Morphology of mitochondria in spatially restricted axons revealed by cryo-electron tomography

Abstract

Neurons project axons to local and distal sites and can display heterogeneous morphologies with limited physical dimensions that may influence the structure of large organelles such as mitochondria. Using cryo-electron tomography (cryo-ET), we characterized native environments within axons and presynaptic varicosities to examine whether spatial restrictions within these compartments influence the morphology of mitochondria. Segmented tomographic reconstructions revealed distinctive morphological characteristics of mitochondria residing at the narrowed boundary between presynaptic varicosities and axons with limited physical dimensions (approximately 80 nm), compared to mitochondria in nonspatially restricted environments. Furthermore, segmentation of the tomograms revealed discrete organizations between the inner and outer membranes, suggesting possible independent remodeling of each membrane in mitochondria at spatially restricted axonal/varicosity boundaries. Thus, cryo-ET of mitochondria within axonal subcompartments reveals that spatial restrictions do not obstruct mitochondria from residing within them, but limited available space can influence their gross morphology and the organization of the inner and outer membranes. These findings offer new perspectives on the influence of physical and spatial characteristics of cellular environments on mitochondrial morphology and highlight the potential for remarkable structural plasticity of mitochondria to adapt to spatial restrictions within axons.

Fig 1. Growth and characterization of primary hippocampal neurons on EM grids.

Hippocampal neurons were isolated from E18 rats and plated on poly-D-lysine-coated Quantifoil 2/1 gold grids. (A) Low-magnification bright-field image showing the typical distribution of neuronal soma and processes after 10 d in culture. (B) Higher-magnification image of the area from the white box in panel (A) with neuronal processes highlighted by white brackets. (C) Wide-field fluorescence image showing immunolabeling of the neuron-specific protein CaMKIIα in green and the presynaptic vesicle–associated protein synapsin 1 in red. The blue color is from a bright-field overlay of the same area that also highlights the bars of the EM grid. (D) Higher-magnification image in a different area of the same immunolabeled grid highlighting the punctate staining of synapsin I (red; arrowheads) along processes typical of en passant varicosities in hippocampal axons. Blue is again from where the grid bars and holes in the Quantifoil grid are apparent. Scale bars in panels A–C = 100 μm and in panel D = 50 μm. (E) Low-magnification montage of 1 area in a cryopreserved grid of hippocampal neurons 10 d post plating, showing the typical distribution of axons and synaptic varicosities. Scale bar = 10 μm. (F) Higher-magnification representation from (E; white box) showing the axon and varicosity distribution overlying carbon (slightly darker areas) and grid holes. Examples of varicosities lying within grid holes are marked with white asterisks, whereas examples lying on the carbon are marked with red asterisks. Axon segments interconnecting the varicosities are highlighted with black brackets. Scale bar = 2 μm. CaMKIIα, calcium/calmodulin-dependent protein kinase II alpha; EM, electron microscopy.

Fig 2. Tomographic reconstruction of a typical presynaptic varicosity and adjoining axon segment.

(A) A 2D slice from the tomographic reconstruction showing the distribution of organelles in the varicosity (Mito = mitochondrion). Scale bar = 200 nm. (B) Segmented representation of the entire 3D tomogram volume shown in (A) revealing the relative size and spatial distribution of the organelle environment in the varicosity and axon segment. PM (dark blue), MT (light blue), mitochondrial outer membrane (“Mito,” dark green), ER (yellow), Ves (dark purple), MVB (light purple), unidentified membrane-bound compartment (pink). Scale bar = 200 nm. ER, endoplasmic reticulum; MVB, multivesicular body; MT, microtubules; PM, plasma membrane; Ves, vesicle.

Fig 3. Mitochondria display atypical morphological features in physically restrictive axons.

(A–C) Three different 3D segmented reconstructions showing representative examples of mitochondria (“Mito1,” “Mito2,” and “Mito3”) residing partially in the varicosity (“Var”) and adjoining axon segments, demonstrating different morphological states at the transition from the varicosity into the restricted space of the axon segment. Additional organelles occupying the axon space are also segmented. For ease of visualization, not all of the organelles and structures in the varicosity are shown. The plasma membrane (dark blue), microtubules (light blue), mitochondrial outer membrane (dark green), endoplasmic reticulum (yellow), and vesicles (dark purple) are highlighted.

Fig 4. Structural features of a mitochondrion captured spanning 2 varicosities (“Var”).

(A) A 10-nm slice through a 3D tomographic reconstruction showing a mitochondrion spanning 2 closely spaced varicosities connected by a short (approximately 100 nm) axon segment. An expanded region of the red box shown in (A) reveals the tubulated nature of the portion of the mitochondrion within the axon segment. Microtubules can be seen running parallel to the tubulated portion of the mitochondrion. (B) shows a surface rendered version highlighting the plasma membrane (purple), microtubules (blue), small segment of ER (yellow), and mitochondria (green). (C) is the same mitochondrion as in (B), displaying distinct segmentation of the outer membrane (green), inner membrane (orange), and cristae (pink).

Fig 5. Mitochondrial membranes display distinct morphological features at the boundaries of varicosities and axons with limited physical dimensions.

(A–C) To highlight the membrane organization of the mitochondria shown in Fig 3A–3C, the cristae and inner and outer membranes of the 3D reconstructions were segmented. (A) IMMs and OMMs remain in close apposition within the narrowed portion of this mitochondrion resident in the axon. (B) OMM is separated from the inner membrane, creating a distinct “matrix-free” compartment in this portion of the mitochondria residing in the axon. (C) Three mitochondria near the varicosity/axon junction show distinct internal membrane organization. The left mitochondrion shows a portion of outer membrane separated from the inner membrane, while the top right mitochondrion is narrowed near the axon junction, but the inner and outer membranes remain in apposition. A short tip of a third mitochondrion is partially captured at the edge of the tomogram that also shows inner and outer membranes together. Plasma membrane (dark blue), OMM (dark green), mitochondrial IBM (orange), cristae (pink). IBM, inner boundary membrane; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane.