Supplementary Materials

Microglia monitor and protect neuronal function via specialized somatic purinergic junctions

Csaba Cserép, Balázs Pósfai, Nikolett Lénárt, Rebeka Fekete, Zsófia I. László, Zsolt Lele, Barbara Orsolits, Gábor Molnár, Steffanie Heindl, Anett D. Schwarcz, Katinka Ujvári, Zsuzsanna Környei, Krisztina Tóth, Eszter Szabadits, Beáta Sperlágh, Mária Baranyi, László Csiba, Tibor Hortobágyi, Zsófia Maglóczky, Bernadett Martinecz, Gábor Szabó, Ferenc Erdélyi, Róbert Szipőcs, Michael M. Tamkun, Benno Gesierich, Marco Duering, István Katona, Arthur Liesz, Gábor Tamás, Ádám Dénes

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Supplementary Text for Main Figs. 1 to 5
  • Figs. S1 to S6
  • Tables S1 to S3
  • Captions for Movies S1 to S7
  • References

Images, Video, and Other Media

Movie S1
In vivo 2P time-lapse imaging shows temporal dynamics of microglia-neuron contacts. A tdTomato expressing neocortical neuron (red) is being contacted by processes of a microglial cell (green) in CX3CR1+/GFP mouse electroporated in utero with pCAG-IRES-tdTomato. The analyzed trajectories of microglial processes contacting the neuron are shown on the right panel, warm colors label trajectories of somatic contacts, while cold colors label trajectories of microglial processes contacting neuronal dendrites. The middle panel shows the trajectories overlaid on the recording.
Movie S2
In vitro CLSM time-lapse imaging of cocultured HEK293 and microglial cells. HEK cells were transfected with GFP-coupled control Kv2.1 construct on the left panel, and with YFP-coupled dominant-negative (DN) Kv2.1 construct on the right panel. Microglia is visualized by Alexa594-conjugated Lectin (red). Microglial processes contact Kv2.1-transfected HEK-cells at the clusters, but not those transfected with a dominant-negative mutant.
Movie S3
Left: stack of electron tomographic 0.5 nm thick virtual sections shows the special nanoarchitecture of a somatic microglia-neuron junction with closely apposed mitochondria, mitochondria-associated membranes (MAMs) and cytoplasmatic structures. The silverintensified P2Y12R-immunogold grains are clearly visible at the cytoplasmatic surface of microglial membrane. Note that large number of gold particles are clustered exactly where the neuronal cytoplasmatic structure is anchored. Right: 3D model of the same tomographic volume, neuronal membrane is magenta, microglial membrane is green, immunogold particles white, mitochondria light blue, MAM light green, cytoplasmatic densities red, vesicle-like structures blue and microglial reticular membrane structures darker green.
Movie S4
Stack of electron tomographic virtual sections shows the special nano-architecture of the core of a somatic microglia-neuron junction. The silver-intensified P2Y12R-immunogold grains are clearly visible at the cytoplasmatic surface of microglial membrane. The tethers between the anchored mitochondria and MAM are clearly visible. The neuronal cytoplasmatic structure (mitochondria, MAM) is anchored to a membrane segment that is precisely facing the high density of P2Y12Rs on the microglial membrane. Note that distance between the neuronal and microglial membrane is the smallest exactly here, and intercellular tethers are also clearly visible.
Movie S5
Left: stack of electron tomographic virtual sections shows the special nano-architecture of a somatic microglia-neuron junction with closely apposed mitochondria, MAMs and cytoplasmatic structures. The silver-intensified P2Y12R-immunogold grains are clearly visible at the cytoplasmatic surface of microglial membrane that touches the neuronal cell body, but are present only at a lower density at membrane segments, where the microglia touches a perisomatic bouton. Right: 3D model of the same tomographic volume, neuronal membrane is magenta, microglial membrane is ocker, immunogold particles white, mitochondria light blue, MAM light green, bouton membrane vivid green.
Movie S6
In vivo 2P imaging of CX3CR1+/GFP mice in utero electroporated with CAG-Mito-R-Geco1 construct. Dashed lines show the outline of a neuron, green microglial processes touch neuronal cell body where somatic mitochondria (red) are present. White arrows indicate the contact sites of microglia.
Movie S7
In vitro CLSM time-lapse imaging of KCl-stimulation of quinacrine-loaded cultured neuron. Left panel shows transmitted channel and superimposed green channel (quinacrine-labeled ATPcontaining vesicles), right panel shows the green channel and the outline of the neuron (white dashed line). White arrows point to vesicles that are released spontaneously, red arrows point to vesicles that are released after 40 mM KCl stimulation. MIP of z-stack (z-range: 2.5 μm), frame dimension: 12.3 x 22.2 μm.