Characterization of Ptpmt1 phosphatase. (A) The human tissue RNA blot (Clontech, Mountain View, CA) was hybridized with [α-³²P]dCTP-labeled human Ptpmt1 cDNA probe by following a standard protocol. The blot was stripped and reprobed with the β-actin probe to check RNA loading. (B) Total RNA was extracted from the indicated mouse tissues. Ptpmt1 mRNA levels in these RNA samples were determined by real-time PCR following standard procedures. (C and D) Glutathione S-transferase (GST)–Ptpmt1 fusion protein was carefully purified and tested for its phosphatase activity using pNPP (OD410 nm, optical density at 410 nm) (C) or the indicated PIPs (Echelon Biosciences, Inc., Salt Lake City, UT) (D) as substrates as we previously reported (38). GST-DUSP23 was included as the positive control (C). (E) MEFs were loaded with MitoTracker Red (Molecular Probe, Eugene, OR) and then immunostained with anti-PI(3,5)P₂ antibody (Echelon Biosciences Inc., Salt Lake City, UT) (upper panel) or the cells were double immunostained with anti-PI(3,5)P₂ and anti-protein disulfide isomerase (PDI; a specific marker for the endoplasmic reticulum) (Stressgen, Ann Arbor, MI) antibodies (lower panel). Images were analyzed with the Zeiss laser scanning microscope LSM510 confocal imaging system. Scale bar = 15 μm.

Global knockout of Ptpmt1 results in postimplantation developmental arrest and lethality in mice. (A) The gene-targeting strategy. Genotyping primers are shown by arrows. Exon 1 containing the start codon ATG to exon 2 was replaced with neo by homologous recombination. The probe used for Southern blotting and two flanking EcoRI sites are also indicated. (B) Genomic DNA extracted from WT and Ptpmt1^+/− ES cells was digested with EcoRI and then analyzed by Southern blotting using the probe labeled with digoxigenin-11-dUTP following standard procedures. (C) Cell lysates prepared from WT and Ptpmt1^+/− ES cells were examined by Western blotting analyses with Ptpmt1 polyclonal antibody. The membrane was stripped and reprobed with anti-α-tubulin antibody to check protein loading. Relative Ptpmt1 levels were determined by densitometry. (D) Embryos produced from intercrosses of Ptpmt1^+/− mice were dissected at various stages and genotyped by PCR or Ptpmt1 immunostaining (E3.5 embryos). Values are numbers of embryos or pups. (E) Embryos produced from the intercrosses of Ptpmt1^+/− mice were dissected at E7.5 and photographed. Arrows point to embryos, while stars indicate placental corns. (F) E7.5 embryos produced from intercrosses of Ptpmt1^+/− mice were immunostained with anti-Ptpmt1 antibody and analyzed with the TUNEL assay. Arrows point to embryos, while stars indicate placental corns. (G) E6.5 deciduas from timed matings of Ptpmt1^+/− mice were dissected in ice-cold PBS, fixed in 10% formalin, dehydrated, paraffin embedded, sectioned, and stained with hematoxylin and eosin.

Inner cell mass (ICM) of Ptpmt1^−/− blastocysts fails to expand, and no Ptpmt1^−/− ES cell clones are derived in vitro. (A) E3.5 blastocysts harvested from Ptpmt1^+/− intercrosses were immunostained with anti-Oct3/4 and anti-Ptpmt1 antibodies, counterstained with the DNA dye DAPI, and analyzed under a confocal microscope. Scale bar = 100 μm. (B) E3.5 blastocysts harvested from Ptpmt1^+/− intercrosses were cultured individually in ES medium without LIF. Each blastocyst was monitored on a daily basis, documented photographically, and subsequently genotyped. (C) A total of 191 E3.5 blastocysts produced from Ptpmt1^+/− intercrosses were cultured on mitomycin C-treated STO feeder cells in ES cell medium containing LIF to generate ES cell clones following standard procedures. The 25 established ES cell clones were genotyped by PCR.

Ptpmt1 ablation decreases cell growth in conditional knockout ES cells without affecting cell survival. (A) E3.5 blastocysts produced from the intercrosses of Ptpmt1^flox/+/ER-Cre⁺ and Ptpmt1^flox/+ mice were used to generate ES cells by following standard procedures. Ptpmt1 deletion from Ptpmt1^flox/flox/ER-Cre⁺ ES cells was induced by 4-OHT (1 μM) treatment. (B) Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ ES cells were treated with 4-OHT or DMSO for 96 h. Cell growth rates were then determined by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. Three experiments were performed, with three independent cell pools for each group. Representative results from one experiment are shown. (C) Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ ES cells were treated with 4-OHT or DMSO and then cultured for the indicated periods of time. Ptpmt1 deletion efficiency in the whole-cell population was determined by real-time PCR quantification of Ptpmt1 mRNA levels. (D) Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ ES cells were treated with 4-OHT or DMSO for 96 h and then cultured in regular ES cell medium for 48 h. Apoptotic cells were assessed as described in Materials and Methods. Representative results from three independent experiments are shown. (E) Ptpmt1^flox/flox/ER-Cre⁺ ES cells were treated with 4-OHT or DMSO for 96 h and then cultured for 48 h. The cells were loaded with the rhodamine-based dye tetramethylrhodamine, ethyl ester (TMRE) (1 mM). Mitochondrial membrane potential was determined by assessing mean fluorescence intensity in the cells using flow cytometry. Experiments were repeated three times. Similar results were obtained.

Ptpmt1 ablation blocks differentiation capabilities in conditional knockout ES cells, and both catalytic activity and mitochondrial localization are required for Ptpmt1 function in ES cell differentiation. (A) Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ ES cells were treated with 4-OHT or DMSO for 96 h and then induced to differentiate into EBs in methylcellulose-Iscove's modified Dulbecco's medium (IMDM) without LIF as previously described (33, 34). After 10 days, resultant EBs were counted under a dissecting microscope. Representative results from three independent experiments are shown. (B) Ptpmt1^flox/flox/ER-Cre⁺ ES cells were treated with 4-OHT or DMSO and induced to differentiate in IMDM without LIF for the indicated periods of time. Resulting cells were harvested and assayed by real-time PCR to determine expression levels of GATA4, GATA6, brachyury, Mef2c, Fgf5, and nestin. Experiments were repeated twice. Similar results were obtained. Representative results from one experiment are shown. (C) Ptpmt1^flox/flox/ER-Cre⁺ ES cells were treated with 4-OHT or DMSO as described above. mRNA levels of the indicated cell cycle regulatory genes were determined by real-time PCR. Representative results from one experiment intriplicate are shown. (D) Ptpmt1^flox/flox/ER-Cre⁺ ES cells were treated with 4-OHT or DMSO as described above. The cells were loaded with 5-bromodeoxyuridine (BrdU; 10 μM) for 30 min, harvested, and fixed with cold 70% ethanol overnight. The cells were then washed with PBS, treated with HCl (2 N), stained with anti-BrdU antibody, and finally resuspended in PBS containing 50 μg/ml propidium iodide (PI). Cell cycle distribution was determined by FACS analyses according to BrdU- and PI-staining profiles. Results shown are means ± standard deviations of three independent experiments. (E) Flag-tagged WT Ptpmt1, catalytically deficient Ptpmt1 C132S, truncated Ptpmt1 (Ptpmt1 Δ37) lacking the mitochondrial localization signal (amino acids 1 to 37), and vector control were transduced into Ptpmt1^flox/flox/ER-Cre⁺ ES cells through retrovirus-mediated gene transfer. Transduced cells were sorted by FACS based on the expression of the GFP marker contained in the retroviral vector MSCV-IRES-GFP (MSCV, murine stem cell virus; IRES, internal ribosome entry site). Sorted cells were expanded for reverse transcription-PCR analyses for expression levels of Ptpmt1 and mutants. (F) WT Ptpmt1, Ptpmt1 C132S, Ptpmt1 Δ37, and vector control were transduced into Ptpmt1^flox/flox/ER-Cre⁺ ES cells, and the cells treated with 4-OHT or DMSO for 96 h. These cells were then induced to differentiate into EBs as described for panel A. After 10 days, resultant EBs were counted under a dissecting microscope and normalized according to the Ptpmt1 deletion efficiency of each line. Experiments were repeated three times with similar results. Representative results from one experiment are shown.

Aerobic metabolism is decreased in Ptpmt1-ablated cells. (A) 4-OHT- or DMSO-treated Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ ES cells were assayed for total cellular ATP levels as described in Materials and Methods. Experiments were performed with three independent cell pools for each group. Results shown are means ± standard deviations of three independent experiments. (B) ROS levels in 4-OHT or DMSO-treated Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ ES cells were determined as described in Materials and Methods. Experiments were performed with 3 cell pools for each group. Results shown are means ± standard deviations. (C) Oxygen consumption rates of intact 4-OHT-treated Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ ES cells were measured following the addition of mitochondrial inhibitor (oligomycin, 5 μM), uncoupling agent (FCCP, 1 μM), and respiratory chain inhibitor (rotenone, 5 μM) as described in Materials and Methods. Experiments were performed with 3 cell pools for each group. Results shown are means ± standard deviations. (D, E) Oxygen consumption (D) and glycolytic activities (E) of intact 4-OHT-treated Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ MEFs were measured following the addition of mitochondrial inhibitor (oligomycin, 1 μM), uncoupling agent (FCCP, 300 nM), and respiratory chain inhibitor (rotenone, 600 nM). (E) Baseline extracellular acidification rates are shown. Experiments were performed with three independent cell pools for each group. Representative results from one pair of cell pools are shown.

Decreased mitochondrial fusion and compromised mitochondrial dynamics in Ptpmt1-depleted cells. (A) Primary Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ MEFs at passage 3 or 4 were treated with 4-OHT for 48 h and then transfected with Mito-DsRed2 plasmid to label mitochondria. Forty-eight hours later, mitochondrial morphology in transfected cells (at least 100 cells were counted for each group) was examined under a fluorescence microscope. Normal mitochondria showed long thread-like tubular structures, whereas fragmented mitochondria were punctate and sometimes rounded. The percentage of cells with fragmented mitochondria was determined. Representative tubular and fragmented mitochondria are shown in the right panel. Experiments were performed three times. Results shown are means ± standard deviations. (B) Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ MEFs were treated with 4-OHT and then transfected with Mito-DsRed2. Fluorescence recovery after photobleaching analyses were carried out as described in Materials and Methods. Time-lapse images were captured using a Zeiss LSM 510 confocal microscope with intervals of 2 s. Regions of interest are indicated with white squares. Cells that failed to show fluorescence recovery after photobleaching were counted. In addition, in the successfully recovered cells, fluorescence recovery efficiencies 40 and 114 s after photobleaching were determined. The relative fluorescence intensity of Mito-DsRed2 recorded during a photobleaching protocol was plotted over time. Experiments were performed two times using different clones, and similar results were obtained in each. (C and D) 4-OHT-treated Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ ES cells (C) or MEFs (D) were separately transfected with Mito-DsRed2 and Mito-AcGFP plasmids to label mitochondria in red and green fluorescence, respectively. Cell lines of the same genotype that express Mito-DsRed2 or Mito-AcGFP were fused using polyethylene glycol 1500. Images of the cells were captured using a fluorescence microscope. Representative pictures are shown. Summarized data are described in the text. (E) Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ MEFs treated with 4-OHT were transfected with Mito-DsRed2. Transfected cells were monitored using a Leica DMI600B inverted microscope equipped with an environmental control chamber. Images were taken every 3 s for 5 min with a Retiga EXI 12-bit camera and analyzed with MetaMorph software. Representative pictures are shown.

PI(3,5)P₂ is accumulated in the mitochondria in Ptpmt1-depleted cells, and overloading of PI(3,5)P₂ results in defective mitochondrial dynamics. (A) 4-OHT-treated Ptpmt1^+/+/ER-Cre⁺ and Ptpmt1^flox/flox/ER-Cre⁺ MEFs were transfected with Mito-DsRed2 and then immunostained with anti-PI(3,5)P₂ antibody. PI(3,5)P₂ was visualized using Alexa Fluor 488-conjugated anti-mouse secondary antibody. Green fluorescence intensity in Mito-DsRed2-positive areas in Ptpmt1^flox/flox/ER-Cre⁺ cells (n = 137) was quantified and normalized against that in the Ptpmt1^+/+/ER-Cre⁺ control (n = 131) using laser scanning cytometric analyses. Results shown are means ± standard deviations of three independent experiments. (B) PI(3,5)P₂ (di-C₁₆) (1 μM) was delivered (shuttled) into Ptpmt1^+/+/ER-Cre⁺ MEFs using Shuttle PIP kits following the protocol provided by the manufacturer (Echelon Biosciences, Inc.). Carrier 2 was used to deliver PI(3,5)P₂. Cells were stained with Mitotracker Red 4 h later. The percentage of the cells with fragmented mitochondria was determined. Representative results of one experiment (average of three different fields, and 200 cells were counted for each field) are shown. Experiments were repeated using two different cell clones, and similar results were obtained.