Cited by 24 publications

(27 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…al., 2013 [19]. dsRNA treatment was performed as described in Kumari et al,2017 [20]. Briefly, 0.5 × 10 6 cells were incubated with 3.75 μg of dsRNA for 96 hours as described in Worby et.…”

Section: Methodsmentioning

confidence: 99%

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

Ghosh

Sharma

Shinde

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In this study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use 18O-ATP to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from both Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity levels from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.

show abstract

Section: Methodsmentioning

confidence: 99%

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

Ghosh

Sharma

Shinde

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Prior studies have demonstrated a role for PI5P4Ka and PI5P4Kb in clathrin-mediated transport and autophagy (Kamalesh et al, 2017;Lundquist et al, 2018), highlighting a possible vesicular distribution for the PI5P4Ks. To investigate their subcellular localization, we performed double immunofluorescence of PI5P4Ka and PI5P4Kb with a panel of subcellular markers.…”

Section: Pi5p4ka and Pi5p4kb Localize To Vesicle Organelles Within Momentioning

confidence: 97%

“…These kinases also regulate cellular growth pathways, including the mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) pathways (Carricaburu et al, 2003;Gupta et al, 2013). Furthermore, they regulate early endosomal homeostasis during clathrin-mediated endocytosis, a key step in synaptic vesicle trafficking (Kamalesh et al, 2017). Of particular interest, these enzymes also play a major role in suppressing the activity of the type 1 phosphatidylinositol-4-phosphate 5-kinases through direct binding to these enzymes (Wang et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Distribution and localization of phosphatidylinositol 5-phosphate, 4-kinase alpha and beta in the brain

Noch

Yim

Milner

et al. 2020

Preprint

View full text Add to dashboard Cite

Phosphatidylinositol-4,5-bisphosphate (PI-4,5-P 2 ) is critical for synaptic vesicle docking and fusion and generation of the second messengers, diacylglycerol and inositol-1,4,5-trisphosphate. PI-4,5-P 2 can be generated by two families of kinases: type 1 phosphatidylinositol-4-phosphate 5kinases, encoded by PIP5K1A, PIP5K1B and PIP5K1C, and type 2 phosphatidylinositol-5phosphate 4-kinases, encoded by PIP4K2A, PIP4K2B, and PIP4K2C. While the roles of the type 1 enzymes in brain function have been extensively studied, the roles of the type 2 enzymes are poorly understood. Using selective antibodies validated by genetic deletion of pip4k2a or pip4k2b in mouse brain, we characterized the location of the enzymes, PI5P4Kα and PI5P4Kß, encoded by these genes. In mice, we demonstrate that PI5P4Kα is expressed in adulthood, whereas PI5P4Kß is expressed early in development. PI5P4Kα localizes to white matter tracts, especially the corpus callosum, and at a low level in neurons, while PI5P4Kß is expressed in neuronal populations, especially hippocampus and cortex. Dual labeling studies demonstrate that PI5P4Kα co-localizes with the oligodendrocyte marker, Olig2, whereas PI5P4Kß co-localizes with the neuronal marker, NeuN. Immunohistochemical subcellular distribution studies demonstrate that PI5P4Kα and PI5P4Kß are expressed in the early endosome system. Ultrastructural analysis demonstrates that both kinases are contained in axon terminals and dendritic spines adjacent to the synaptic membrane, which support a potential role in synaptic transmission. Immunohistochemical analysis of macaque and human brain tissue demonstrate a conserved pattern for PI5P4Kα and PI5P4Kß. These results highlight the diverse cell-autonomous expression of PI5P4Kα and PI5P4Kß and support further exploration into their role in synaptic function in the brain.

show abstract

“…A recent study indicated that in Drosophila photoreceptors, dPIP4K regulates 145 clathrin-mediated endocytosis (CME) from the plasma membrane, and its absence 146 causes increased CME uptake, leading to an increased number of Rh1-loaded early 147 endocytic vesicles (Kamalesh et al, 2017). This cytoplasmic accumulation of Rh1 in 148 dPIP4K-deficient photoreceptors is light-dependent, likely because of light-stimulated 149 endocytosis, and this phenotype is rescued by the inhibition of endocytosis by 150 Rab5RNAi.…”

Section: Pcs-deficiency 144mentioning

confidence: 99%

A Rab11-GEF Parcas recruits Rab11 onto recycling endosomes for rhodopsin transport in Drosophila photoreceptors

Otsuka

Satoh

Nakayama

et al. 2019

Preprint

View full text Add to dashboard Cite

Rab11 and its effectors dRip11 and MyoV are essential for polarized post-Golgi vesicle trafficking to photosensitive membrane rhabdomeres in Drosophila photoreceptors. Here, we found that Parcas (Pcs), recently shown to have guanine-nucleotide-exchange (GEF) activity toward Rab11, co-localizes with Rab11 on the trans-side of Golgi units and post-Golgi vesicles at the base of the rhabdomeres in pupal photoreceptors. Pcs fused with the EM-tag APEX2 localizes on 150-300 nm vesicles at the trans-side of Golgi units, which are presumably fly recycling endosomes (RE). Loss of Pcs impairs Rab11 localization on the trans-side of Golgi units and induces the cytoplasmic accumulation of post-Golgi vesicles bearing rhabdomere proteins, as observed in Rab11-deficiency. In contrast, loss of the specific subunits of TRAPPII, another known Rab11-GEF, does not cause any defects on the eye development nor the transport of rhabdomere proteins, however, simultaneous loss of TRAPPII and Pcs shows severe defects on eye development. These results indicated that in pupal photoreceptors, Pcs is the predominant Rab11-GEF, and TRAPPII performs a function that is redundant but subsidiary to that of Pcs.

show abstract

Phosphatidylinositol 5-phosphate 4-kinase regulates early endosomal dynamics during clathrin-mediated endocytosis

Cited by 24 publications

References 54 publications

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

Distribution and localization of phosphatidylinositol 5-phosphate, 4-kinase alpha and beta in the brain

A Rab11-GEF Parcas recruits Rab11 onto recycling endosomes for rhodopsin transport in Drosophila photoreceptors

Contact Info

Product

Resources

About