Enforced expression of phosphatidylinositol 4-phosphate 5-kinase homolog alters PtdIns(4,5)P2 distribution and the localization of small G-proteins

Abstract

The generation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) is essential for many functions including control of the cytoskeleton, signal transduction, and endocytosis. Due to its presence in the plasma membrane and anionic charge, PtdIns(4,5)P2, together with phosphatidylserine, provide the inner leaflet of the plasma membrane with a negative surface charge. This negative charge helps to define the identity of the plasma membrane, as it serves to recruit or regulate a multitude of peripheral and membrane proteins that contain polybasic domains or patches. Here, we determine that the phosphatidylinositol 4-phosphate 5-kinase homolog (PIPKH) alters the subcellular distribution of PtdIns(4,5)P2 by re-localizing the three PIP5Ks to endomembranes. We find a redistribution of the PIP5K family members to endomembrane structures upon PIPKH overexpression that is accompanied by accumulation of PtdIns(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). PIP5Ks are targeted to membranes in part due to electrostatic interactions; however, the interaction between PIPKH and PIP5K is maintained following hydrolysis of PtdIns(4,5)P2. Expression of PIPKH did not impair bulk endocytosis as monitored by FM4-64 uptake but did result in clustering of FM4-64 positive endosomes. Finally, we demonstrate that accumulation of polyphosphoinositides increases the negative surface charge of endosomes and in turn, leads to relocalization of surface charge probes as well as the polycationic proteins K-Ras and Rac1.

Label-free proteomic comparison reveals ciliary and non-ciliary phenotypes of IFT-A mutants

“…In our initial experiments, we found that Pip5kl1 robustly localizes to cilia of Xenopus MCCs (Fig 4E). Pip5kl1 is poorly characterized, but the few studies that have been performed show that while it lacks kinase activity, it regulates membrane phospholipid content by controlling the localization of other, enzymatically active PIP5Ks (Yang et al ., 2019). Interestingly, Pip5kl1 localization extended to the full length of the cilium as marked by membrane-RFP, in contrast to AK8 (Fig.…”

Clarification on the Gassing Behavior of Carbon‐Coated Li4Ti5O12 at Elevated Temperature: Importance of Coating Coverage

“…The magnified image of the 111 peak (Figure 3b) shows a shift in LTO/C‐3 h due to the oxygen vacancy. Since the coating process takes place in a reducing atmosphere, which is also favorable for oxygen vacancy formation on the surface of the LTO [32,50,51] . The Raman spectra of LTO contains a set of six bands (Figure 3c) with major bands centered at 231 cm −1 (which is F 2g vibrations in TiO 6 octahedral), 341 cm −1 (F 2g vibrations in LiO 6 octahedral), 421 cm −1 (E g vibrations in LiO 4 tetrahedral), and 668 cm −1 (A 1g vibrations in TiO 6 octahedral).…”

“…Since the coating process takes place in a reducing atmosphere, which is also favorable for oxygen vacancy formation on the surface of the LTO. [32,50,51] The Raman spectra of LTO contains a set of six bands (Figure 3c) with major bands centered at 231 cm À 1 (which is F 2g vibrations in TiO 6 octahedral), 341 cm À 1 (F 2g vibrations in LiO 6 octahedral), 421 cm À 1 (E g vibrations in LiO 4 tetrahedral), and 668 cm À 1 (A 1g vibrations in TiO 6 octahedral). In comparison, the two minor bands are located at 258 cm À 1 (attributed to F 2g vibrations in TiO 6 octahedral) and 737 cm À 1 (so-called A 1g shoulder).…”

Phase transformation and interface crystallography between TiO2 and different TinO2n−1 phases

“…The first endothermic peak located at around 85 °C with a weight loss of 3.4% corresponds to the dehydration process of the water adsorbed at the surface of the sample. 42 By increasing the temperature to 195 °C, the second endothermic peak is closely related to the phase transformation from H 2 Ti 3 O 7 to TiO 2 (B). 40 The corresponding weight loss for this process is 8.0% (11.4-3.4%), which is very close to the theoretical value (7.0%).…”