Identifying the mechanisms through which calcineurin regulates proportional growth control.

• Applicant: Dr. Christopher Antos
• Subject Area: Developmental Biology ; Evolutionary Cell and Developmental Biology (Zoology) ; Cell Biology
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 283635409

• Final Report Year: 2022

Final Report Abstract

We identified that calcineurin regulated Kcnk5b activity by electrophysiological patch-clamp experiments and determined that transgenic Kcnk5b up-regulation in adult zebrafish fins led to the upregulation of shh, lef1 and adhl1a2 in the adult fin, but led to minimal up-regulation of pea3, an Fgf-responsive gene, or no up-regulation other members of the developmental program. Only chronic expression of Kcnk5b led to the up-regulation of these other genes. We found that transgenic up-regulation of Kcnk5b in zebrafish larva, also increased the expression of shh and lef1. From patch-clamp experiments, we found that serine 345 in the cytoplasmic C-terminal tail of Kcnk5b regulates Kcnk5b channel activity and that calcineurin no longer affected the channel when this serine is mutated; whereas, calcineurin still regulated the activity of the channel when other adjacent serines in its C-terminus were mutated and that mutations of these other serines had no effect on channel activity. We transferred these findings into transgenic experiments in vivo to show that mimicking post-translational modifications on serine 345 in the channel directly correlates with the increasing or decreasing the proportional growth of the entire adult fin. We set up in vitro Fluorescence Lifetime Microscopy to visualize the reduction of intracellular K+ when Kcnk5b was expressed in cells and used this in experiments of the eLIFE paper. The additional attribution of this method is because electrophysiological research has been hampered by the limitations on assessing the electrophysiology of cell and tissues in vivo. The important reason for setting up fluorescence lifetime microscopy is the translation of this application into a use with live zebrafish; consequently, we will provide a method to perform future electrophysiological studies in vivo.

Publications

• (2017) Chronic loss of inhibitor-1 diminishes cardiac RyR2 phosphorylation despite exaggerated CaMKII activity. Naunyn-Schmiedeberg's Archives of Pharmacology; 390(8):857-862
  Neef S , Brammen CAA, Wittköpper K, Heijman Jr, Antos CL, Otte C, Seitz T, Dewenter M, Saadatmand AR, Meyer-Roxlau S, Vettel C, Weber S, Backs J, Hasenfuss G, Dobrev D, Maier LS, El-Armouche A
  (See online at https://doi.org/10.1007/s00210-017-1376-1)
• (2017) Human TAUP301L overexpression results in TAU hyperphos-phorylation without neurofibrillary tangles in adult zebrafish brain. Science Reports 7(1):12959
  Cosacak MI, Bhattarai P, Bocova L, Dzewas T, Mashkaryan V, Papadimitriou C, Brandt K, Hollak H, Antos CL, Kizil C
  (See online at https://doi.org/10.1038/s41598-017-13311-5)
• (2017) Making human cardiomyocytes up to date: Derivation, maturation state and perspectives. International Journal of Cardiology 241:379-386
  Kolanowski T, Antos CL and Guan K
  (See online at https://doi.org/10.1016/j.ijcard.2017.03.099)
• (2018) 3D Culture Method for Alzheimer's Disease Modeling Reveals Interleukin-4 Rescues Aβ42-Induced Loss of Human Neural Stem Cell Plasticity. Developmental. Cell 46:85-101
  Papadimitriou C, Celikkaya H, Cosacak MI, Mashkaryan V, Bray L, Bhattarai P, Brandt K, Hollak H, Chen X, He S, Antos CL, Lin W, Thomas AK, Dahl A, Kurth T, Friedrichs J, Zhang Y, Freudenberg U, Werner C, Kizil C
  (See online at https://doi.org/10.1016/j.devcel.2018.06.005)
• (2020) Asymmetric stratification-induced Polarity loss and coordinated individual cell movements drive directional migration of vertebrate epithelium. Cell Reports 33(2):108246
  Lu Y, Deng R, You H, Xu Y, Antos CL, Sun J, Klein OD, Lu P
  (See online at https://doi.org/10.1016/j.celrep.2020.108246)
• (2021) A calcineurin-mediated scaling mechanism that controls a K+- leak channel to regulate morphogen and growth factor transcription. Elife
  Yi C, Spitters TW, Al-Far EEA, Wang S, Xiong T, Cai S, Yan X, Guan K, Wagner M, El- Armouche A, Antos CL
  (See online at https://doi.org/10.7554/elife.60691)
• (2021) Discovery of a subtype-selective, covalent inhibitor against palmitoylation pocket of TEAD3. Acta Pharmaceutica Sinica
  Lu T, Li Y, Lu W, Spitters TWGM, Wang J, Cai S, Gao J, Zhou Y, Duan Z, Xiong H, Liu L, Li Q, Jiang H, Chen K, Zhou H, Lin H, Feng H, Zhou B, Antos CL, Luo C
  (See online at https://doi.org/10.1016/j.apsb.2021.04.015)