Rapid and intermittent cotransport of slow component-b proteins

Abstract

After synthesis in neuronal perikarya, proteins destined for synapses and other distant axonal sites are transported in three major groups that differ in average velocity and protein composition: fast component (FC), slow component-a (SCa), and slow component-b (SCb). The FC transports mainly vesicular cargoes at average rates of approximately 200-400 mm/d. SCa transports microtubules and neurofilaments at average rates of approximately 0.2-1 mm/d, whereas SCb translocates approximately 200 diverse proteins critical for axonal growth, regeneration, and synaptic function at average rates of approximately 2-8 mm/d. Several neurodegenerative diseases are characterized by abnormalities in one or more SCb proteins, but little is known about mechanisms underlying SCb compared with FC and SCa. Here, we use live-cell imaging to visualize and quantify the axonal transport of three SCb proteins, alpha-synuclein, synapsin-I, and glyceraldehyde-3-phosphate dehydrogenase in cultured hippocampal neurons, and directly compare their transport to synaptophysin, a prototypical FC protein. All three SCb proteins move rapidly but infrequently with pauses during transit, unlike synaptophysin, which moves much more frequently and persistently. By simultaneously visualizing the transport of proteins at high temporal and spatial resolution, we show that the dynamics of alpha-synuclein transport are distinct from those of synaptophysin but similar to other SCb proteins. Our observations of the cotransport of multiple SCb proteins in single axons suggest that they move as multiprotein complexes. These studies offer novel mechanistic insights into SCb and provide tools for further investigating its role in disease processes.

Figure 1.

Punctate nonsynaptic localization of endogenous α-synuclein particles in axons and characterization of mRFP::α-SYN. A, Cultured hippocampal neurons immunostained with MAP2 (green) and α-synuclein (red) with corresponding overlay and phase images. Although the bulk of α-synuclein localizes to synapses (yellow; along MAP2-positive dendrites in overlay image), punctate nonsynaptic α-synuclein (red; arrowheads along MAP2-negative axons) is evident. B, Cultured hippocampal neurons transfected with mRFP::α-SYN (red) and subsequently immunostained with the presynaptic marker synaptophysin (green) shows that the bulk of the fusion protein localizes to presynaptic sites (arrowheads), similar to the wild-type protein. Perikarya in A and B are denoted by an asterisk.

Figure 2.

Punctate α-synuclein particles are transported bidirectionally in axons. A, Axonal transport of transfected mRFP::α-SYN particles in 10–14 DIV hippocampal neurons. Selected video frames show mRFP::α-SYN particles transported anterogradely (single arrowheads) and retrogradely (double arrowheads) in axons; elapsed time (in seconds) is shown on the left of each frame. (For the corresponding video, see supplemental video 1, available at www.jneurosci.org as supplemental material.) Several immobile particles are shown (one marked by asterisk), and particle movements are quantified in kymographs on the right. Lines on the kymograph marked by the asterisk and the arrowheads represent the transport of similarly marked particles in the video frames to the left. Note that the movement is rapid, with pauses (arrow) during transit. Scale bars for the kymographs are at the bottom left of kymograph(s). Scale bar, 5 μm.

Figure 3.

Axonal transport dynamics of synaptophysin compared with those of α-synuclein. A, Axonal transport of SYP::GFP particles in cultured hippocampal neurons. Several anterograde particles are observed, three of which are marked by colored arrowheads (red, blue, and yellow). The corresponding kymograph is shown on the right. Lines on the kymograph marked by colored arrowheads represent transport of similarly marked particles in the video frames to the left. (For the corresponding video, see supplemental video 2, available at www.jneurosci.org as supplemental material.) B, Representative kymographs obtained from mRFP::α-SYN (left) and SYP::GFP (right) imaging. In the kymographs below, anterogradely moving particles are overlaid with red or green lines representing movement of α-synuclein and synaptophysin, respectively. Note the infrequent and rapid mRFP::α-SYN movements, interrupted by pauses (arrowheads), unlike the frequent, rapid, and persistent movements of SYP::GFP. (For videos corresponding to these kymographs, see supplemental videos 3 and 4, available at www.jneurosci.org as supplemental material.) Scale bars for the kymographs are at the bottom left of kymograph(s). Scale bar, 5 μm.

Figure 4.

Frequency histograms of transported α-synuclein and synaptophysin. A, Frequency histograms of average anterograde and retrograde velocities of moving mRFP::α-SYN and SYP:GFP particles (excluding pauses) show that they both move rapidly at a broad range of velocities, and the groups are statistically similar (p > 0.05; Mann–Whitney test, two tailed).

Figure 5.

Simultaneous visualization of transport kinetics of α-synuclein and synaptophysin in the same axon. Cultured hippocampal neurons were double-transfected with mRFP::α-SYN and SYP::GFP and visualized by simultaneous dual-color video microscopy. A, Selected frames from a time-lapse sequence shows a single mRFP::α-SYN particle (red) transported anterogradely (arrowhead). In the same axon, several SYP::GFP particles (green) are seen, including those being transported (marked with asterisks, also see the corresponding kymograph). (For corresponding video, see supplemental Video 5, available at www.jneurosci.org as supplemental material.) B–D, Kymographs from dual-cam experiments wherein each three-frame panel shows the original mRFP::α-SYN kymograph (left), SYP::GFP kymograph (middle), and a pseudocolor overlay of anterograde tracings (mRFP::α-SYN in red and SYP::GFP in green) from the two kymographs (right). The kymograph in B is from the time-lapse shown above. Note the different transport frequencies and cotransport (yellow diagonal lines in merged kymographs) of mRFP::α-SYN and SYP:GFP. Retrograde mRFP::α-SYN particles were often cotransported with SYP::GFP; the asterisk in D marks one such particle. Scale bars for the kymographs are at the bottom left of the kymograph(s). Scale bar, 5 μm.

Figure 6.

Simultaneous visualization of SCb proteins reveal that they are cotransported anterogradely. Cultured hippocampal neurons were double transfected with mRFP::α-SYN and either GFP::SYS-I or GFP::GAPDH (all SCb proteins) and visualized by simultaneous dual-color video microscopy. A–F show selected frames and kymographs from dual-cam imaging. Each set of frames shows α-synuclein (red) and synapsin-I or GAPDH (green), with the corresponding overlay image (yellow). Each three-frame kymograph panel shows the mRFP::α-SYN kymograph (left), GFP::SYS-I or GFP::GAPDH kymograph (middle), and a pseudocolor overlay of anterograde tracings from the respective kymographs (right). The yellow lines in overlaid kymographs represent cotransport. A, B, Cotransport of an object (arrowhead) positive for mRFP::α-SYN and GFP::SYS-I; the asterisk marks a stationary object. Arrowheads and asterisks in B represent movement of correspondingly marked particles in the frames above. C, Additional kymograph showing cotransport and pausing of an anterograde mRFP::α-SYN/GFP::SYS-I object. D, E, Cotransport of an object (arrowhead) positive for mRFP::α-SYN and GFP::GAPDH with corresponding kymographs. F, Additional examples of mRFP::α-SYN/GFP::GAPDH cotransport. (For corresponding videos, see supplemental videos 6 and 7, available at www.jneurosci.org as supplemental material.) Scale bars for the kymographs are at the bottom left of the kymograph(s). Scale bar, 5 μm.