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. 2022 Aug 16;135(16):jcs259436. doi: 10.1242/jcs.259436

Fig. 2.

Fig. 2.

Live imaging reveals cotransport of mRNA with mitochondria. (A) Scheme of mRNA imaging system. Top, schematic representation of the genetic constructs encoding target coding sequence (CDS) and the 3′ UTR of Cox7c and Cryab genes, which were cloned with multiple MS2 aptamers and CFP. Bottom, scheme of mRNA imaging in primary motor neurons. MS2-loop-containing constructs are introduced into motor neurons together with a plasmid expressing fluorescent protein-tagged MS2-binding protein (MCP-FP). Cells are grown in microfluidic chambers and signals are quantified along axonal extensions. (B) Top, representative images of MS2–mRNA constructs expressed in N2a cells together with a vector expressing MS2-binding protein fused to YFP (MCP–FP; green) colocalized with mitochondria staining (red). Scale bars: 5 μm. Bottom, quantification of the mRNA signal overlapping with mitochondria. Each data point represents a frame. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the minimum and maximum values; n=19, 20, 20 cells from 15, 12, 14 frames for MS2 only, Cox7c and Cryab, respectively. n.s (non-significant) P>0.05, ***P<0.001 (Mann–Whitney test). (C) Representative images (top) and kymographs (bottom) of primary motor neurons infected with viral vectors expressing MCP–GFP and Cox7c or Cryab MS2–mRNA constructs (green), showing colocalization with axonal mitochondria (Mitotracker, red) and acidic compartments (Lysotracker, magenta). Arrowheads indicate colocalization of mRNA and mitochondria. Scale bars: 10 μm. (D) Quantification of axonal cotransport between mRNA and the mitochondria separated to moving and static mRNA. Error bars are s.d.; n=12 and 12 axons for Cox7c and Cryab, respectively, from three independent biological cultures. ***P<0.001 (two-way ANOVA with Holm–Sidak correction). (E) Quantification of axonal cotransport between mRNA and acidic compartments separated to moving and static mRNA. Error bars are s.d.; n=12 and 12 axons for Cox7c and Cryab, respectively, from three biological independent cultures. ***P<0.001 (two-way ANOVA with Holm–Sidak correction).