Dataset Information

PKC? diffusion and translocation are independent of an intact cytoskeleton.

ABSTRACT: Translocation of cytosolic cPKC to the plasma membrane is a key event in their activation process but its exact nature is still unclear with particular dispute whether sole diffusion or additional active transport along the cell's cytoskeleton contributes to cPKC's dynamics. This was addressed by analyzing the recruitment behavior of PKC? while manipulating the cytoskeleton. Photolytic Ca²⁺ uncaging allowed us to quantify the kinetics of PKC? redistribution to the plasma membrane when fused to monomeric, dimeric and tetrameric fluorescence proteins. Results indicated that translocation kinetics were modulated by the state of oligomerization as expected for varying Stokes' radii of the participating proteins. Following depolymerization of the microtubules and the actin filaments we found that Ca²⁺ induced membrane accumulation of PKC? was independent of the filamentous state of the cytoskeleton. Fusion of PKC? to the photo-convertible fluorescent protein Dendra2 enabled the investigation of PKC?-cytoskeleton interactions under resting conditions. Redistribution following spatially restricted photoconversion showed that the mobility of the fusion protein was independent of the state of the cytoskeleton. Our data demonstrated that in living cells neither actin filaments nor microtubules contribute to PKC?'s cytosolic mobility or Ca²⁺-induced translocation to the plasma membrane. Instead translocation is a solely diffusion-driven process.

SUBMITTER: Hui X

PROVIDER: S-EPMC5428563 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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PKCα diffusion and translocation are independent of an intact cytoskeleton.

Hui Xin X Sauer Benjamin B Kaestner Lars L Kruse Karsten K Lipp Peter P

Scientific reports 20170328 1

Translocation of cytosolic cPKC to the plasma membrane is a key event in their activation process but its exact nature is still unclear with particular dispute whether sole diffusion or additional active transport along the cell's cytoskeleton contributes to cPKC's dynamics. This was addressed by analyzing the recruitment behavior of PKCα while manipulating the cytoskeleton. Photolytic Ca<sup>2+</sup> uncaging allowed us to quantify the kinetics of PKCα redistribution to the plasma membrane when ...[more]

PMID: 28352102

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Project description:Intratympanic drug administration depends on the ability of drugs to pass through the round window membrane (RW) at the base of the cochlea and diffuse from this location to the apex. While the RW permeability for many different drugs can be promoted, passive diffusion along the narrowing spiral of the cochlea is limited. Earlier measurements of the distribution of marker ions, corticosteroids, and antibiotics demonstrated that the concentration of substances applied to the RW was two to three orders of magnitude higher in the base compared to the apex. The measurements, however, involved perforating the cochlear bony wall and, in some cases, sampling perilymph. These manipulations can change the flow rate of perilymph and lead to intake of perilymph through the cochlear aqueduct, thereby disguising concentration gradients of the delivered substances. In this study, the suppressive effect of salicylate on cochlear amplification via block of the outer hair cell (OHC) somatic motility was utilized to assess salicylate diffusion along an intact guinea pig cochlea in vivo. Salicylate solution was applied to the RW and threshold elevation of auditory nerve responses was measured at different times and frequencies after application. Resultant concentrations of salicylate along the cochlea were calculated by fitting the experimental data using a mathematical model of the diffusion and clearing of salicylate in a tube of variable diameter combined with a model describing salicylate action on cochlear amplification. Concentrations reach a steady-state at different times for different cochlear locations and it takes longer to reach the steady-state at more apical locations. Even at the steady-state, the predicted concentration at the apex is negligible. Model predictions for the geometry of the longer human cochlea show even higher differences in the steady-state concentrations of the drugs between cochlear base and apex. Our findings confirm conclusions that achieving therapeutic drug concentrations throughout the entire cochlear duct is hardly possible when the drugs are applied to the RW and are distributed via passive diffusion. Assisted methods of drug delivery are needed to reach a more uniform distribution of drugs along the cochlea.

Dataset Information

PKC? diffusion and translocation are independent of an intact cytoskeleton.

Publications

PKCα diffusion and translocation are independent of an intact cytoskeleton.

Similar Datasets

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