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. 2023 Dec 15:11:1302585.
doi: 10.3389/fcell.2023.1302585. eCollection 2023.

Toolkit for cellular studies of mammalian mitochondrial inorganic polyphosphate

Vedangi Hambardikar  1 , Yaw A Akosah  2 , Ernest R Scoma #  1 , Mariona Guitart-Mampel #  1 , Pedro Urquiza #  1 , Renata T Da Costa #  1 , Matheus M Perez #  1 , Lindsey M Riggs  1 , Rajesh Patel  3 , Maria E Solesio  1
Affiliations

Toolkit for cellular studies of mammalian mitochondrial inorganic polyphosphate

Vedangi Hambardikar et al. Front Cell Dev Biol. .

Abstract

Introduction: Inorganic polyphosphate (polyP) is an ancient polymer which is extremely well-conserved throughout evolution, and found in every studied organism. PolyP is composed of orthophosphates linked together by high-energy bonds, similar to those found in ATP. The metabolism and the functions of polyP in prokaryotes and simple eukaryotes are well understood. However, little is known about its physiological roles in mammalian cells, mostly due to its unknown metabolism and lack of systematic methods and effective models for the study of polyP in these organisms. Methods: Here, we present a comprehensive set of genetically modified cellular models to study mammalian polyP. Specifically, we focus our studies on mitochondrial polyP, as previous studies have shown the potent regulatory role of mammalian polyP in the organelle, including bioenergetics, via mechanisms that are not yet fully understood. Results: Using SH-SY5Y cells, our results show that the enzymatic depletion of mitochondrial polyP affects the expression of genes involved in the maintenance of mitochondrial physiology, as well as the structure of the organelle. Furthermore, this depletion has deleterious effects on mitochondrial respiration, an effect that is dependent on the length of polyP. Our results also show that the depletion of mammalian polyP in other subcellular locations induces significant changes in gene expression and bioenergetics; as well as that SH-SY5Y cells are not viable when the amount and/or the length of polyP are increased in mitochondria. Discussion: Our findings expand on the crucial role of polyP in mammalian mitochondrial physiology and place our cell lines as a valid model to increase our knowledge of both mammalian polyP and mitochondrial physiology.

Keywords: bioenergetics; inorganic polyphosphate; mammalian cells; mitochondria; polyP; toolkit.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Confocal microscopy images confirm the mitochondrial expression of GFP in mitochondria from MitoPPX and MitoPPN SH-SY5Y cells, as well as the expression of GFP in ER from ER-PPX SH-SY5Y cells. (A). Representative confocal images of Wt, MitoGFP, MitoPPX, MitoPPN, and ER-PPX cells. ER was labeled using ER-Tracker Red, while mitochondria were labeled using TMRM. Images that show the overlap of the GFP signal and ER-Tracker Red or TMRM are included. Magnifications (×3) of significant areas obtained from the overlay images are also included. Scale bar = 50 µm for all the images except for the magnifications. In that case, scale bar = 17 µm. (B). Densitometry conducted along the lines that are marked in the magnification images included in Figure 1A. Green: GFP, Red: specific markers of mitochondria or ER.
FIGURE 2
FIGURE 2
PPX is expressed in mitochondria from MitoPPX, cytoplasm from CytoPPX, and ER from ER-PPX SH-SY5Y cells. (A). Representative immunoblots showing the presence of the PPX-GFP complex (70 kDa) in the mitochondrial fraction of MitoPPX cells, as well as the absence of the same complex in the mitochondrial fractions of Wt and MitoGFP SH-SY5Y cells. Please note that the levels of GFP were not assayed in this figure. TOMM20 levels indicate mitochondrial enrichment in the assayed fractions; while low β-actin levels indicate decreased presence of cytoplasmic proteins in the mitochondrial fractions. MitoPPN cells were not assayed because no antibodies for the PPN protein are available. (B). Representative immunoblots that show the presence of the PPX protein (45 kDa) in the cytoplasmic fractions of CytoPPX cells, and its absence in the corresponding fractions of Wt and MitoGFP SH-SY5Y cells. Note that the CytoPPX construct does not contain the sequence for the expression of GFP. β-actin signal indicates cytoplasmic enrichment in the assayed fractions. (C). Representative immunoblots that show the presence of the PPX-GFP protein complex (70 kDa) in the ER fraction of ER-PPX SH-SY5Y cells, as well as the absence of the same complex in the ER fraction of the Wt cells. Calreticulin presence indicates ER enrichment in the assayed fractions, and low β-actin signal indicates decreased presence of cytoplasmic proteins.
FIGURE 3
FIGURE 3
Depletion of mammalian polyP causes structural mitochondrial changes. (A). Representative images of Wt, MitoGFP, MitoPPX, MitoPPN, ER-PPX and CytoPPX cells. These images were obtained using EM. Arrows point towards significant mitochondria. Scale bar = 500 nm. (B). Quantification of the images. To conduct this quantification, mitochondria from all the conditions were classified as normal, electron lucent, and electron dense. MitoPPX and MitoPPN cells showed a clear increase in the number of electron lucent mitochondria, while CytoPPX cells showed increased electron dense mitochondria. Graphs represent average ±SEM of four independent images. Statistical analysis conducted by unpaired t-test with α = 0.05 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).
FIGURE 4
FIGURE 4
(A). PPX and PPN enzymes are active in mutant SH-SY5Y cells. (A). Graph showing the kinetics of the PPX and PPN enzymes in the different subcellular compartments in SH-SY5Y cells. To conduct these studies, we treated the cells with exogenous PPX and AP. Subsequently, we assayed DAPI-polyP fluorescence. Note the sharp decrease in the fluorescence levels of DAPI-polyP after treatment with PPX and AP, in both mitochondria isolated from Wt cells and exogenous polyP. Graphs represent average ±SEM of three independent experiments. Statistical analysis was conducted by unpaired t-test. α = 0.05 (* p ≤ 0.05, *** p ≤ 0.001). (B). Cell lysates obtained from Wt, MitoPPX, MitoPPN, ER-PPX, and CytoPPX were incubated with exogenous, short chain polyP (concentration is expressed in terms of Pi) and DAPI. Enzymatic activity was assayed by quantification of DAPI-polyP fluorescence over 12 h. PolyP treated with the scPPX enzyme was used as control. Measurements were normalized to the Wt signal at each time point. Graph shows representative enzymatic activity assay.
FIGURE 5
FIGURE 5
The effects of PPX in SH-SY5Y cells are specific to the subcellular location where the enzyme is expressed. By measuring DAPI-polyP fluorescence, we assayed the levels of polyP in mitochondrial fraction from (A). MitoPPX, and (B). MitoPPN and CytoPPX SH-SY5Y cells. Using the same method, polyP levels were also assayed in (C). Cytoplasmic fraction from CytoPPX, and (D). the ER fraction from ER-PPX cells. Corresponding Wt fractions are used as control in each of the experiments, and polyP levels were normalized to the values obtained in Wt cells. Graphs represent average ±SEM of three independent experiments. Statistical analysis was conducted by unpaired t-test. α = 0.05 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).
FIGURE 6
FIGURE 6
Targeted expression of PPX in SH-SY5Y cells affects gene expression. Expression of some of the main genes involved in mitochondrial physiology was assayed in all mutant cells. Expression levels of mutant cells were normalized with the values obtained in the Wt samples. (A). MitoPPX, and (B). MitoPPN cells showed increased expression of SOD2 and SIRT3. (C). ER-PPX cells showed increased expression of SIRT3 and decreased expression of MFN2 and PRKN. (D). CytoPPX cells showed decreased expression of MFN2. Graphs represent average ±SEM of three independent experiments. Statistical analysis was conducted by one-way ANOVA with Tukey’s post hoc analyses. α = 0.05 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).
FIGURE 7
FIGURE 7
Decreased levels of polyP affect the status of OXPHOS in SH-SY5Y cells. (A). Seahorse measurements that show OCR profiles in MitoPPN, MitoPPX, ER-PPX, and CytoPPX SH-SY5Y cells. Quantification of the Seahorse results showed the effects of the depletion of polyP in OCR in basal respiration, ATP-linked respiration, maximal respiration, proton leak, non-mitochondrial respiration, and spare capacity in (B). MitoPPX and MitoPPN SH-SY5Y cells, and (C). ER-PPX and CytoPPX cells. Wt cells were used as control in all the cases. Note that the depletion of mitochondrial polyP decreased OCR in all the cases; when the enzyme was expressed in ER or cytoplasm, this effect was less dramatic and, in some cases, even opposite. Graphs represent average ±SEM of three independent experiments. Statistical analysis conducted by one-way ANOVA and Tukey’s post hoc with α = 0.05 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).
FIGURE 8
FIGURE 8
Mitochondrial expression of PPK deleteriously affects the viability of SH-SY5Y cells. Fluorescence images obtained using transmitted light and a GFP filter of MitoPPK cells. In this case, DAPI was visualized in the standard spectrum, as we used this dye to label the nuclei. (A). on day one post-transfection, and (B). on day seven post-transfection. MitoGFP cells were used as control. Note that the expression of MitoPPK deleteriously affects the viability of SH-SY5Y cells. Scale bar = 100 µm.
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