Engineered FKBP** permits rapid, reversible and tunable regulation of PTEN function in cellular models.

<p><b>(A)</b> FKBP**-PTEN is reversibly regulated in response to Shld1 treatment in cells. U87MG cells stably expressing FKBP**-PTEN were treated with vehicle or with Shld1 (1μM) for 24 h. One set of cells was then washed thoroughly with DMEM and cultured in Shld1-free culture media for further 24hs. The expression level of FKBP**-PTEN was examined by immunofluorescence staining. <b>(B)</b> upper panel, U87MG cells stably expressing FKBP**-PTEN were treated with Shld1 (1 μM) for different time periods; lower panel, Shld1 (1 μM) was removed after 24 h treatment, and cultured in Shld1 free culture media for different time periods as indicated. <b>(C)</b> PTEN protein levels and activity can be maintained at specific concentrations over a prolonged period of time in dependence of Shld1. U87MG cells stably expressing FKBP**-PTEN were cultured in media containing indicated concentrations of Shld1 for 24 h. <b>(D)</b> Stable FKBP**-PTEN expressing U87MG cells were analyzed in cell invasion (left) and colony formation (right) assays. In the cell invasion assay, data are shown as the mean ±SEM of the number of cells migrated in three independent experiments. Plating efficiency refers to the ratio of the number of colonies to the number of cells seeded, shown as mean ±SEM of three independent experiments.</p

Engineered FKBP** permits regulation of PTEN function in the zebrafish.

<p><b>(A)</b> The migration and growth of stable FKBP**-PTEN expressing U87MG cell clones injected into zebrafish embryos can be inhibited by Shld1. Prior to microinjection into the perivitelline cavity of 2 days post fertilization (dpf) zebrafish embryos, cells were pre-stained with DiI. Embryos were then transferred into fish water with (n = 11) or without (n = 13) Shld1 (4 μM) and maintained for 24h. Images were taken immediately after injections (0 days post injections, dpi) and after 24 h (1dpi). <b>(B)</b> The migration indices and the increase in tumor mass size of transplanted cells were quantified. ** p<0.01; ***p<0.001.</p

Codon optimization of FKBP** establishes a conditional fine-tuning protein regulation system.

<p><b>(A)</b> The transcription of the destabilization cassette FKBP**-PTEN was subcloned downstream of LoxP-Stop-LoxP; transcription was driven by the ubiquitous CMV-enhanced chicken beta actin promoter (CAG). Presence of Cre induces FKBP**-PTEN gene transcription; however, the translated FKBP**-PTEN will be rapidly degraded. Addition of Shld1 confers FKBP**-PTEN stabilization in a tunable and reversible manner. <b>(B)</b> Cre-mediated cleavage of LoxP-Stop-LoxP (LSL) created a truncated FKBP**-PTEN fusion protein. Mouse forebrain neurons were nucleofected with LSL-FKBP**-PTEN and Cre-IresRFP (Cre (+)), or RFP (Cre (-)). Cells were treated with Shld1 (or control vehicle) overnight, before analyses of cell lysates using indicated antibodies. <b>(C)</b> Sequence alignment of FKBP** and LoxP. The third ATG codon region sequence (TATGCTA) of FKBP** (M3L^cta) is identical to the linker region of the LoxP stem-loop sequence (TATGCTA). Codon optimization of CTA with TTG (both code for amino acid Leu) will destroy the potential pseudo-cleavage site of Cre on FKBP**. <b>(D)</b> Codon optimization of M3L^cta to M3L^ttg in FKBP** abolished the Cre-dependent FKBP**-PTEN truncation and produced a Shld1 dependent PTEN fusion protein. The M3L^ttg modified LSL-FKBP**-PTEN construct was co-expressed with Cre-IresRFP (Cre (+)), or RFP (Cre (-)) in mouse forebrain neurons as before. <b>(E)</b> Combinatorial use of the Cre-LoxP system with the FKBP**-PTEN/Shld1 chemical-genetic protein control system in PTEN^flox/flox cells. Constructs were nucleofected into <i>PTEN</i>^{<i>loxp/loxp</i>} mouse primary neurons as before. Note that nucleofection of primary cells occurs with efficiencies at approx. 80%, and result in a residual endogenous PTEN signals detected by western blotting. <b>(F)</b> The generated system is able to couple PTEN-loss directly with the expression of tunable FKBP**-PTEN. Upon Cre-mediated recombination, <i>PTEN</i>^{<i>loxp/loxp</i>} cells will lose PTEN and, at the same time, activate expression of FKBP**-PTEN. In essence, endogenous PTEN is replaced by tuneable PTEN, which will enable to test whether PTEN-loss induced phenotypes can be rescued at different time-points (<i>t</i>_Shld1) and/or at different PTEN-concentrations (<i>d</i>_Shld1).</p

Consequence of linker optimization on PTEN activity of the FKBP*-PTEN fusion protein.

<p>(<b>A)</b> Rationale of the DD-technology: Fusion of PTEN with a modified human FKBP variant harboring the F36V and L106P point mutations (FKBP*) leads to degradation of the fusion protein. Presence of the FKBP* ligand Shld1 confers stabilisation of FKBP*-PTEN and results in inhibition of PI3K/Akt signaling. (<b>B)</b> The fusion protein FKBP*-PTEN shows no PTEN activity. PTEN-deficient U87MG cells were transiently co-transfected with FKBP*-PTEN and AKT-GFP. After one day, cells were treated with Shld1 for 12 hours at 1μM before cell lysis and samples analyses by western blotting using indicated antibodies. <b>(C)</b> Consequence of linker optimization on PTEN activity of the FKBP*-PTEN fusion protein. Schematic representation of linker optimization. FKBP*-linker-PTEN variants with different linker regions (A-E) were generated. Linker A—short flexible linker; Linker B—a helix forming linker. Linker C—long flexible linker. Linker D—long flexible linker containing the PWR motif. Linker E—the original linker present in the enzymatic active GFP-PTEN. <b>(D)</b> Analyses of the effect of different linkers on PTEN activity in FKBP*-PTEN fusion variants in U87MG cells. Constructs expressing FKBP*-PTEN variants or GFP-PTEN were co-transfected with AKT-GFP into a PTEN null U87MG cells. Stabilization of FKBP*-PTEN or GFP-PTEN in presence or absence of Shld1 was monitored by PTEN antibody. The phosphorylation level of AKT-GFP at Ser473 served as a fast and effective experimental protocol to control for PTEN activity towards PIP3-dependent signaling in transfected cells, only. Only constructs 4 and 5, which contain proline in the linker sequence, showed moderate PTEN activity towards PI3K signaling.</p

FKBP surface engineering restores PTEN activity of FKBP**-PTEN fusion protein.

<p><b>(A)</b> Electrostatic potential map of PTEN (PDB ID: 1d5r) and FKBP (PDB ID: 1BL4). Positively charged binding pocket of the PTEN PIP3 substrate is circled with a white dashed line; the negatively charged lump formed by E31/D32 residues on the FKBP surface is circled by a black dashed line. <b>(B)</b> Schematic representation of amino acid residue substitutions of FKBP* protein surface at E31/D32. <b>(C)</b> Different FKBP*-PTEN mutants were transiently co-tansfected with AKT-GFP into U87MG cells, the expression of fusion protein and pAKT were examined by Western blotting. <b>(D)</b> E31S/D32S amino acid substitutions on FKBP* (FKBP**) restores PTEN activity of the FKBP**-PTEN fusion protein. FKBP**-PTEN (or GFP/GFP-PTEN) was transiently co-expressed with AKT-GFP in U87MG cells. Cells were treated with Shld1 and analyzed as before.</p

Altering levels of drebrin and pS142 phosphorylation affects neuroblast migration along the RMS.

<p>P2 mice were electroporated <i>in vivo</i> with plasmids encoding YFP, or YFP-tagged wt, S142A, or S142D drebrin. Fluorescently-labelled RMS neuroblasts were imaged 5 days later in acute brain slice cultures over a period of 3 hours. (A) Representative migration paths of neuroblasts expressing YFP empty vector, YFP-tagged wt, S142A, or S142D drebrin. Yellow asterisks mark the location of the OB. Quantitative tracking analysis reveals that neuroblasts overexpressing any one of the drebrin versions display a lower migratory distance (B), displacement (C), speed (D) and a trend towards a lower migratory index (E) compared to control cells expressing only YFP (mean ± SEM; n = 6 brains for control; n = 5 brains for wt; and n = 4 brains for S142A and n = 5 for S142D; **<i>P</i><0.01, ***<i>P</i><0.001).</p

pS647-Drebrin is negatively regulated by PTEN in neurons independently of PI3K.

<p>(A) Cortical neurons isolated from <i>PTEN</i>^{<i>flox/flox</i>} mice were transduced with increasing concentrations of Cre-expressing viruses at 13 DIV. Neuronal cell lysates were analysed at 19 DIV. Bar graph represents the relative band density of pS647-DBN and PTEN in three independent experiments with maximal cre-virus titer used, <u>+</u> sem. *p<0.05, *** < 0.001. (B) Rat cortical neurons were treated with the PI3K inhibitor LY294002 at indicated concentrations for 60 minutes before analyses of neuronal cell lysates. Bar graph represents the average band density of pS647-DBN in three independent experiments. (C) Hippocampal neurons were cultured for 1.5 DIV and labelled with anti-DBN, anti-pS647-DBN and anti-PTEN antibodies. (D) 18 DIV hippocampal neurons were stained with anti-DBN, anti-pS647-DBN and anti-PTEN. Top images show a specimen images at higher magnification. DBN is highly enriched in dendritic spines, but also found - albeit at lower levels - in the dendrite and the soma, whereas phosphorylation at S657 is more confined to the dendritic spine compartment. (E) Hippocampal neurons were cultured for 18 DIV and stained with anti-pS647-DBN and anti-PTEN antibodies. In dendritic spines (<b>arrows</b>), DBN is highly phosphorylated on pS647, whereas PTEN is mainly present in the dendrite process. Sporadically, PTEN is found in spines (<b>star</b>), which coincides with depleted pS647-Drebrin labeling. (F) Cortical neurons isolated from PTEN floxed/floxed mice were nucleofected with RFP or Cre-RFP and cultured for DIV3 before staining for pS647-DBN. Scale bars C, D, F: 10 µm; E: 2 µm.</p

Drebrin is highly expressed in SVZ-derived migratory neuroblasts.

<p>(A) Immunostained sagittal brain sections from P7 (left) and P90 (right) mice show strong drebrin expression (brown) in the subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB). (B) Confocal images from P7 mice SVZ sagittal sections showing that drebrin immunostaining overlaps with DCX+ migrating neuroblasts (top row), but is almost completely excluded from GFAP+ stem cells and astrocytes (middle row). Very little colocalization is observed with Mash-1+ transit-amplifying progenitors (bottom row). Merge panels include DAPI staining to visualize cell nuclei and higher magnification insets. Scale bars: (A), 200 μm; (B), 10 μm; insets, 5 μm.</p

Altering levels of drebrin and pS142 phosphorylation of drebrin affects neuroblast distribution in the OB.

<p>P2 mice were electroporated in the lateral ventricle with plasmids encoding YFP or YFP-tagged wt, S142A or S142D drebrin. Sagittal brain slices were prepared 14 days later and immunostained for YFP. (A, top row) Representative confocal projections of sagittal brain slices showing overall labeling in the OB and overlaid with the mask used for analysis (area A, inner OB; area B, outer OB). High magnification pictures of the OB insets are shown in the bottom row. (B) Schematic diagrams of a typical quantification analysis using all OB sections from one brain for each condition. Cell bodies in areas A and B are represented as dots. (C) Overexpression of wt, S142A and S142D drebrin caused a significant cell accumulation in the inner OB compared to GFP (mean ± SEM; n = 3 brains for each construct; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). Scale bars: (A, top row), 500 μm; (A, bottom row), 100 μm.</p

Drebrin phosphorylation on S142 regulates neuroblast morphology and orientation <i>in vivo</i>.

<p>(A) Sagittal brain slices were immunostained for YFP 5 days after <i>in vivo</i> electroporation of plasmids encoding YFP-tagged wt, S142A or S142D drebrin and imaged with a confocal microscope. The empty vector encoding only YFP served as control. The majority of control cells display a single leading process oriented towards the OB (yellow asterisk). Many neuroblasts overexpressing wt drebrin display a bipolar morphology with two diametrically opposite protrusions (one towards the OB and one towards the SVZ) (wt, arrowheads and D). Overexpression of S142A or S142D drebrin significantly increases the percentage of misoriented cells (pointing towards the SVZ instead of the OB, arrowheads) compared to control or wt drebrin (C). (B) Quantitative morphological analysis also shows a decrease in leading process length for wt, S142A and S142D compared to the control (mean ± SEM; n = 8 brains for empty vector; n = 5 brains for wt, S142A, and S142D drebrin; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). Scale bar: 50 μm.</p