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

Journal of Negative Results in Biomedicine

Mark J Zylka²  Joseph E Rittiner²  Brendan J Fitzpatrick²  Samuel B Snider²  Jaeda C Coutinho-Budd¹ 

[1] Curriculum in Neurobiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA;Department of Cell Biology & Physiology, UNC Neuroscience Center, University of North Carolina at Chapel Hill, NRB 5109D, CB #7545, 115 Mason Farm Road, Chapel Hill, NC 27599, USA

关键词: Dorsal root ganglia;    FKBP12;    Inp54p;    Rapamycin;    PIP2;    Phosphatidylinositol 4,5-bisphosphate;   

Others  :  812424
DOI  :  10.1186/1477-5751-12-13

 received in 2013-05-30, accepted in 2013-09-05,  发布年份 2013

【 摘 要 】

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-FRB^PLF (a destabilized FRB mutant) to the ubiquitously expressed Rosa26 locus, generating a Rosa26-FRB^PLF 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 FRB^PLF-expressing DRG neurons (in vitro or in vivo). Moreover, rapamycin treatment did not alter PIP₂-dependent thermosensation in vivo. Instead, rapamycin treatment stabilized FRB^PLF in cultured DRG neurons, suggesting that rapamycin promoted dimerization of FRB^PLF 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 FRB^PLF, hence limiting the use of rapamycin-inducible systems to cells with low levels of endogenous FKBP12.

【 授权许可】

   
2013 Coutinho-Budd et al.; licensee BioMed Central Ltd.

【 图 表 】

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