Identification of an axonal kinesin-3 motor for fast anterograde vesicle transport that facilitates retrograde transport of neuropeptides - PubMed (original) (raw)
Identification of an axonal kinesin-3 motor for fast anterograde vesicle transport that facilitates retrograde transport of neuropeptides
Rosemarie V Barkus et al. Mol Biol Cell. 2008 Jan.
Abstract
A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism.
Figures
Figure 1.
Influence of unc-104 mutations on CSP distribution in larval axons. CSP, a vesicle associated protein, was immunolocalized in wild-type (A, D, and G), unc-104O1.2/unc-104P350 mutant (B, E, and H), and Khc6/Khc27 mutant (C, F, and I) third instars. (A–C) Segmental nerves. (D–F) Motor axon terminals on muscle 6/7. (G–I) Motor axon terminals on muscles 12/13. Note that unc-104 mutations did not cause CSP accumulation in the sort of focal axonal swellings that are caused by Khc mutations (short arrows in C). However, unc-104 mutations did seem to cause a reduction in terminal size that was particularly noticeable for the terminals on muscles 12/13 (G–I). Bar, 10 μm.
Figure 2.
Effects of unc-104 mutations on organelle distribution in the ventral ganglion and motor axons. Wild-type (wt) (A and B), unc-104O1.2/unc-104P350 (1.2) (C and D), and unc-104O3.1/unc-104P350 (3.1) (E and F) larvae, in which D42-GAL4 induced expression of either a DCV tag (ANF::GFP; green in A, C, and E) or a mitochondrial tag (mitoGFP; green in B, D, and F). To allow imaging of axons independent of GFP presence, neuromuscular systems were fixed and immunostained with antibodies to syntaxin (red). Motoneuron cell bodies are located in the ventral ganglion (left side of each panel) and axons are in segmental nerves (extending to the right). Bar, 10 μm.
Figure 3.
Effects of unc-104 mutations on GFP-tagged organelle distribution in motor axon terminals. Wild-type (wt) (A and B) and unc-104O1.2/unc-104P350 mutant (1.2) (C and D) larvae with D42-GAL4–driven expression of ANF::GFP (green in A and C) or mitoGFP (green in B and D). Immunostaining with anti-syntaxin highlights axon plasma membranes (red). Portions of muscle 12/13 axon terminals in segment A4 are shown. Note in unc-104 mutants the absence of DCVs (ANF::GFP) and the aberrant terminal structure. Mitochondria remained abundant. Bar, 10 μm.
Figure 4.
Live transport behavior of organelles in unc-104 mutant axons. Each panel, extracted from a time-lapse movie of GFP-organelles in motor axons of a larval segmental nerve, shows a kymograph representation of fluorescent organelle positions as a function of time. Anterograde movements have negative slopes, whereas retrograde movements have positive slopes. Stationary organelles appear as vertical streaks. Before each movie, the field of view was photobleached, which reduced signal from stationary organelles, allowing better contrast for organelles that subsequently moved into the bleached area. (A and B) ANF::GFP shows DCV behavior in wild-type (wt) and unc-104O1.2/unc-104P350 (1.2) axons. Note the lower abundance of anterograde DCVs and their slower movements (larger negative slopes) compared with wild type. (C and D) MitoGFP shows mitochondrial behavior. Intact time-lapse movies of organelle transport can been seen in Supplemental Movies S1–S6.
Figure 5.
Influence of unc-104 mutations on axonal organelle flux and net transport rates. (A) Mean flux values (±SEM) for DCVs (ANF::GFP) and mitochondria (mitoGFP) were estimated by counting in one segmental nerve per larva (n = 5 larvae per genotype) the number of clearly defined organelles per unit time that entered the field of view moving in either the anterograde (charted above the origin) or retrograde (below the origin) directions. Because of the low abundance of distinct syt::GFP punctae, flux approximations were made by dividing the total number of directionally transported organelles seen anywhere in the nerve by the total time of imaging. Genotypes were wild-type (+), unc-104O1.2/unc-104P350 (1.2), and unc-104O3.1/unc-104P350 (3.1). (B) Net velocity for a single organelle is a summation of all its position changes divided by total time. Means (±SEM) were determined for five organelles in each direction from five larvae for each genotype, except for STVs in which all distinct organelles were tracked (see Table 1 for STV sample sizes). For both A and B, differences between wild-type (unshaded bars) and unc-104 mutant (shaded bars) means were assessed using F-tests for variance followed by two tailed t tests at either equal or unequal variance with 95% confidence intervals. Significant differences for a given organelle type are indicated by asterisks (p < 0.05).
Figure 6.
Accumulation of axonal transport cargoes at a physical blockade. Live wild-type larvae with expression of ANF::GFP driven in motoneurons by D42-Gal4 were ligated with a fine thread for 2 h and subsequently dissected, fixed, and stained with antibodies to endogenous synaptotagmin (anti-Syt). Motoneuron cell bodies were to the left (proximal) and terminals were to the right (distal). Note that both proteins accumulated on both sides of the ligation-induced segmental nerve constriction, but although synaptotagmin seemed relatively balanced, ANF::GFP accumulated more heavily on the proximal side. Bar, 10 μm.
Figure 7.
Changes in run behaviors for different organelles in unc-104 mutant axons. Runs were defined as periods of uninterrupted organelle motion in one direction bounded by pauses or reverse runs. Means (±SEMs) are shown for run velocities (A) and run lengths (B) for DCVs, STVs, and mitochondria (Mito). Sample sizes and additional data can be found in Table 1. Wild-type (unshaded) and unc-104 mutant (shaded) values were compared using linear contrast. Significant differences are noted by an asterisk (p < 0.05). The genotypes tested were wild-type (+), unc-104O1.2/unc-104P350 (1.2), and unc-104O3.1/unc-104P350 (3.1).
References
- Allen R. D., Metuzals J., Tasaki I., Brady S. T., Gilbert S. P. Fast axonal transport in squid giant axon. Science. 1982;218:1127–1129. - PubMed
- Brand A. H., Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–415. - PubMed
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