Signal transduction in astrocytes: Localization and release of inorganic polyphosphate

Abstract

Inorganic polyphosphate (polyP) is present in every cell and is highly conserved from primeval times. In the mammalian cells, polyP plays multiple roles including control of cell bioenergetics and signal transduction. In the brain, polyP mediates signaling between astrocytes via activation of purinergic receptors, however, the mechanisms of polyP release remain unknown. Here we report identification of polyP-containing vesicles in cortical astrocytes and the main triggers that evoke vesicular polyP release. In cultured astrocytes, polyP was localized predominantly within the intracellular vesicular compartments which express vesicular nucleotide transporter VNUT (putative ATP-containing vesicles), but not within the compartments expressing vesicular glutamate transporter 2 (VGLUT2). The number of lysosomes which contain polyP was dependent on the conditions of astrocytes. Release of polyP from a proportion of lysosomes could be induced by calcium ionophores. In contrast, polyP release from the VNUT-containing vesicles could be triggered by various physiological stimuli, such as pH changes, polyP induced polyP release and other stimuli which increase [Ca²⁺ ] _i . These data suggest that astrocytes release polyP predominantly via exocytosis from the VNUT-containing vesicles. © 2018 Wiley Periodicals, Inc.

Keywords: VNUT; astrocytes; inorganic polyphosphate; lysosomes; mitochondria.

Figure 1

Localization of inorganic polyphosphate to mitochondria and lysosomes. (a) inorganic polyphosphate is found in mitochondria (polyP, JC‐D8, blue; mitochondria, TMRM, red). (b) Highly specific polyP signal (JC‐D7, blue) is colocalized with lysosomal (CD63‐mKate, red) signal. (c) DAPI‐polyP can also be used to track the localization of inorganic polyphosphate in lysosomes. d, Bar charts summarizing the quantification of colocalization (Mander's coefficient) of polyP signals with fluorescence signal out of mitochondria and lysosomes [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Localization of inorganic polyphosphate in healthy and stressed astrocytes. (a) Wildtype healthy astrocyte, labeled for lysosomal tetraspanin CD63 (LAMP‐3), fused with red fluorescent probe mKate and labeled for polyP with the JC‐D7 indicator. (Ai) colocalization profile of CD63‐mKate and JC‐D7. (b) Stressed wildtype astrocyte, expressing CD63‐mKate and labeled for polyP with DAPI‐polyP. Note the redistribution of the lysosomal signal. Colocalization of polyP (JC‐D8, blue) and lysosomes (LysoTracker Red, red) in cultured astrocytes from Parkinson's disease model LRRK2 knockout, known to have impaired lysosomal morphology and function, (c,d) histogram depicting the distribution of polyP in lysosomes from wildtype healthy (black bar), wildtype stressed (red bar) and LRRK2 knockout (blue bar) astrocytes. **p < .001 [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

Lack of localization of polyP to glutamate‐containing vesicles (VGLUT2) in astrocytes from mixed primary culture. (a) Fluorescent image of an astrocyte, transduced to express eGFP–VGLUT2 and labeled with JC‐D8. (b) Colocalization image depicting the intensities of enhanced eGFP–VGLUT2 (green) and polyP indicator JC‐D7 (blue). Note lack of colocalization of the two signals. (c) Bar chart quantifying the colocalization coefficient (Mander's) of polyP and VGLUT signals [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 4

Localization of inorganic polyphosphate to ATP‐containing vesicles (expressing VNUT). (a,b) JC‐D8 and DAPI‐polyP fully colocalize with eGFP–VNUT signals in cultured astrocytes see summary in (c). (Bi) Colocalization profile of DAPI‐polyP and eGFP–VNUT. (d,e) JC‐D7 and DAPI‐polyP do not colocalize with the eGFP–TMPAP signal in rat astrocytes [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 5

Total internal reflection microscopy (TIRF) imaging of lysosomal vesicle polyP release. Representative images (a) and traces (measurement of polyP in individual vesicles) (A1) depicting partial release of polyP from lysosomes upon application of calcium ionophore ionomycin (1 μM). Partial lysosomal polyP release upon acidification of the cytosol with NH4Cl (b,b1). (c,c1) Application of glycyl‐l‐phenylalanine‐β‐naphthylamide (GPN, 100 μM) results in the collapse of lysosomes and release of polyP to the cytoplasm (see increase of DAPI‐polyP fluorescence (blue) in cytosol). (d) Summary data showing release of polyP from the lysosomes upon different stimuli [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Release of polyphosphate from ATP‐containing (expressing VNUT) vesicles. TIRF microscopy reveals that various stimuli trigger fusion of VNUT‐expressing vesicles: (a) changes of the intracellular pH upon application of ammonium chloride (NH4Cl induced drop of polyP signal in vesicles; (b) exogenously applied medium‐chain polyphosphate (polyP M); (c,d) ferutinin, an electrogenic calcium ionophore (30 μM). (e) Quantification histogram depicting release of polyP from the VNUT‐containing vesicles upon different stimuli; (f) release of polyP from VNUT vesicles in response to short episode of hypoxia [Color figure can be viewed at http://wileyonlinelibrary.com]