title: PKCα 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 PKCα while manipulating the cytoskeleton. Photolytic Ca2+uncaging allowed us to quantify the kinetics of PKCα 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 PKCα was independent of the filamentous state of the cytoskeleton. Fusion of PKCα to the photo-convertible fluorescent protein Dendra2 enabled the investigation of PKCα-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 PKCα's cytosolic mobility or Ca2+-induced translocation to the plasma membrane. Instead translocation is a solely diffusion-driven process.
authors.affiliation: Institute for Molecular Cell Biology, Research Centre for Molecular Imaging and Screening, Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, Homburg/Saar, Germany.
authors.forename: Xin
authors.initials: X
authors.lastname: Hui
authors.affiliation: Institute for Molecular Cell Biology, Research Centre for Molecular Imaging and Screening, Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, Homburg/Saar, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Sauer
authors.affiliation: Institute for Molecular Cell Biology, Research Centre for Molecular Imaging and Screening, Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, Homburg/Saar, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Kaestner
authors.affiliation: NCCR Chemical Biology, Departments of Biochemistry and Theoretical Physics, University of Geneva, 30 quai Ernest-Ansermet, 1211, Geneva, Switzerland.
authors.forename: Karsten
authors.initials: K
authors.lastname: Kruse
authors.affiliation: Institute for Molecular Cell Biology, Research Centre for Molecular Imaging and Screening, Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, Homburg/Saar, Germany. peter.lipp@uks.eu.
authors.forename: Peter
authors.identifier: 0000-0003-4728-9174
authors.initials: P
authors.lastname: Lipp
mesh_terms: D000199:Actins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D055503:Protein Multimerization; D021381:Protein Transport
chemical_list: D000199:Actins; C496628:PRKCA protein, human; D051571:Protein Kinase C-alpha; D002118:Calcium