Characteristics of EYFP-actin and visualization
of actin dynamics during ATP depletion and
repletion
Stefan
Herget-Rosenthal1,2,
Melanie
Hosford1,
Andreas
Kribben2,
Simon J.
Atkinson1,
Ruben M.
Sandoval1, and
Bruce A.
Molitoris1
1 Indiana Center for Biological Microscopy, Division of
Nephrology, Department of Medicine, Indiana University School of
Medicine and Roudebush Veterans Affairs Medical Center,
Indianapolis, Indiana 46202; and 2 Division of Nephrology,
Department of Medicine, University Hospital Essen, Essen,
Germany
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ABSTRACT |
Disruption of the
actin cytoskeleton in proximal tubule cells is a key pathophysiological
factor in acute renal failure. To investigate dynamic alterations of
the actin cytoskeleton in live proximal tubule cells,
LLC-PK10 cells were transfected with an enhanced yellow
fluorescence protein (EYFP)-actin construct, and a clone with stable
EYFP-actin expression was established. Confluent live cells were
studied by confocal microscopy under physiological conditions or during
ATP depletion of up to 60 min. Immunoblots of stable
transfected LLC-PK10 cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogenous cytosolic distribution of EYFP-actin indicated
colocalization with G-actin as well. Beyond previous findings, we
observed differential subcellular disassembly of F-actin structures:
stress fibers tagged with EYFP-actin underwent rapid and complete
disruption, whereas cortical and microvillar actin disassembled at
slower rates. In parallel, ATP depletion induced the formation of
perinuclear EYFP-actin aggregates that colocalized with F-actin. During
ATP depletion the G-actin fraction of EYFP-actin substantially
decreased while endogenous and EYFP-F-actin increased. During
intracellular ATP repletion, after 30 min of ATP depletion, there was a
high degree of agreement between F-actin formation from EYFP-actin and
endogenous actin. Our data indicate that EYFP-actin did not alter the
characteristics of the endogenous actin cytoskeleton or the morphology
of LLC-PK10 cells. Furthermore, EYFP-actin is a suitable
probe to study the spatial and temporal dynamics of actin cytoskeleton
alterations in live proximal tubule cells during ATP depletion and ATP repletion.
actin cytoskeleton; green fluorescent protein; live imaging; renal
proximal tubule cell; enhanced yellow fluorescent protein
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INTRODUCTION |
ISCHEMIA IN
VIVO and cellular ATP depletion in vitro severely disrupt the
actin cytoskeleton of renal proximal tubule cells (1, 12, 14, 15,
20). Filamentous actin structures within the microvilli, in the
meshwork beneath the junctional complexes and in actin stress fibers,
are fragmented (1). Disruption of the actin cytoskeleton
initiates further structural cellular changes, such as loss of surface
membrane polarity and microvilli, membrane destruction, opening of
tight junctions, dissociation of junctional complexes, and detachment
of cells from the basement membrane (13, 17, 18). These
alterations are detrimental to the reabsorbtive and secretory function
of proximal tubule cells since their physiological function depends on
an intact polarized structure (18, 26). In addition, these
cellular alterations have been related to abnormalities in
tubuloglomerular feedback, backleak of glomerular filtrate, and tubular
obstruction, as mechanisms for decreased glomerular filtration rate and
acute renal failure (19, 23). Therefore, disruption of the
actin cytoskeleton in proximal tubule cells is a key factor in the
pathophysiology of acute renal failure.
Despite the important insights provided by previous studies, the
precise dynamics of actin cytoskeletal alterations in proximal tubule
cells during ATP depletion and repletion are poorly understood. Previous studies of actin cytoskeletal alterations in proximal tubule
cells have predominantly been of static nature, i.e., performed on
fixed samples (1, 11, 12, 17, 20). To analyze the spatial
and temporal details of highly dynamic, actin cytoskeletal alterations,
studies in live cells are of great benefit (2, 9, 25).
Dynamic studies with microinjection of fluorescently labeled actin have
been limited by the short experimental time due to proteolysis of
labeled actin, by mechanical injury to cells, and by the small number
of cells microinjected. Fusion of proteins with green fluorescent
protein (GFP) has become a useful method to observe proteins in living
cells (10, 16). GFP fusion proteins enable direct
visualization of protein localization and dynamics in a large number of
unimpaired cells in real time (2, 5, 8, 9, 25, 27).
However, GFP fusion proteins need to be characterized before use, as
they may display nonphysiological properties and may impair the
properties of endogenous proteins (27, 29).
Therefore, the purpose of the present study was to evaluate the
application of enhanced yellow fluorescent protein (EYFP)-actin fusion
protein as a probe for actin in live proximal tubule cells. EYFP is a
mutant form of GFP with enhanced fluorescent intensity. We hypothesized
that EYFP-actin would colocalize with endogenous F-actin and G-actin,
show the characteristics of these actin fractions, and provide
visualization of spatial and temporal alterations of different actin
cytoskeletal structures during ATP depletion and repletion.
Furthermore, EYFP-actin would not interfere with the behavior of the
endogenous actin cytoskeleton.
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METHODS |
Cell culture, reagents, and ATP depletion and
repletion.
LLC-PK10 cells, a clone of porcine proximal tubule
LLC-PK1 cells, were utilized for all studies.
LLC-PK10 cells were maintained in 1:1 DMEM/Ham's F-12
medium (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, 14 mM NaHCO3,
and 12.5 mM HEPES in a 5% CO2 incubator at 37°C. For live imaging and transfection, cells were plated on
poly-D-lysine-coated glass coverslips mounted on culture
dishes. For imaging of fixed cells, LLC-PK10 cells were
grown on glass coverslips. Experiments were performed on confluent
cells. Reagents were from Sigma (St. Louis, MO), unless otherwise
indicated. For ATP depletion, LLC-PK10 cells were incubated
with substrate-free medium (no glucose, amino acids, pyruvate, FCS, or
geneticin) containing 0.1 µM antimycin A (17). To allow
ATP repletion, substrate-free medium was replaced with complete medium
containing FCS.
Transfection procedures and selection of
EYFP-actin-expressing cells.
LLC-PK10 cells were plated at 5 × 104
cells/plate and transfected 48 h later with 1 µg of purified
plasmid DNA encoding for EYFP or with 1 µg of plasmid DNA encoding
for EYFP-tagged
-actin (both from Clontech, Palo Alto, CA), mixed
with 2 µg Novafector reagent (Venn Nova, Pompano Beach, FL) per dish
in 200 µl of FCS-free DMEM. EYFP is linked via its COOH-terminal end
to the NH2-terminal end of actin with a seven-amino-acid
linker. Cells were incubated with the DNA-Novafector mixture for 6 h at 37°C, and then complete medium was added. A cell population
entirely expressing EYFP-actin was selected with medium containing
geneticin (200 µg/ml; GIBCO BRL). Two consecutive selection steps
were performed, and EYFP-actin expression was confirmed by fluorescence
microscopy. This cell population was maintained in geneticin-containing
medium. Transfection procedures for transient transfections were
performed similarly. Experiments were performed 24-96 h after transfection.
Fluorescent staining.
Cells were fixed in 4% paraformaldehyde in PBS overnight at 4°C,
permeabilized in 0.1% Triton X-100 in PBS for 10 min, and blocked in
PBS containing 2% BSA for 1 h at room temperature. F-actin was
labeled with 0.1 µg/ml Texas red or Alexa-647-conjugated phalloidin
(Molecular Probes, Eugene, OR) for 1 h at room temperature. G-actin was labeled with the G-actin-specific monoclonal mouse antibody
JLA-20 (1:100; obtained from Developmental Studies Hybridoma Bank,
University of Iowa, Iowa City, IA) (4) for 1 h at
room temperature. This was followed by a 1-h incubation with Cy-5
conjugated goat anti-mouse IgM (Jackson Immuno Research, West Grove,
PA). The samples were mounted in 50% glycerol/PBS with 100 mg/ml
1,4-diamino-bicyclo[2,2,2]octane (Sigma).
Immunofluorescence microscopy.
Images were collected with an MRC-1024 laser scanning confocal
microscope (Bio-Rad, Hercules, CA) on a Nikon Diaphot 200 inverted stand using ×40 numerical aperture (NA) 1.3 or ×100 NA 1.4 oil-immersion objectives (Nikon, Melville, NY). During live studies
temperature was kept constant at 37°C with a warm stage and pH 7.4 was maintained by gassing with 5% CO2. To avoid possible
spectral overlap, all signals were excited and acquired sequentially.
Through focus, optical series were collected from entire cell volumes
with separations of 0.2-0.4 µm between focal planes. Images were
processed with Metamorph 4.01 imaging software (Universal Imaging, West
Chester, PA). We quantified stress fibers in live cells under
physiological conditions and during ATP depletion by measuring the mean
EYFP-actin fluorescence intensity from representative stress fibers,
summed from four basal planes, according to an established protocol
(21). Background fluorescence from cytoplasmic actin was
subtracted from EYFP-actin fluorescence. A threshold function was
applied to the resulting image and converted to a binary mask.
An erode function was applied to remove small particulate structures
while retaining filamentous structures. The mask was then multiplied against the original image, with the result that stress fiber fluorescence was retained with pixel values unaltered, whereas nonstress fiber fluorescence was removed. For all consecutive measurements during one time sequence, the fluorescence intensity was
determined within the same area of the respective stress fiber.
Microscopic colocalization of EYFP-actin and
Alexa-647 phalloidin.
Corresponding images from EYFP-actin and Alexa-647
phalloidin acquired simultaneously were processed using Metamorph
software v 4.1 (Universal Imaging). Because the question of
colocalization of the two signals was the main issue to be addressed,
the images were first enhanced in contrast and brightness using the
"autoenhance" function. Next, a 3 × 3 low-pass filter was
applied. The average intensity of the EYFP signal was calculated. The
Alexa-647 phalloidin image was then subtracted from the EYFP-actin, and
the average intensity from the subtracted image was calculated.
Background readings were taken from several subconfluent areas, and the
values were averaged and then subtracted from both average intensity readings to yield a background corrected value. For each corresponding image, intensity was normalized to the EYFP-actin fluorescence. A value
for percent colocalization was derived by subtracting the residual
intensity from the EYFP-actin intensity.
SDS-PAGE and immunoblotting.
To recover cell homogenate for total actin, cells were extracted in hot
SDS buffer (1% SDS, 10 mM Tris, pH 7.5, 2 mM EDTA), and lysates were
boiled for 3 min and sonicated. To recover supernatant samples for
G-actin, cells were extracted with a PBS extraction buffer containing
0.1% Triton X-100, 10 mM EDTA, 2 mM MgSO4, 0.5 mM
phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol, and 10 µg/ml
each of chymostatin, leupeptin, aprotinin, and pepstatin A for 30 s on ice. Cells were scraped and lysates centrifuged for 15 min
at 12,000 g at 4°C. Samples were resuspended in equal volumes of 2× sample buffer (4% SDS, 20% glycerol, 10%
-mercaptoethanol, 0.5 M Tris, pH 6.8, containing bromophenol blue)
and immediately frozen at
20°C. We measured total protein
concentrations by bicinchoninic acid assay (Pierce, Rockford, IL).
Equal quantities of total protein were loaded on each lane, and
proteins were electrophoretically separated on 14% polyacrylamide
gels. Proteins were transferred to polyvinylidene difluoride membranes
(Millipore, Bedford, MA) in a buffer containing 10% methanol,
0.1% SDS, 40 mM glycine, and 120 mM Tris, pH 8.2. Membranes were
blocked in wash buffer (0.1 M NaCl, 0.01 M Tris, 0.05% Tween 20, pH
7.4) containing 10% newborn calf serum at 4°C overnight and
incubated with the monoclonal panactin antibody C4 (1:2,000; Roche,
Indianapolis, IN) or a monoclonal GFP antibody (1:3,000; BABCO,
Richmond, CA) for 2 h at room temperature. Incubation with
horseradish perioxidase-conjugated goat anti-mouse IgG (1:40,000;
Southern Biotechnology, Birmingham, AL) for 1 h at room
temperature followed. The antigen-antibody complexes were detected with
enhanced chemiluminescence (Pierce) and exposed to film (Eastman Kodak,
Rochester, NY). For quantification, films were scanned using a
Silverscanner III (LaVie, Beaverton, OR) and analyzed using Bio Image
Intelligent Quantifier software (BI Systems, Ann Arbor, MI). The
concentration of endogenous actin concentration in supernatant and
homogenate was determined by densitometrically quantifying the
respective bands and comparing them to actin standards. The
concentration of EYFP-actin in supernatant and homogenate was
approximated by densitometrically quantifying the respective bands and
comparing them to rGFP standards (Clontech).
F-actin determination.
F-actin content was determined in confluent cells grown on 96-well
plates and fixed and stained with 0.1 mg/ml
tetramethylrhodamine isothiocyanate (TRITC)-phalloidin and
50 µg/ml 4',6-diamidino-2-phenylindole (DAPI; procedures as
above) (3). TRITC fluorescence intensity was
measured on the Cytofluor II fluorescence plate reader (PerSeptive Biosystems, Framingham, MA). TRITC fluorescence intensity was corrected
for cell number by division by DAPI fluorescence intensity of the same sample.
Statistics.
Data are presented as means ± SD. Results are expressed either as
absolute values or as percent of the control levels. A minimum of four
values were collected for each condition in each experiment. Differences between groups were evaluated using ANOVA, and significance was defined as P < 0.05.
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RESULTS |
EYFP-actin expression and labeling of the actin
cytoskeleton in LLC-PK10 cells.
EYFP-actin expression was initially detectable 8 h after
transfection of LLC-PK10 cells with pEYFP-actin.
We obtained maximum expression of EYFP-actin, monitored by fluorescence
microscopy, 24-96 h after transfection. Under physiological
conditions, distinct fluorescent structures were present in the
cytoplasm but not in the nucleus of LLC-PK10 cells (Fig.
1, A and B), and
the fluorescence intensity differed between
EYFP-actin-expressing cells. We noted less difference of EYFP-actin
expression levels in cells with stable EYFP-actin expression (Fig.
1B). In contrast to the fluorescence pattern of EYFP-actin,
LLC-PK10 cells transfected with pEYFP showed a more
homogenous distribution of EYFP (Fig. 1C). EYFP was present in all cell compartments, including the nucleus but excluding some
vesicular cytoplasmic structures, while filamentous actin was not
labeled by EYFP.

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Fig. 1.
Localization of enhanced yellow fluorescent protein
(EYFP)-actin or EYFP in live LLC-PK10 cells under
physiological conditions by fluorescence microscopy. Emission from
EYFP-actin expressed in transient transfected LLC-PK10
cells (A) and in cells of the stably transfected
LLC-PK10 subclone (B) is shown. Approximately
only 10% of the transient transfected cells expressed EYFP-actin,
whereas EYFP-actin was expressed by nearly all cells of the
LLC-PK10 subclone. EYFP-actin was distributed in the entire
cell excluding the nucleus. Emission from EYFP expressed in transient
transfected LLC-PK10 cells is shown in C. EYFP
was distributed over the entire cell including the nucleus. Bars in
A and B = 30 µm; bar in C = 20 µm.
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Imaging at higher magnification revealed that EYFP-actin was primarily
located to stress fibers, to cortical actin, and to the actin core
within microvilli (Fig. 2,
A-C). As shown in middle (Fig. 2B) and
apical planes (Fig. 2C) of LLC-PK10 cells, a
less intense fluorescence signal from EYFP-actin was present in the cytoplasm. This cytoplasmic distribution pattern of EYFP-actin was
diffuse and almost homogenous.

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Fig. 2.
Localization of EYFP-actin in stable transfected live
LLC-PK10 cells under physiological conditions. The
localization of EYFP-actin is shown in 3 representative focal planes of
the same image field: basal (A), middle (B), and
apical (C). Note predominant labeling of EYFP-actin of
stress fibers (A, arrowheads), of cortical actin
(B), and microvilli (C). In addition, a
homogenous cytoplasmic fluorescence signal was present that resembled
the distribution of G-actin. Again the nucleus showed no labeling with
EYFP-actin. Bars = 10 µm.
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To confirm these observations, we compared the localization of
EYFP-actin to the localization of G- and F-actin. On basal planes
EYFP-actin colocalized predominantly with F-actin (Fig. 3, A and C). In
apical planes, the close colocalization of EYFP-actin and F-actin was
also present, as seen with cortical actin and microvillar actin (Fig.
3, B and D). The diffuse fluorescence distribution of EYFP-actin resembled the pattern of endogenous G-actin.
The cytoplasmic localization of EYFP-actin and G-actin was similar in
middle and apical planes (Fig. 3, D and F).
Somewhat different from G-actin, EYFP-actin demonstrated a more
homogenous cytoplasmic distribution. The exclusion of EYFP-actin
fluorescence from the nucleus was in agreement with both the G- and the
F-actin staining. The expression of EYFP-actin did not appear to affect the morphology of LLC-PK10 cells, and the incorporation of
EYFP-actin into the cytoskeleton did not alter the organization or
distribution of the actin cytoskeleton compared with endogenous F-actin
or G-actin in nontransfected cells (data not shown; actin not labeled with EYFP is referred to as endogenous actin).

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Fig. 3.
Localization of EYFP-actin (A, B), F-actin
(C, D), and G-actin (E, F) in confluent
LLC-PK10 cells under physiological conditions on
representative basal (A, C, E) and apical focal plane
(B, D, F). Colocalization of EYFP-actin with F-actin in
stress fibers (A and C, arrows). Apically,
EYFP-actin colocalized with F-actin in microvilli (B and
D, arrowheads) and cortical F-actin (arrow, D),
and there was cytoplasmic colocalization of EYFP-actin with G-actin
(B and F). Bars = 20 µm.
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The content of total and globular endogenous actin and of total and
globular EYFP-actin was estimated by immunobloting techniques in
nontransfected cells, transiently transfected cells, and cells with
stably transfected EYFP-actin under physiological conditions (Fig.
4 and Table
1). Total endogenous actin and total
EYFP-actin were determined from cell homogenates and from endogenous
and EYFP-labeled G-actin from cell supernatants. When quantified, nontransfected cells, transiently transfected cells, and cells with
stable expression of EYFP-actin did not significantly differ in total endogenous actin. The difference of endogenous G-actin between
nontransfected and stable transfected cells was significant (P < 0.05), but no difference was noted between
nontransfected and transiently transfected cells (Table 1). The
monoclonal antibody against G-actin did not detect EYFP-actin;
therefore, an antibody to GFP was used to quantify GFP-actin. As
expected, stable transfected cells expressed the greatest amount of
total EYFP-actin and the G-actin fraction from EYFP-actin. Markedly
smaller amounts were expressed in transiently transfected cells, as
determined by immunoblot analysis with a monoclonal antibody against
GFP. Total EYFP-actin accounted for ~4% of total cellular actin in
cells with stable expression of EYFP-actin. A ratio of ~2:1 was
observed for endogenous F-actin to G-actin in nontransfected cells and
in transiently and stable transfected cells. A similar ratio was found
for F-actin to G-actin fractions of EYFP-actin in transient and stable
transfected cells expressing EYFP-actin.

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Fig. 4.
Immunoblot of endogenous actin
and EYFP-actin in LLC-PK10 cells. Ten micrograms of total
protein from the supernatant of Triton X-100-solubilized cells or total
cell homogenate from nontransfected cells, transient transfected cells,
and cells of the stable subclone expressing EYFP-actin were separated
by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes,
and immunoblotted to detect actin or EYFP-actin as described in
METHODS. Expression of EYFP-actin reduced total cellular
endogenous actin and G-actin in the supernatant in transient
transfected cells. This suppression was more marked in cells of the
stable subclone expressing EYFP-actin compared with nontransfected
cells. Cells of the stable subclone showed higher EYFP-actin than
transient transfected cells.
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Effect of ATP depletion and repletion on
EYFP-actin and endogenous actin.
The effect of various time periods of ATP depletion and repletion on
EYFP-actin and endogenous actin was evaluated in LLC-PK10 cells with stable expression of EYFP-actin. These cells maintained constant amounts of both total EYFP-actin and total endogenous actin
during ATP depletion as well as ATP repletion, compared with controls
(Fig. 5, A and B).
ATP depletion resulted in a marked decrease of the G-actin fraction of
EYFP-actin (Fig. 6A). The largest decrease occurred during the first 5 min of ATP depletion to
levels significantly lower than control levels (P < 0.01). The G-actin fraction of EYFP-actin remained at low levels
throughout 60 min of ATP depletion. After 4 h of ATP repletion the
amount of unpolymerized EYFP-actin had slightly increased again.
Twenty-four hours of ATP repletion were required for the G-actin
fraction of EYFP-actin to return to baseline levels. As demonstrated in Fig. 6B, ATP depletion resulted in a substantial increase of
the combined F-actin fractions of EYFP-actin and endogenous actin in
LLC-PK10 cells. F-actin had increased significantly after
15 min (P < 0.05) and 60 min of ATP depletion
(P < 0.01). However, after 4 h of ATP repletion,
F-actin levels were still significantly higher compared with control
levels (P < 0.05). By 24 h of ATP repletion, the
combined F-actin fractions of EYFP-actin and endogenous actin had
returned to the F-actin level found during physiological conditions.

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Fig. 5.
Effect of ATP depletion and repletion on total endogenous actin and
total EYFP-actin. A: total cell homogenates from stable
transfected LLC-PK10 cells were separated by SDS-PAGE,
transferred to PVDF membranes, and immunoblotted to detect actin or
EYFP-actin as described in METHODS. These 2 immunoblots are
representative of 6 separate experiments. B: total
endogenous actin and total EYFP-actin levels from total cell homogenate
of stably transfected LLC-PK10 cells were quantitated
densitometrically from immunoblots. Values are expressed as a
percentage of the control values and are means ± SD of 6 separate
experiments. Samples were taken under physiological conditions
(control), after 5, 15, 30, and 60 min of ATP depletion, and 4 and
24 h after ATP repletion succeeding 60 min of ATP depletion. As
demonstrated in A and B, total endogenous actin
and total EYFP-actin levels were unaltered by ATP depletion and ATP
repletion compared with physiological conditions.
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Fig. 6.
Effect of ATP depletion and repletion on G-actin
fraction of EYFP-actin and total endogenous and EYFP-labeled F-actin.
A: determination of G-actin fraction of EYFP-actin from
supernatant of Triton X-100 solubilized, stably transfected
LLC-PK10 cells by immunoblot (see METHODS)
during ATP depletion and repletion. B: quantification of
total endogenous and EYFP-labeled F-actin in stably transfected
LLC-PK10 cells. Samples in A and B
were measured under physiological conditions (control), after 5, 15, 30, and 60 min of ATP depletion, and 4 and 24 h after ATP
repletion succeeding 60 min of ATP depletion. Values in A
and B are expressed as a percentage of the control values.
Values are means ± SD of 8 (A) or 16 (B)
separate experiments. *P < 0.05 and **
P < 0.01 compared with control groups.
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Dynamics of EYFP-actin during ATP
depletion.
To visualize the dynamics of actin cytoskeletal alterations, we
observed live LLC-PK10 cells with stable expression of
EYFP-actin over a period of 60 min with or without prior ATP depletion.
Because alterations of the actin cytoskeleton were most prominent in
stress fibers, basal images were obtained, and the fluorescence
intensity of stress fibers were quantitatively analyzed. The time
sequence in Fig. 7,
A-D, provides representative images of the
alterations of stress fibers. After 15 min of ATP depletion (Fig.
7B), the fluorescence intensity of stress fibers with
EYFP-actin was markedly reduced and the stress fibers were severely
disrupted. Disruption of stress fibers became more pronounced after 30 min of ATP depletion (Fig. 7C). By 60 min of ATP depletion
(Fig. 7D), stress fibers had almost completely disintegrated
and could hardly be detected by immunofluorescence microscopy. Control
cells showed no alterations of stress fibers under physiological
conditions during 60 min of repeated observations (data not shown).

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Fig. 7.
Dynamics of stress fiber alterations during ATP
depletion. Representative basal images from the same live, stably
transfected LLC-PK10 cells under physiological conditions
(A), and after 15 min (B), 30 min (C),
and 60 min (D) of ATP depletion. Images were taken from one
basal focal plane, maintaining the same microscopy and laser settings.
Note the marked disruption of stress fibers labeled with EYFP-actin,
which was present at 15 min and proceeded over the entire time
sequence. Bars = 10 µm. Alterations of stress fibers were
quantified from their fluorescence on confocal micrographs
(E). Fluorescence of the same individual stress fibers was
measured in live, stably transfected LLC-PK10 cells at 0, 15, 30, and 60 min under physiological conditions (control), and during
ATP depletion. Values are means ± SD of 120 (ATP depletion) or
100 (control) separate stress fibers imaged over the same time
sequence. *P < 0.05 and **P < 0.01 compared with the control group.
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To quantify these changes, the mean EYFP-actin fluorescence intensity
of stress fibers was consecutively measured within the same defined
cell regions over a 60-min time course (Fig. 7E). The
fluorescence intensity of stress fibers decreased substantially during
ATP depletion. The most pronounced decrease of stress fiber fluorescence occurred during the first 15 min of ATP depletion. In the
next consecutive 45-min interval, fluorescence intensity diminished
further but at a slower rate than before. In parallel to qualitative
fluorescence image analysis, control cells without ATP depletion
exhibited no marked change in stress fiber fluorescence intensity
during the 60 min of observation (Fig. 7E).
Besides disruption of stress fibers and cortical and microvillar actin,
multiple EYFP-actin aggregates accumulated in live LLC-PK10
cells during ATP depletion (Fig. 8,
A and B). These aggregates formed a
globular, perinuclear pattern and colocalized with F-actin stained with Texas red phalloidin. No characteristic F-actin
structures, such as stress fibers or microvillar bundles, were visible
in the aggregates. We initially observed perinuclear F-actin after as
little as 10 min of ATP depletion. The size and the number of
aggregates increased continuously to a maximum at 60 min of ATP
depletion.

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Fig. 8.
Dynamics of actin cytoskeleton alterations during ATP
depletion. A and B: basal images from the same
live, stably transfected LLC-PK10 cells under physiological
conditions (A) and after 30 min of ATP depletion
(B). Besides the disruption of stress fibers, EYFP-actin was
incorporated into multiple, cytosolic aggregates (arrowheads) formed
during ATP depletion. These aggregates were widely distributed in the
cells and demonstrated high fluorescence intensity. C and
D: apical images from live, stably transfected
LLC-PK10 cells under physiological conditions
(C) and after 60 min of ATP depletion (D). The
microvillar actin core (arrows) was lost during ATP depletion. Images
were taken from the same basal (A and B) or
apical (C and D) focal plane, respectively,
maintaining the same microscopy and laser settings. Bars = 10 µm.
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Little alteration of microvillar and cortical actin was present
during this early time period (15 min) of ATP depletion. Apical planes
of EYFP-actin labeling in the microvilli core were present with only
moderate decreases in fluorescence intensity for 45 min of ATP
depletion (Fig. 8C). However, we hardly detected any microvillar EYFP-actin after 60 min of ATP depletion, and no actin was
detected. Only a weak homogeneous cytoplasmic distribution of
EYFP-actin remained on apical planes in the former location of the
actin microvillar cores (Fig. 8D). In addition, the apical cell membrane of these cells extended outward but was still
intact after 60 min ATP depletion. In parallel to microvillar F-actin, the fluorescence intensity of EYFP-actin incorporated in cortical actin
diminished later compared with that of stress fibers. We observed a
decrease of cortical actin only after 60 min of ATP depletion. Unlike
stress fiber and microvillar actin, the fluorescence intensity of
cortical EYFP-actin never decreased substantially.
Microscopic colocalization of EYFP-actin and
Alexa-647 phalloidin.
To address the issue of utilization of EYFP-actin as a marker of
endogenous actin, colocalization studies were conducted in LLC-PK10 cells with stable expression of EYFP-actin using
Alexa-647 phalloidin, a far-red-emitting fluorophore. The use of the
Alexa-647 fluorophore enabled the simultaneous acquisition of
EYFP-actin and phalloidin signals without the possibility of spectral
emission overlap. Cells stained with Alexa-647 phalloidin after 30 min of ATP depletion (Fig. 9A)
showed excellent correlation with the corresponding EYFP-actin signal
(Fig. 9B). There was ~83 ± 13.0% colocalization of
EYFP-actin and filamentous actin structures, occurring as either stress
fibers or actin aggregates (arrows in Fig. 9, A and
B). The number of aggregates previously seen in
abundance during 30 min of depletion, are greatly reduced after 4 h of ATP repletion in both the EYFP-actin (Fig. 9C) and
Alexa-647-phalloidin (Fig. 9D) channels. More filamentous actin was
seen in both channels as either stress fibers (arrows) or cortical
actin (arrowheads in Fig. 9, C and D). The
average colocalization between the two channels falls to ~74 ± 16.5% after the 4 h of ATP repletion. The reduction in
colocalization, compared with controls, is in part due to our inability
to factor out EYFP-G-actin from filamentous actin.

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|
Fig. 9.
Colocalization of EYFP-actin with Alexa-647 phalloidin
during ATP depletion and repletion. Basal images from stably
transfected LLC-PK10 cells after 30 min of ATP depletion
showing EYFP-actin (A) and Alexa-647 phalloidin
(B) fluorescence. Quantitative colocalization yielded an
overlap value of 83 ± 13.0% between the 2 signals at 30 min of
ATP depletion. EYFP-actin aggregates (A, open arrow) show
good colocalization with the signal from filamentous actin stained with
phalloidin (B, open arrow). A general breakdown of the
stress fibers and cortical actin was seen in both photomicrographs. In
cells allowed to recover for 4 h after ATP depletion, both
EYFP-actin (C) and filamentous actin fluorescence
(D) exhibited reformation of both stress fibers (thin
arrows) and the cortical actin network (arrowheads). Colocalization
values for the EYFP-actin and phalloidin channels for this group was
74 ± 16.5%. Bars = 10 µm.
|
|
 |
DISCUSSION |
Our study demonstrates that the fusion protein EYFP-actin
expressed in LLC-PK10 cells exhibits major structural and
functional characteristics similar to endogenous F-actin and G-actin.
EYFP-actin incorporated well into the endogenous actin cytoskeleton and
did not disturb its characteristic structural properties. Our
quantitative and qualitative data indicate that EYFP-actin is a
suitable probe for the actin cytoskeleton in live proximal tubule
cells. Compared with previous studies with cells fixed and stained for
actin, or microinjected with fluorescently labeled actin, we present more detailed data of spatial and temporal dynamics of actin
cytoskeletal alterations in proximal tubule cells during ATP depletion
and repletion over an extended observation period. The actin
cytoskeleton in epithelial cells is a highly dynamic structure that
undergoes rapid and frequent alterations (7, 19, 30).
Ischemia and reperfusion are potent causes of actin
cytoskeletal alterations in proximal tubule cells (1, 13, 14,
20-22). LLC-PK10 cells are a valid model to
study the actin cytoskeleton in proximal tubule cells (1, 6, 11,
20, 24) with ischemia mimicked by antimycin A-induced
ATP depletion (17).
EYFP-actin predominantly colocalized with F-actin in
LLC-PK10 cells and incorporated especially into microvilli,
stress fibers, and the cortical actin ring. This result was in
agreement with recent studies showing the distribution of GFP-actin in
other mammalian cells (2, 5, 9, 25). Besides labeling
F-actin structures, EYFP-actin also localized diffusely in the
cytoplasm. This diffuse cytoplasmic pattern of EYFP-actin resembled the
distribution of endogenous G-actin and presumably represents the
distribution of EYFP-actin as monomers (4). In contrast,
EYFP alone was not associated with cytoskeletal structures but
distributed diffusely in the cytoplasm and nucleus of
LLC-PK10 cells. This indicates that EYFP itself does not
specifically label actin cytoskeletal structures.
In the subpopulation of LLC-PK10 cells with stable
EYFP-actin expression, EYFP-actin accounted for ~4% of total
intracellular actin. This result was in agreement with the amount of
5-6% GFP-actin of total actin, which has been reported recently
for other cells (5, 27). Besides qualitative
characteristics of F- and G-actin fractions, EYFP-actin also
demonstrated quantitative characteristics of both endogenous actin
fractions. Under physiological conditions, the distribution between
endogenous F-actin and G-actin in epithelial cells was tightly
regulated, with a ratio of ~2:1 (F-actin to G-actin)
(19). This ratio was reflected correctly by the F- to
G-actin ratio of EYFP-actin in our study. In LLC-PK10 cells expressing EYFP-actin, the ratio of endogenous F-actin to G-actin of
2:1 was also preserved under physiological conditions. Additionally, total endogenous actin concentrations did not markedly differ between
nontransfected LLC-PK10 cells and those expressing
EYFP-actin.
Although tagging a protein with EYFP may interfere with the protein's
structure and/or function (16, 27, 29), EYFP-actin did not
seem to impair the endogenous actin cytoskeleton under physiological
conditions. No major differences were present between entirely
endogenous F-actin structures and F-actin with EYFP-actin incorporated
in LLC-PK10 cells. We observed no differences in form,
organization, or distribution of the actin cytoskeleton between
transfected and nontransfected LLC-PK10 cells, comparing our data with previous actin cytoskeleton studies (13, 18, 20,
21). The overexpression of EYFP-actin did not result in overt
cytotoxic effects, since the morphology of transfected and nontransfected LLC-PK10 cells did not differ.
The behavior of EYFP-actin was consistent with known actin cytoskeleton
alterations during ATP depletion in live proximal tubule cells
(1, 12, 14, 28). However, our results extend previous
findings, because we obtained more detailed immunofluorescent analysis
of the spatial and temporal dynamics of actin disassembly with
EYFP-actin. A short period of ATP depletion caused dramatic disruption
of stress fibers in live LLC-PK10 cells, while no changes in microvilli and cortical actin were apparent at this time point. In
parallel to further shortening of stress fibers, we observed destruction of microvilli and cortical actin as the time of
ATP depletion was extended. After an extended period of ATP
depletion, stress fibers and microvilli were disassembled while
cortical actin was only moderately affected by disassembly. The
perinuclear aggregation of EYFP-actin, observed during ATP depletion,
was consistent with previously described F-actin structures (15, 20, 28). Differential subcellular disassembly of F-actin
structures may be due to differences in activity of different
actin-binding proteins. During ATP depletion, actin-severing proteins
are possibly recruited to different subcellular regions. Therefore,
differential disassembly of stress fibers and cortical and microvillar
actin tagged with EYFP-actin may illustrate spatially and temporally separate regulatory mechanisms of different actin-binding proteins present in proximal tubule cells. EYFP-actin incorporation into cellular F-actin structures during ATP repletion was also consistent with the behavior of endogenous actin, as shown by the high degree of
colocalization of EYFP- and phalloidin-labeled F-actin. This was not so
apparent for the intracellular aggregates of EYFP-actin during ATP
depletion. However, this may relate to excessive actin depolymerizing
factor (ADF) binding and inhibition of phalloidin binding
(22).
The incorporation of the EYFP-actin into the actin cytoskeleton did not
affect characteristics of endogenous actin during ATP depletion and
repletion. During ATP depletion, transfected LLC-PK10 cells
maintained constant amounts of total endogenous actin and EYFP-actin
during ATP depletion and repletion. Meanwhile, the concentration of
F-actin and G-actin, as endogenous and EYFP-actin, varied
simultaneously. Endogenous F-actin and EYFP-actin in the F-actin state
increased, while endogenous G-actin and EYFP-actin in the G-actin state
decreased, and these changes reversed during ATP repletion.
The strong emission of EYFP-actin permitted monitoring of the actin
cytoskeleton alterations in LLC-PK10 cells for an extended period. Maximal fluorescence intensity of EYFP-actin was observed in
LLC-PK10 cells over 48 h, and experiments can be
performed at least for that time period. The stable emission of
EYFP-actin permits serial excitation without marked quenching of the
EYFP-actin fluorescence intensity. Therefore, diminished fluorescence
signal intensity indicates true changes of the actin cytoskeleton and seems not to be due to photobleaching. This further underscores the
usefulness of EYFP-actin as a marker for actin dynamics.
In summary, our data indicate that EYFP-actin is a suitable probe for
actin in live proximal tubule cells. EYFP-actin incorporates into all
components of the actin cytoskeleton and demonstrates the
characteristics of endogenous F-actin and G-actin without altering the
endogenous actin cytoskeleton. EYFP-actin provides detailed information
regarding spatial and temporal dynamics of actin cytoskeletal
alterations in live proximal tubule cells during ATP depletion and
repletion. Furthermore, EYFP-actin enables one to quantify these
alterations, with stress fibers undergoing the most rapid and complete
disassembly during ATP depletion and rapid reassembly during ATP repletion.
 |
ACKNOWLEDGEMENTS |
The monoclonal antibody JLA-20, developed by Dr. J. J. Lin,
was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human
Development and maintained by the University of Iowa, Department of
Biological Sciences, Iowa City, IA 52242.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: B. A. Molitoris, Division of Nephrology, Indiana Univ. School of Medicine, 1120 South Dr., FH 115, Indianapolis, IN 46202-5116.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 April 2001; accepted in final form 24 August 2001.
 |
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