Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for depleting PIP2 in dorsal root ganglia neurons Public Deposited

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Creator
  • Coutinho-Budd, Jaeda C.
    • Affiliation: School of Medicine, UNC Neuroscience Center, Neuroscience Curriculum, Neuroscience Center
  • Zylka, Mark J.
    • Affiliation: School of Medicine, UNC Neuroscience Center, Neuroscience Curriculum, Neuroscience Center, Department of Cell Biology and Physiology
  • Rittiner, Joseph E.
    • Affiliation: School of Medicine, Neuroscience Center, Department of Cell Biology and Physiology
  • Fitzpatrick, Brendan J.
    • Affiliation: School of Medicine, Neuroscience Center, Department of Cell Biology and Physiology
  • Snider, Samuel B.
    • Affiliation: School of Medicine, Neuroscience Center, Department of Cell Biology and Physiology
Abstract
  • Abstract: Background: Rapamycin-induced translocation systems can be used to manipulate biological processes with precise temporal control. These systems are based on rapamycin-induced dimerization of FK506 Binding Protein 12 (FKBP12) with the FKBP Rapamycin Binding (FRB) domain of mammalian target of rapamycin (mTOR). Here, we sought to adapt a rapamycin-inducible phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphatase (Inp54p) system to deplete PIP2 in nociceptive dorsal root ganglia (DRG) neurons. Results: We genetically targeted membrane-tethered CFP-FRBPLF (a destabilized FRB mutant) to the ubiquitously expressed Rosa26 locus, generating a Rosa26-FRBPLF knockin mouse. In a second knockin mouse line, we targeted Venus-FKBP12-Inp54p to the Calcitonin gene-related peptide-alpha (CGRPα) locus. We hypothesized that after intercrossing these mice, rapamycin treatment would induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in CGRP+ DRG neurons. In control experiments with cell lines, rapamycin induced translocation of Venus-FKBP12-Inp54p to the plasma membrane, and subsequent depletion of PIP2, as measured with a PIP2 biosensor. However, rapamycin did not induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in FRBPLF-expressing DRG neurons (in vitro or in vivo). Moreover, rapamycin treatment did not alter PIP2-dependent thermosensation in vivo. Instead, rapamycin treatment stabilized FRBPLF in cultured DRG neurons, suggesting that rapamycin promoted dimerization of FRBPLF with endogenous FKBP12. Conclusions: Taken together, our data indicate that these knockin mice cannot be used to inducibly deplete PIP2 in DRG neurons. Moreover, our data suggest that high levels of endogenous FKBP12 could compete for binding to FRBPLF, hence limiting the use of rapamycin-inducible systems to cells with low levels of endogenous FKBP12.
Date of publication
Identifier
  • 24010830
  • doi:10.1186/1477-5751-12-13
Resource type
  • Article
Rights statement
  • In Copyright
Rights holder
  • Jaeda C Coutinho-Budd et al.; licensee BioMed Central Ltd.
License
Journal title
  • Journal of Negative Results in BioMedicine
Journal volume
  • 12
Journal issue
  • 1
Page start
  • 13
Language
  • English
Is the article or chapter peer-reviewed?
  • Yes
ISSN
  • 1477-5751
Bibliographic citation
  • Journal of Negative Results in BioMedicine. 2013 Sep 08;12(1):13
Access
  • Open Access
Publisher
  • BioMed Central Ltd
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