Reversible translocation and activity-dependent localization of the calcium-myristoyl switch protein VILIP-1 to different membrane compartments in living hippocampal neurons.
Visinin-like protein-1 (VILIP-1) belongs to the family of neuronal calcium sensor (NCS) proteins, a neuronal subfamily of EF-hand [corrected] calcium-binding proteins that are myristoylated at their N termini. NCS proteins are discussed to play roles in calcium-dependent signal transduction of physiological and pathological processes in the CNS. The calcium-dependent membrane association, the so-called calcium-myristoyl switch, localizes NCS proteins to a distinct cellular signaling compartment and thus may be a critical mechanism for the coordinated regulation of signaling cascades. To study whether the biochemically defined calcium-myristoyl switch of NCS proteins can occur in living neuronal cells, the reversible and stimulus-dependent translocation of green fluorescent protein (GFP)-tagged VILIP-1 to subcellular targets was examined by fluorescence microscopy in transfected cell lines and hippocampal primary neurons. In transiently transfected NG108-15 and COS-7 cells, a translocation of diffusely distributed VILIP-1-GFP but not of myristoylation-deficient VILIP-1-GFP to the plasma membrane and to intracellular targets, such as Golgi membranes, occurred after raising the intracellular calcium concentration with a calcium ionophore. The observed calcium-dependent localization was completely reversed after depletion of intracellular calcium by EGTA. Interestingly, a fast and reversible translocation of VILIP-1-GFP and translocation of endogenous VILIP-1 to specialized membrane structures was also observed after a depolarizing stimulus or activation of glutamate receptors in hippocampal neurons. These results show for the first time the reversibility and stimulus-dependent occurrence of the calcium-myristoyl switch in living neurons, suggesting a physiological role as a signaling mechanism of NCS proteins, enabling them to activate specific targets localized in distinct membrane compartments.
Neuroscience Research Center-Institute for Physiology of the Charite, Humboldt University Berlin, Signal Transduction Research Group, D-10117 Berlin, Germany.
Christina
C
Spilker
Thomas
T
Dresbach
Karl-Heinz
KH
Braunewell
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation / Q000502:physiology*; D002460:Cell Line; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000378:metabolism*; D020407:Internet; D007424:Intracellular Fluid / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D009226:Myristates / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D051596:Neurocalcin; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D044169:Receptors, Calcium-Sensing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D014741:Video Recording
D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D009226:Myristates; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D044169:Receptors, Calcium-Sensing; D011993:Recombinant Fusion Proteins; C496689:Vsnl1 protein, mouse; C496690:Vsnl1 protein, rat; D049452:Green Fluorescent Proteins; D002118:Calcium