Dramatic changes in mitochondrial subcellular location and morphology accompany activation of the CO2 concentrating mechanism

Abstract

Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for ~50% of global photosynthesis, compartmentalize their Ribulose 1,5 Bisphosphate Carboxylase/Oxygenase (Rubisco) into a specialized structure known as the pyrenoid when the cells experience limiting CO₂ conditions; this compartmentalization is a component of the CO₂ Concentrating Mechanism (CCM), which facilitates photosynthetic CO₂ fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO₂ limitation, although a role for this reorganization in CCM function remains unclear. We used the green microalga Chlamydomonas reinhardtii to monitor changes in mitochondrial position and ultrastructure as cells transition between high CO₂ and Low/Very Low CO₂ (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral cell location and orient in parallel tubular arrays that extend along the cell's apico-basal axis. We show that these ultrastructural changes correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membranes, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, with the latter involved in membrane-microtubule interactions. Furthermore, blocking mitochondrial respiration in VLC-acclimated cells reduces the affinity of the cells for Ci. Overall, our results suggest that mitochondrial repositioning functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

Keywords: CO2 concentrating mechanism; Chlamydomonas; fluorescence microscopy; microalgae; mitochondria.

Fig. 1.

Modes of CO₂ delivery to Rubisco. At HC, CO₂ passively diffuses to Rubisco (green arrow). At LC, Ci is trapped in the stroma as HCO₃⁻, which is facilitated by LCIB activity (yellow arrow). At VLC, HCO₃⁻ is directly taken up from the medium by the plasma membrane transporter HLA3 and then channeled from the cytoplasm to the stroma by LCIA (red arrow). Stromal HCO₃⁻ is channeled into the thylakoid lumen through BST1-3 and delivered as CO₂ to the pyrenoid through CAH3 activity. Solid lines: diffusion/transport through membrane protein; dashed lines: diffusion through lipid membrane. Acronyms: CO₂: Carbon dioxide; HCO₃⁻: bicarbonate; HLA3: High Light-Activated 3; LCIA: Low CO₂ Induced A; LCIB: Low CO₂ Induced B; BST1-3: Bestrophin 1-3; CAH3: Carbonic Anhydrase 3.

Fig. 2.

Physiological conditions and mitochondrial ultrastructure. (A) Photoautotrophically grown cells were sparged with HC (~2% in air), LC (0.04%), or VLC (<0.02%) in Tris-Phosphate (TP) buffered medium. Chlorophyll autofluorescence (red) marks the chloroplast while the Clover signal (yellow) marks the positions of the mitochondrial membranes. A weak Clover signal can be observed in the cytosol, which might be a consequence of mistargeting of the protein fusion. (Scale bar: 10 µm.) (B) Localization of a LCIB-mCherry fusion (magenta) monitored together with the mitochondria (yellow) in cells sparged with HC, LC, or VLC. The pyrenoid is shown in brightfield for a single cell. (Scale bar: 10 µm.) (C) Mitochondria localization was monitored in cells grown without aeration (shaken in flask, 120 rpm), in acetate-supplemented liquid medium (TAP) under LL (30 µmol photons m⁻² s⁻¹) or HL (500 µmol photons m⁻² s⁻¹), as indicated, or sparged with VLC in LL. (Scale bar: 10 µm.) (D) Localization of a LCIB-mCherry fusion (magenta) monitored together with the position of the mitochondrial membranes (yellow) in cells grown mixotrophically as described in (C). Fluorescence from the Chl channel was filtered out. (Scale bar: 10 µm.) (E) Mitochondrial membrane locations in cells and their arrangement. Cells were layered on a poly-lysine-coated slide, topped with TP solid medium (1.5% low melting point agarose) and acclimated to HC or VLC conditions for 6 h. Dotted lines highlight the cells’ apico-basal axis as observed in cross-sections. (Scale bar: 10 µm.) Fluorescence microscopy images are representative of two experiments. (F) Cryo-EM tomogram of HC- or VLC-grown cells showing typical positions of mitochondrial membranes and distances between these membranes (mito) and the chloroplast (cp) or plasma membrane (pm). Red rectangles designate areas of very close proximity of the chloroplast envelope to the mitochondrial outer membrane. (Scale bar: 200 nm.)

Fig. 3.

Involvement of cytoskeleton components on relocation of mitochondrial tubules. (A) Effect of Latrunculin B (LatB) on the nap1-1 mutant. Mitochondrial relocation induced by HL was examined in the nap1-1 background, in the absence (only DMSO 0.1%) and presence (LatB 10 µM in DMSO 0.1%) of the actin inhibitor LatB. (Scale bar: 10 µm.) (B) Effect of APM on mitochondrial location and membrane tubule organization. Mitochondrial relocation was examined in WT cells in the absence (only DMSO 0.1%) and presence of APM (APM 10 µM in DMSO 0.1%); cortical sections showing mitochondrial membrane organization near the plasma membrane and the maximal projections (cells immobilized on 1.5% TP agar) showing the whole cell mitochondrial signal. (Scale bar: 10 µm.) (C) Effect of absence of MIRO1 upon HL-induced relocation and the organization of mitochondrial membranes. Mixotrophically grown WT (CC-125) and mutant (miro1) cells were exposed to HL to induce mitochondria relocation. Cortical sections and maximal projections show the organization of the mitochondrial network. (Scale bar: 10 µm.) Fluorescence microscopy images are representative of two experiments.

Fig. 4.

Effect of mutations that block photosynthesis on mitochondrial relocation/reorganization. (A) Effect of PSII and PRK mutations: mutant strains (F64 and prk) and their corresponding parental strains (CC-124 and CC-5325) were mixotrophically grown in very low light (VLL, <5 µmol photons m⁻² s⁻¹) before being assayed for mitochondrial membrane rearrangement in HL. (B) Effect of CO₂ depletion on the position of mitochondria in the F64 and prk mutants: mixotrophically grown cells were sparged with CO₂-depleted air for 6 h and then assayed for their capacity for mitochondrial relocation under VLC conditions. (Scale bar: 10 µm.) Fluorescence microscopy images are representative of two experiments.

Fig. 5.

Relationship of mitochondrial relocation to the CCM. (A) Effect of transcription and translation inhibitors. Cells were grown in TAP LL before HL treatment in the presence of ethanol (EtOH 0.1%), the inhibitor of nuclear transcription, actinomycin D (Act D), or the inhibitor of translation on 80S ribosomes, cycloheximide (CHX). Control cells were incubated in LL in the presence of the drugs. (Scale bar: 10 µm.) (B) Induction of CCM genes under conditions that cause movement of mitochondria to the cell periphery. The level of induction of genes encoding HLA3, LCIA, CAH4, and CCP1 in cultures sparged with HC (2% CO₂), LC (air), and VLC (CO₂-depleted air; <0.02%); all cultures were exposed to 100 µmol photons m⁻² s⁻¹. (C) Dependence of mitochondrial relocation on CIA5. Wild-type (CC-125), the mutant (cia5), and the complemented (cia5-C1) cells were grown photoautotrophically in HC and tested for mitochondrial relocation following 4 h of VLC treatment. (Scale bar: 10 µm.) Fluorescence microscopy images are representative of two experiments.

Fig. 6.

Analysis of mitochondrial inhibitors and mutants on the affinity of the cells for Ci (pH 7.8). (A) Ci affinity in WT cells grown in HC, LC, and VLC conditions (n = 3). (B) Effect of the inhibitors SHAM and MX, separately and simultaneously on Ci affinity in cells grown in VLC and LC conditions. Inhibitors were added just prior to the assay (n = 3). (C) Effect of the aminotransferase inhibitor AOA (1 mM) on Ci affinity at VLC and LC. Cells were preincubated for 30 min in the presence of AOA (n = 3). (D) Ci affinity in the cah4/5 mutants grown in LC and VLC conditions, compared to the WT parental strain (CC-125) (VLC, n = 4; LC, n = 3). *P value < 0.05. **P value < 0.005.

Fig. 7.

Possible roles of mitochondrial relocation to the cell cortex. (i) Mitochondrial CAH4/5 could capture CO₂ that leaks from the chloroplast and channel it back into the chloroplast. (ii) Mitochondrial respiration can provide energy in the form of ATP for the active uptake of HCO₃⁻ at the plasma membrane. (iii) Mitochondria can intercept glycolate and metabolize it through the photorespiratory pathway to limit the loss of Ci.