Lymphocyte-specific protein tyrosine kinase (Lck) interacts with CR6-interacting factor 1 (CRIF1) in mitochondria to repress oxidative phosphorylation

Cell lines and reagents

The human T-cell line Jurkat clone E6.1 and its Lck-deficient derivative Jcam clone
1.6 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).
Jurkat E6.1, Jcam 1.6 and the mouse LSTRA leukemia cell lines were maintained as described
previously 39]. CRIF1 knock-down stable cell lines were generated in Jcam using lentiviral transduction.
CRIF1 shRNA (sc-97804-V) and scrambled shRNA control (sc-108080) lentiviral particles
were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). After 24-h starvation,
10
⁴
Jcam cells were harvested and resuspended in 50 ?l of freshly thawed virus mixture
(2 × 10
⁵
infectious units of virus). After 6-h incubation, 500 ?l of complete RPMI were added.
After one day of recovery, puromycin was added to a final concentration of 14 ?g/ml
to select for stably transduced cells. Efficiency of CRIF1 knock-down was evaluated
by Western blot and real-time PCR analyses.

Subcellular fractionation

Mitochondrial fraction was isolated by hypotonic lysis and differential centrifugation
as described previously 39]. Briefly, cells were washed in phosphate-buffered saline (PBS) and then homogenized
by passing through a 27-gauge needle in ice-cold hypotonic buffer. Light microscopy
was used to ensure cell rupture before proceeding to the next step. Mitochondria-enriched
heavy membrane fraction (mitochondrial fraction) was collected by differential centrifugation.
Fraction purity was verified by immunoblotting of specific markers.

Immunoprecipitation and immunoblotting

Whole cell lysates were prepared by solubilizing cell pellets in RIPA buffer 39]. Target proteins were either immunoprecipitated or directly detected from whole cell
lysates after SDS-PAGE using specific antibodies according to manufacturers’ instructions.
Mitochondrial proteins were extracted from heavy membrane pellets using either high
salt buffer for co-immunoprecipitation 40] or 1 % NP-40 lysis buffer for direct immunoblotting. Antibodies specific for Lck
and CRIF1 were purchased from Santa Cruz Biotechnology. Antibodies specific for VDAC1
(voltage-dependent anion channel 1), ND1 (NADH dehydrogenase subunit 1), COI (cytochrome
c oxidase subunit I) and Tid1 were purchased from Abcam (Cambridge, MA, USA). Anti-COIV
(cytochrome c oxidase subunit IV) antibody was purchased from Bethyl Laboratories (Montgomery,
TX, USA). Antibodies specific for phospho-Src family (Tyr416) and GAPDH (glyceraldehyde
3-phosphate dehydrogenase) were purchased from Cell Signaling Technology (Danvers,
MA, USA). Anti-phosphotyrosine antibody (clone 4G10) was purchased from EMD Millipore
(Billerica, MA, USA). Appropriate secondary antibodies conjugated with horseradish
peroxidase were used in enhanced chemiluminescence system to detect signals. Conformation-specific
antibodies that do not recognize heavy chains in the immunoprecipitates (from Affymetrix
eBioscience, San Diego, CA, USA) were also used to minimize interference in detecting
Lck signals. For signal quantitation, the bands were digitalized using the AlphaImager
2200 (ProteinSimple, San Jose, CA, USA) and analyzed by the ImageJ software.

Confocal immunofluorescence microscopy

Live cells were incubated with 100 nM of MitoTracker Deep Red (Life Technologies,
Grand Island, NY, USA) for 20 min under regular culture condition or left unstained
as a negative control. Stained cells were washed with PBS, adhered to 10-well slides,
fixed, and permeabilized as previously described 40]. Cells were blocked with Image-iT FX signal enhancer (Life Technologies) for 15 min
at room temperature, and then either singly or doubly stained with primary antibodies.
Subsequent labeling with Alexa Fluor-conjugated secondary antibodies and DAPI counterstain
(Life Technologies) were performed to visualize primary antibodies and nuclei, respectively.
Stained cells were viewed using the Olympus FV10i fluorescence confocal microscope.
Images were analyzed using the Fluoview software (Olympus, Melville, NY, USA).

In situ proximity ligation assay (PLA) microscopy

PLA was performed using the DuoLink PLA Kit (Sigma-Aldrich, St. Louis, MO, USA) to
detect close-range protein-protein interactions under a fluorescence microscope according
to manufacturer’s protocol. Briefly, 10
⁴
cells were seeded on each well of 10-well slides. Adhered cells were fixed with 4 %
paraformaldehyde for 15 min at room temperature, and then permeabilized with 0.2 %
Triton X-100. After treatment with DuoLink blocking buffer for 30 min at 37 °C, cells
were incubated with diluted primary antibodies from two different species for another
hour at 37 °C. After washing, cells were incubated with species-specific PLA probes
and two additional oligonucleotides under conditions that facilitate hybridization
only in close proximity (16 nm). A ligase was added to join the hybridized oligonucleotides
to form a closed circle. A rolling-circle amplification step with polymerase was then
performed to generate a concatemeric product extending from the oligonucleotide arm
of the PLA probe. The amplified product can be visualized with fluorophore-labeled
oligonucleotides after hybridization as distinct fluorescent dots under a fluorescence
microscope. For negative controls, samples were treated as described above, except
that no primary antibodies were added. Slides were also counterstained with DAPI to
visualize the nuclei.

Quantitative real-time PCR analysis

Total RNAs were extracted by TRIzol (Life Technologies), treated with RQ1 RNase-free
DNase (Promega, Madison, WI, USA), and then reverse transcribed using High Capacity
cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) into cDNAs.
Quantitative real-time PCR using SYBR Green chemistry (Applied Biosystems) was performed
according to standard protocol using an annealing temperature of 60 °C for all primer
sets. Relative fold values were obtained using ??CT method by normalization to ?-actin.
Primers for various human genes are itemized below.

ND1 (forward): 5?-GAGCAGTAGCCCAAACAATCTC-3?

ND1 (reverse): 5?-AAGGGTGGAGAGGTTAAAGGAG-3?

COI (forward): 5?-CAATATAAAACCCCCTGCCATA-3?

COI (reverse): 5?-GCAGCTAGGACTGGGAGAGATA-3?

COIV (forward): 5?-TGGATGAGAAAGTCGAGTTG-3?

COIV (reverse): 5?-CTTCTGCCACATGATAACGA-3?

CRIF1 (forward): 5?-GGTGGTCCCCGGTTCGTTATGG-3?

CRIF1 (reverse): 5?-CTCGCGCCTCCTTCTTCCGTTTCT-3?

Actin (forward): 5?-CGCAGAAAACAAGATGAGATTG-3?

Actin (reverse): 5?-ACCTTCACCGTTCCAGTTTTTA-3?

Mass spectrometry

Whole cell pellet of LSTRA was solubilized with 1 % NP-40 lysis buffer. Lysates with
500 ?g of proteins were immunoprecipitated with 2 ?g of anti-Lck antibody or control
mouse IgG overnight. Immunoprecipitates were resolved using 4–20 % gradient SDS-PAGE
(Bio-Rad, Hercules, CA, USA) and visualized with Coomassie blue staining. A total
of eight bands specifically present in the Lck immunoprecipitates, but not in the
IgG control, were cut out from the gel. Proteins extracted from gel slices were analyzed
by mass spectrometry using liquid chromatography-electrospray ionization-tandem mass
spectrometry (LC-ESI-MS/MS) based approach at the Midwest Proteome Center, Rosalind
Franklin University of Medicine and Sciences (RFUMS).

Electron microscopy

Jurkat and Jcam cells were washed with warm PBS and prefixed in 0.2 % paraformaldehyde
and 0.25 % glutaraldehyde for 15 min at room temperature. Prefixed cells were centrifuged
and resuspended in ice-cold fixation solution (2 % paraformaldehyde and 2.5 % glutaraldehyde)
overnight. Cell pellets were washed in 0.1 M Sorensen’s sodium phosphate buffer (SPB),
pH 7.4 at room temperature for 15 min, followed by post-fixation with 1 % OsO4 and
1.5 % K4Fe(CN) in SPB for 1 h. After washing, cell pellets were dehydrated through
an ascending ethanol series and embedded in Epon 812 resin. Ultra-thin sections were
cut with a diamond knife and Leica UC-6 ultramicrotome, and collected onto 200-mesh
grids. Sections on grids were contrasted using Reynolds’ lead citrate stain and then
viewed using a JEOL JEM-1230 transmission electron microscope (Peabody, MA, USA).
Digital images were collected using a Hamamatsu Orca high resolution CCD camera.

Oxygen consumption analysis

Oxygen consumption rate was measured using a Clark-type electrode equipped with the
782 oxygen meter (Strathkelvin Instrument, North Lanarkshire, Scotland) with a water
circulation system to maintain the reaction condition at 37 °C. Cells were washed
with warm PBS and then adjusted to a final concentration of 10
⁷
cells per ml in TD assay buffer (0.137 M NaCl, 5 mM KCl, 0.7 mM Na
₂
HPO
₄
, 25 mM Tris, pH 7.4) 41]. Five million cells were transferred to water-jacked chamber MT-200 (Strathkelvin
Instrument) to record their oxygen consumption rate. Homogenous distribution of cells
was maintained throughout the recording process by constant magnetic stirring. Other
than the measurement of basal oxygen consumption rates, oligomycin (Cayman Chemical,
Ann Arbor, MI, USA) was also added to the same chamber at a final concentration of
500 nM to determine the oxygen consumption rates independent of ATP. Data were analyzed
using the SI 782 Oxygen System software version 3.0 (Warner Instruments LLC, Hamden,
CT, USA) and normalized to cell number.

Mitochondrial superoxide measurement

Mitochondrial superoxide was measured using MitoSOX Red (Life Technologies) according
to manufacturer’s protocol. MitoSOX Red is a fluorescent dye that targets mitochondria
in live cells and is specifically oxidized by superoxide. Approximately 10
⁶
cells were stained with 1 ?M MitoSOX Red for 20 min at 37 °C, washed in warm PBS and
then analyzed by the LSR II flow cytometer (BD Bioscience, San Jose, CA, USA). Mean
fluorescence intensity from oxidized MitoSOX (ex/em 510/580) positively correlates
with mitochondrial superoxide levels.

Mitochondrial membrane potential measurement

Mitochondrial membrane potential was measured using tetramethylrhodamine, ethyl ester
(TMRE) according to manufacturer’s protocol. Mitochondrial membrane potential drives
the accumulation of TMRE, a fluorescent dye, within the inner membrane region. Approximately
10
⁶
cells were harvested and washed with warm plain RPMI media and then resuspended in
TMRE solution (Life Technologies) at the final concentration of 25 nM. After incubation
at 37 °C for 30 min, cells were washed and then analyzed by flow cytometry. Mean fluorescence
intensity (ex/em 549/575) positively correlates with mitochondrial membrane potential.

Statistical analysis

Data are presented as mean?±?S.E. from at least three independent experiments. The
significance of differences was analyzed by Student’s t-test (SigmaPlot 11, Chicago, IL, USA). Differences were considered significant when
p??0.05.