FIGURE 1

Expression and dynamics of mCherry and mCherry-Smn in SH-SY5Y cell culture. (A) Expression of mCherry and mCherry-Smn in SH-SY5Y cells 2 days after transfection. Scale bar = 10 μm. (B) Representative autocorrelation function (ACF) and fitted ACFs of mCherry and mCherry-Smn in the cytoplasm. ACF for mCherry is fitted to 3D 1p1t while ACF for mCherry-Smn is fitted to 3D 2p1t diffusion model. (C) Mean diffusion coefficients, D1 (μm2/s) in cytoplasm and nucleus for mCherry, mCherry-Smn and mCherry-Smn: Smn (mCherry-Smn co-transfected with untagged Smn). (D) Mean diffusion coefficients, D2 (μm2/s) in cytoplasm and nucleus. Error bars represent the SD. (∗∗P < 0.01, ****P < 0.0001). (E) Brightness analysis of mCherry and mCherry-Smn in cytoplasm by plotting average intensity vs. number of particles. mCherry-Smn particles are brighter than mCherry. (F) Brightness analysis of mCherry-Smn vs. mCherry-Smn with untagged Smn in the cytoplasm. With presence of untagged Smn, brightness of mCherry-Smn goes down. The numbers above the graphs indicate the number of cells (number of points) the measurements were taken in.
FIGURE 2

Expression and diffusion coefficients of FPs and FP-tagged Smn in zebrafish motor neurons. (A) Expression of eGFP, eGFP-Smn, mCherry, and mCherry-Smn in cell body and axon of motor neurons at 2 dpf. Images of cell body and axon are taken from different motor neurons. Scale bar = 5 μm. Red arrow indicates representative position where FCS measurement was taken in axon. (B) Average diffusion coefficients, D1 (μm2/s) of eGFP, eGFP-Smn, mCherry, mCherry-Smn in cell body. (C) Average diffusion coefficients, D2 (μm2/s) of eGFP-Smn, mCherry-Smn in cell body. (D) Average diffusion coefficients, D1 (μm2/s) of eGFP, eGFP-Smn, mCherry, mCherry-Smn in axons. (E) Average diffusion coefficients, D2 (μm2/s) of eGFP-Smn, mCherry-Smn in axons. Error bars represent the SD. (n.s.P > 0.05, *P < 0.05). The numbers above the graph indicate the numbers of fish (number of points) the measurements were taken in.
FIGURE 3

Diffusion coefficient of mCherry-Smn in motor neurons under smn MO conditions. (A,B) Average diffusion coefficients, D1, D2 (μm2/s) of mCherry-Smn in motor neuron cell bodies under uninjected control and smn MO conditions. D1 was not observed for mCherry-Smn under smn MO conditions. (C,D) Average diffusion coefficients, D1, D2 (μm2/s) of mCherry-Smn in axons for uninjected control and smn MO conditions. Error bars represent the SD. (n.s.P > 0.05, ****P < 0.0001). The numbers above the graph indicate the number of fish (number of points) the measurements were taken in.
FIGURE 4

Expression and dynamics of eGFP- Smn Δex6,7 in zebrafish motor neurons. (A) Expression of eGFP- SmnΔex6,7 in motor neuron cell body and axon at 2 dpf. It is expressed strongly in the nucleus and weakly in axon and cytoplasm of cell body. (B) Representative ACF and fitted ACF of eGFP, eGFP-Smn and eGFP-SmnΔex6,7. (C) Average diffusion coefficients, D1 (μm2/s) of eGFP, eGFP-Smn, eGFP-SmnΔex6,7 in cell body. (D) Average diffusion coefficients, D2 (μm2/s) of eGFP-Smn, eGFP-SmnΔex6,7 in cell body. (E) Average diffusion coefficients, D1 (μm2/s) of eGFP, eGFP-Smn, eGFP- SmnΔex6,7 in axons. (F) Average diffusion coefficients, D2 (μm2/s) of eGFP-Smn, eGFP-SmnΔex6,7 in axons. Error bars represent the SD. (n.s.P > 0.05, ∗P < 0.05, ∗∗∗P < 0.001). Measurements grouped under Case 1 were fitted to 3D 1-particle diffusion model, found in 6 out of 14 measurements, indicating a loss of the second component. Case 2 refers to measurements fitted to 3D 2-particle diffusion model, found in 8 out of 14 measurements. The numbers above the graph indicate the numbers of fish (number of points) the measurements were taken in.
FIGURE 5

Summary of FCS observations. (A) Freely diffusing mCherry. (B) Two components of freely diffusing mCherry-Smn and mCherry-Smn with endogenous Smn in complexes that are distinct for cell body and axon (oligomers of mCherry-Smn and Smn are indicated by tetramers). (C) Truncated eGFP-Smn concentrates in nucleus, freely diffuses in cytoplasm. Complexes consist of only endogenous Smn. (D) Depletion of endogenous Smn led to a depletion of freely diffusing mCherry-Smn and mCherry-Smn replacing endogenous Smn in complexes. For details, see main text.
Acknowledgments
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