A new organellar complex in rat sympathetic neurons

Abstract

Membranous compartments of neurons such as axons, dendrites and modified primary cilia are defining features of neuronal phenotype. This is unlike organelles deep to the plasma membrane, which are for the most part generic and not related directly to morphological, neurochemical or functional specializations. However, here we use multi-label immunohistochemistry combined with confocal and electron microscopy to identify a very large (approximately 6 microns in diameter), entirely intracellular neuronal organelle which occurs singly in a ubiquitous but neurochemically distinct and morphologically simple subset of sympathetic ganglion neurons. Although usually toroidal, it also occurs as twists or rods depending on its intracellular position: tori are most often perinuclear whereas rods are often found in axons. These 'loukoumasomes' (doughnut-like bodies) bind a monoclonal antibody raised against beta-III-tubulin (SDL.3D10), although their inability to bind other beta-III-tubulin monoclonal antibodies indicate that the responsible antigen is not known. Position-morphology relationships within neurons and their expression of non-muscle heavy chain myosin suggest a dynamic structure. They associate with nematosomes, enigmatic nucleolus-like organelles present in many neural and non-neural tissues, which we now show to be composed of filamentous actin. Loukoumasomes also separately interact with mother centrioles forming the basal body of primary cilia. They express gamma tubulin, a microtubule nucleator which localizes to non-neuronal centrosomes, and cenexin, a mother centriole-associated protein required for ciliogenesis. These data reveal a hitherto undescribed organelle, and depict it as an intracellular transport machine, shuttling material between the primary cilium, the nematosome, and the axon.

Figure 1. The loukoumasome.

A, Loukoumasomes and their morphological variants in whole-mounted pelvic ganglion (confocal stack). ax = axon. B, The loukoumasome contains γ-tubulin (γT). Glial centrosomes are apparent as small dots (pelvic ganglion, standard epifluorescence, z-stack, maximum projection). Inset: confocal orthogonal view showing distribution of SDL.3D10 and γ-tubulin immunoreactivity. MAP2: microtubule-associated protein 2. C, Unlike centrioles (arrow, single confocal slice), loukoumasomes are pericentrin-negative (Inset: confocal stack). Scale bar units: µm.

Figure 2. Distribution of the loukoumasome among neurons.

A, Loukoumasome-containing neurons are TH-positive (pelvic ganglion, standard epifluorescence). B, In the stellate ganglion, loukoumasome-containing neurons are larger, more densely packed, and situated near the exit point of the cardiac nerve (CN). MAP2: microtubule-associated protein 2. B′ and B″ indicate the regions enlarged in the lower panels (confocal stacks, maximum projections). C, Loukoumasomes occur exclusively in a subset of neurons expressing both neuropeptide Y (NPY) and calbindin-d28k (Calb) (stellate ganglion, single confocal slice). D, Neurons with loukoumasomes (+louk) in the stellate ganglion have fewer processes than those without (-louk). Insets: confocal slices at the depths indicated. Coloured arrows indicate the same processes in confocal slices and 3D reconstructions. See also Videos S1 and S2. Scale bar units: µm.

Figure 3. Subcellular distribution of the loukoumasome.

A,B, Relationship to the trans-Golgi network (TGN), which defines perinuclear and sub-cortical compartments. Tori occur primarily in the perinuclear compartment, linearized variants more peripherally and often in the axon (arrow in A), and figure-eights straddle the TGN. γT: γ-tubulin. Pelvic ganglion, confocal stack. See also Videos S3 and S4. Scale bar units: µm.

Figure 4. Loukoumasomes contain non-muscle heavy-chain myosin (mIIb).

mIIb co-localizes with sub-plasmalemmal F-actin, as expected, but also to the F-actin-negative loukoumasome (arrow). Stellate ganglion, single confocal slice. Scale bar units: µm.

Figure 5. Loukoumasomes associate non-randomly with the primary cilium.

A, Pericentrin-positive centrioles close to the loukoumasome in a binucleate pelvic ganglion neuron (single confocal slice). B, Examples of ‘ciliated’ loukoumasomes (stellate ganglion, single confocal slices). ACIII: adenylyl cyclase III. C, surface-rendered, cutaway reconstruction of a loukoumasome with an embedded primary cilium. Interior surfaces are coloured magenta (loukoumasome) and cyan (primary cilium). Plane of slice indicated by green borders. See also Video S5. D, The loukoumasome contains cenexin, a mother centriole-related protein. Scale bar units: µm.

Figure 6. Loukoumasomes associate non-randomly with nematosomes.

A, The loukoumasomal hole is occupied by an F-actin sphere. Left panel: confocal stack plus orthogonal views. Right panel, high-power view (single confocal slice). B, C, Loukoumasomal ultrastructure. N: nematosome; L: loukoumasome; mvb: multivesicular body; ly: lysosome; rer: rough endoplasmic reticulum; g: Golgi apparatus. Schematic: origins of sections shown in B and C. C, Contact between the nematosome and the loukoumasome (region indicated enlarged in inset). Note the similarity between the electron-lucent material within the nematosome and the loukoumasome. µt: microtubule. D, Loukoumasome-associated nematosomes are γ-tubulin-positive (γT) (single confocal slice). E, Immunoelectron microscopy confirming γT-immunoreactivity of the loukoumasome-nematosome complex (arrows: 10 nm gold particles). F, Schematic illustrating the composition of the loukoumasome/cilium and loukoumasome/nematosome complexes. L: loukoumasome; C: primary cilium; N: nematosome. Scale bar units: µm.