Visualization and quantification of endoplasmic reticulum Ca²⁺ in renal cells using confocal microscopy and Fluo5F

Abstract

Sarcoplasmic/endoplasmic reticulum (ER) Ca²⁺ is the most abundant store of intracellular Ca²⁺, and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca²⁺ in toxicant-induced renal proximal tubular cell (RPTC) death. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F, a low affinity Ca²⁺ indicator, to directly monitor changes in RPTC ER Ca²⁺. Fluo5F staining reflected ER Ca²⁺, resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca²⁺ pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15 min, respectively, whereas A23187, a Ca²⁺ ionophore caused more rapid ER Ca²⁺ release (55% and 75% decrease in fluorescence at 5 and 15 min).

Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, added at the end of the experiment, further decreased ER fluorescence after thapsigargin treatment, revealing that thapsigargin did not release all ER Ca²⁺. In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca²⁺ release. ER Ca²⁺ release in response to A23187 or thapsigargin resulted in a modest but significant decrease in mitochondrial membrane potential. These data provide evidence that confocal microscopy and Fluo5F are useful and effective tools for directly monitoring ER Ca²⁺ in intact cells.

INTRODUCTION

Ca²⁺ acts as a universal second messenger and regulates numerous cellular functions including metabolism, motility, and transport[1]. Loss of Ca²⁺ homeostasis is critical to many disease processes and is a major component of cell death pathways including necrosis, apoptosis, and autophagy [2,3,4,5,6,7,8]. Sarcoplasmic/endoplasmic reticulum (ER) Ca²⁺ is the most abundant store of intracellular Ca²⁺ and its disruption often initiates the deleterious cascade of events leading to cell death and dysfunction [5,6,7,8,9,10,11].

Traditional methods of measuring of ER Ca²⁺ are indirect or require difficult probe loading techniques (i.e. membrane permeabilization, microinjection, or fused cell hybrids). For example, human embryonic kidney cells require 1 h of dye loading (2 μM Fluo-3 at room temperature) and extracellular Ca²⁺ chelation using BAPTA or EGTA, just to monitor increases in cytosolic Ca²⁺ as an indirect measure of ER Ca²⁺ stores [12]. To monitor ER Ca²⁺ directly, Montero and Robert et al. engineered an ER-targeted aequorin chimera, a Ca²⁺-sensitive photoprotein with a lower affinity for Ca²⁺. However, in HeLa and skeletal muscle cells, the aequorin chimera was rapidly saturated by the high Ca²⁺ concentrations within the ER and required a non-physiological reduction of ER Ca²⁺ to detect changes in Ca²⁺ stores [13,14].

Primary cultures of renal proximal tubular cells (RPTC) are highly aerobic, gluconeogenic, and exhibit robust transport capacity, making them ideal for the study of kidney tubular function and injury [15,16]. Previous work in our laboratory revealed the importance of ER Ca²⁺ in toxicant-induced kidney injury; although, the mechanism by which Ca²⁺ plays such a pivotal role is not completely understood [5,7,9,10,17,18].

Confocal microscopy is a useful method for the visualization and quantification of fluorophores at the subcellular level in living cells, and is compatible with most Ca²⁺ indicators. Chemical fluorescent (UV and visible-wavelength excitation fluorescent indicators) and bioluminescent calcium indicators (Ca²⁺ binding photoproteins and GFP-based Ca²⁺ indicators) differ in their characteristics (loading requirements, excitation/emission spectrum, permeability, compartmentalization, relative brightness, and Ca²⁺ affinity) and have inherent drawbacks (i.e., dye leakage, cytotoxicity, bleaching, autofluorescence, intracellular buffering, and lack of ion specificity)[19]. Fluo5F is a chemical fluorescent Ca²⁺ indicator with a lower affinity for Ca²⁺ (K_d ~2.3 μM), limited cytoxicity and bleaching, and high Ca²⁺ specificity, making it ideal for studying ER Ca²⁺. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F for directly monitoring changes in ER Ca²⁺ in RPTC.

MATERIALS AND METHODS

Materials

Female New Zealand White rabbits (1.5–2.0 kg) were purchased from Myrtle’s Rabbitry (Thompson Station, TN). Tetramethyl rhodamine methyl ester (TMRM) and Fluo-5F-AM were purchased from Molecular Probes, Invitrogen (Carlsbad, CA). All other chemical and materials were obtained from Sigma Chemical (St. Louis, MO).

Isolation of Rabbit RPTC and Culture Conditions

Rabbit RPTC were isolated using the iron oxide perfusion method and grown to confluence in 35-mm tissue culture dishes under improved conditions as previously described [15,16]. The culture medium was a 1:1 mixture of Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (without glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM L-glutamine, 1 μM pyridoxine HCl, 15 mM sodium bicarbonate, and 6 mM lactate. Hydrocortisone (50 nM), selenium (5 ng/ml), human transferrin (5 μg/ml), bovine insulin (10 nM), and L-ascorbic acid-2-phosphate (50 μM) were added daily to fresh culture medium.

RPTC Loading

RPTC were loaded with 2 μM Fluo-5F-AM (Fluo5F) and 100 nM TMRM for 20 min at 37° C, washed twice with 37° C phosphate buffered saline, and media was replaced. Then, 1 μM Fluo5F and 40 nM TMRM were added to the media to maintain dye equilibrium and incubated for 30 min at 37° C prior to imaging. The TMRM loading protocol was modified from Lemasters and Ramshesh [20].

Microscopic Imaging and Analysis

RPTC were imaged on a Leica Microsystems, TCS SP2 AOBS laser scanning confocal microscope (LSCM) using standardized pinhole, gain, and black level settings. A 63×0.9 NA water-immersion objective was used in an upright microscope configuration. All images were acquired at 8-bit resolution and at 1024×1024 with a line averaging of two.

RPTC were monitored for 10 min prior to treatment to establish baseline. Then, RPTC were treated and monitored an additional 15 min prior to Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 1 μM) addition to depolarize mitochondria. Images were acquired in 1-min intervals during the course of experiments. To quantify data, images were analyzed using Adobe Photoshop. Mean intensities of images in the green and red channel after background subtraction were interpreted as a quantitative measure of ER Ca²⁺ and mitochondrial membrane potential, respectively. Values are graphed as the percent mean intensity of each subsequent image vs. mean intensity of the image taken at time zero. Images comprise of 7–10 cells.

Statistical Analysis

RPTC isolated from one animal represents one experiment (N=1). Data are presented as means ± SE, and multiple means were compared using Student-Newman-Keuls test at each time point. Means with values p<0.05 were considered statistically significant.

RESULTS AND DISCUSSION

To investigate the use of confocal microscopy to monitor ER Ca²⁺ dynamics in RPTC, Fluo5F fluorescence was monitored as described above (Fig. 1a). Fluo5F fluorescence revealed a reticular pattern around nuclei void of staining. The peripheral granularity of staining and scarcity of staining at the cell margins provided evidence that the probe is not fluorescing in the cytosol. To confirm that Fluo5F was not in the mitochondria, RPTC were loaded with tetramethyl rhodamine methyl ester (TMRM), a marker of polarized mitochondria. TMRM staining (Fig. 1b) had a punctuate/tubular pattern unlike Fluo5F staining with no co-localization (Fig. 1c), confirming that Fluo5F does not reflect mitochondrial Ca²⁺, the second most significant storage/buffering compartment for intracellular Ca²⁺[4].

Figure 1. Morphology of Renal Proximal Tubule Cells (RPTC) co-loaded with Fluo5F and TMRM using confocal microscopy.

RPTC were co-loaded with Fluo5F (2 μM) and TMRM (100 nM) as described in the materials and methods. Green fluorescence of Fluo5F and red fluorescence of TMRM were imaged by laser scanning confocal microscopy. The white bar represents 10 μm. Fluo5F fluorescence (A) represents endoplasmic reticulum (ER) Ca²⁺, TMRM fluorescence (B) represents polarized mitochondria, and there was no co-localization (C).

We investigated the use of Fluo5F to monitor real-time changes in RPTC ER Ca²⁺. TMRM was used to measure mitochondrial membrane potential. In control cells, both Fluo5F and TMRM fluorescence intensity, morphology, and resolution were maintained over time (Fig. 2). Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, was added at the end of each experiment to cause mitochondrial depolarization and indirectly ER Ca²⁺ release, due to ATP depletion and cessation of sarcoplasmic/ER Ca²⁺ ATPase (SERCA) activity.

Figure 2. ER Ca²⁺ release RPTC exposed to A23187 and thapsigargin.

RPTC were loaded with Fluo5F and TMRM (see in Fig. 1), monitored for 10 min to establish baseline, exposed to diluent (DMSO, Control), A23187 (10 μM), or thapsigargin (10 μM) for 15 min, and then exposed to FCCP (1 μM), a mitochondrial uncoupler. Open arrows represent time of A23187 or thapsigargin treatment and closed arrows represent the addition of FCCP. Green fluorescence (A) and red fluorescence (B) represents ER Ca²⁺ and mitochondrial polarization, respectively. Note the more rapid ER Ca²⁺ release (A) in response to A23187 exposure compared to thapsigargin and the rapid loss of mitochondrial polarization (B) in response to FCCP.

We then explored the effects of two pharmacological agents known to alter ER Ca²⁺. RPTC were treated with the SERCA inhibitor, thapsigargin, and the Ca²⁺ ionophore, A23187. Thapsigargin decreased ER Ca²⁺ over several min (Fig. 2A), which was more indicative of ER Ca²⁺ leakage than receptor-dependent ER Ca²⁺ release that is characterized by DAG, ryanodine, and IP₃ receptor sensitive stores [1,21,22]. In contrast, A23187 caused a more rapid release of ER Ca²⁺, an anticipated finding due to its ionophore activity.

Temporal and incremental changes in ER Ca²⁺ release and mitochondrial membrane potential were quantified (Fig. 3). A23187 decreased fluorescence by 50% and 70% at 5 and 15 min after treatment, respectively, and FCCP addition did not decrease fluorescence further. Thapsigargin decreased fluorescence by 25% and 55% at 5 and 15 min after treatment, respectively. In contrast to A23187, FCCP addition further decreased fluorescence after thapsigargin, providing evidence that thapsigargin did not completely release all ER Ca²⁺ stores under these conditions.

Figure 3. Quantification of A23187- and thapsigargin- induced ER Ca²⁺ release in RPTC.

RPTC were co-loaded with Fluo5F and TMRM, treated as described in fig. 2, and images acquired as described in Materials and Methods. Values are mean intensities of images in the green channel ± SE and loss of intensity represents ER Ca²⁺ release. The open arrow represents exposure to A23187, thapsigargin, or diluent control and the closed arrow represents the addition of FCCP. Note the more rapid ER Ca²⁺ release after A23187 exposure compared to thapsigargin and an additional loss of fluorescence with FCCP following thapsigargin exposure.

The advantage of this approach is that ER Ca²⁺ stores are measured directly, allowing observation of spatiotemporal differences between A23187- and thapsigargin-induced ER Ca²⁺ release that would otherwise be masked by measuring cytosolic free Ca²⁺. Interestingly, Jiang et al. reported a higher rate and magnitude of cytosolic Ca²⁺ increase in response to A23187 over thasigargin using Fluo-3, but this approach required chelation of extracellular Ca²⁺ prior to treatment [12].

The effect of ER Ca²⁺ release on mitochondrial polarization was monitored using TMRM (Fig. 4). A23187- and thapsigargin-induced ER Ca²⁺ release decreased TMRM fluorescence by approximately 10%. In contrast, FCCP, the mitochondrial uncoupler added at the end of each experiment, decreased TMRM fluorescence by 60%, reflecting complete mitochondrial depolarization. Together, these data reveal that ER Ca²⁺ release produced by A23187 or thapsigargin partially decreases mitochondrial polarization and is consistent with previous reports [23].

Figure 4. Quantification of A23187- and thapsigargin-induced mitochondrial depolarization in RPTC.

See above for experimental details. Values are mean intensities of images in the red channel ± SE and loss of intensity represents mitochondrial depolarization. The open arrow represents exposure to A23187, thapsigargin, or diluent control and the closed arrow represents the addition of FCCP. Note the decrease in mitochondrial polarization following A23187 or thapsigargin exposure compared with control and the rapid loss of mitochondrial polarization following FCCP exposure in all groups.

In conclusion, confocal microscopy and Fluo5F are valuable tools for visualization of spatiotemporal dynamics of ER Ca²⁺ release in complex cellular systems in real time and will aid in our understanding of cellular dynamics and death.

RESEARCH HIGHLIGHTS.

• A new method of monitoring ER Ca²⁺ directly in intact cells

• Confocal microscopy and low affinity Ca²⁺ indicator, Fluo5F, reveals ER Ca²⁺ morphology

• Fluo5F fluorescence shows no co-localization with mitochondria or cytosol

• Stimulation of ER Ca²⁺ release reveal spatiotemporal differences in ER Ca²⁺ release dynamics

Abbreviations

ER

endoplasmic reticulum

EGTA

ethylene glycol tetraacetic acid

RPTC

renal proximal tubular cell

TMRM

tetramethyl rhodamine methyl ester

Fluo5F

Fluo-5F-AM

LSCM

laser scanning confocal microscopy

FCCP

Carbonylcyanide-p-trifluoromethoxyphenylhydrazone

SERCA

sarcoplamic/endoplasmic reticulum ATPase

DAG

diacylglycerol

IP3

inositol triphosphate

DMSO

dimethyl sulfoxide