Two kinesins drive anterograde neuropeptide transport

Abstract

Motor-dependent anterograde transport, a process that moves cytoplasmic components from sites of biosynthesis to sites of use within cells, is crucial in neurons with long axons. Evidence has emerged that multiple anterograde kinesins can contribute to some transport processes. To test the multi-kinesin possibility for a single vesicle type, we studied the functional relationships of axonal kinesins to dense core vesicles (DCVs) that were filled with a GFP-tagged neuropeptide in the Drosophila nervous system. Past work showed that Unc-104 (a kinesin-3) is a key anterograde DCV motor. Here we show that anterograde DCV transport requires the well-known mitochondrial motor Khc (kinesin-1). Our results indicate that this influence is direct. Khc mutations had specific effects on anterograde run parameters, neuron-specific inhibition of mitochondrial transport by Milton RNA interference had no influence on anterograde DCV runs, and detailed colocalization analysis by superresolution microscopy revealed that Unc-104 and Khc coassociate with individual DCVs. DCV distribution analysis in peptidergic neurons suggest the two kinesins have compartment specific influences. We suggest a mechanism in which Unc-104 is particularly important for moving DCVs from cell bodies into axons, and then Unc-104 and kinesin-1 function together to support fast, highly processive runs toward axon terminals.

FIGURE 1:

Kinesin-1 influences the distribution and flux of axonal DCVs. A Gal4-UAS controlled ANF::GFP that concentrates in DCVs was expressed in neurons of control and Khc mutant (Khc⁶/Khc²⁷) larvae using the P{GawB}D42 Gal4 driver. (A) Confocal images of fixed control and Khc mutant segmental nerves passing through segments A4–A5 showing distributions of ANF::GFP (green) and an antibody to CSP (red), which is a vesicle associated synaptic protein. In this and subsequent figures, the ventral ganglion (motor neuron cell bodies) is to the left. Note the shift from a finely punctate GFP signal in control nerves to large focal accumulations of signal in the mutant axons (scale bar = 12 μm). (B) Synaptic terminals on muscles 6 and 7 of control and Khc mutant larvae in segments A4–A5 (scale bar = 12 μm). (B′) Higher magnification of the boxed areas in B showing the DCV signal alone (scale bar = 3 μm). Note the scarcity of DCVs in the Khc mutant boutons. (C) Kymographs of ANF::GFP signal created from 100 s time-lapse image series (2 frames/s) of a control or a Khc mutant segmental nerve. Each kymograph shows DCV positions (x-axis, bar = 5 μm) as a function of time (y-axis, time = 0 s at top). Negative slopes, positive slopes, and vertical lines show anterograde, retrograde, and stationary DCVs, respectively. (D) Quantification of DCV flux (number of DCVs observed moving past a perpendicular line in a segmental nerve per minute). Bars show mean ± SE for n = 10 animals per genotype (one nerve per animal). Brackets show significant differences between Khc mutant and control values as determined by a Student’s t test (***p ≤ 0.001).

FIGURE 2:

Axonal mitochondria depletion by Milton knockdown does not alter anterograde DCV transport. Expression of a Milton shRNAi transgene was induced in motor neurons of animals along with either mito-GFP or ANF::GFP, using the OK³⁷¹-GAL4 driver. (A) Confocal images of fixed segmental nerves in control and Milton RNAi (Milt) larvae showing mitochondrial-GFP with enhanced sensitivity using anti-GFP (green) and anti-CSP (red) (scale bar = 20 μm). Note that, even with the anti-GFP enhancement, axonal mitochondria were not observed in distal axon regions (A5–A8) of Milton RNAi larvae. (B) Kymographs of time-lapse image series in more proximal segments (A2–A3) where more mitochondria were present show that Milton RNAi inhibited most mitochondria transport in motor axons (x-axis bar = 5 μm; y-axis t = 0 s at top and 100 s at bottom; 1 frame/s). (C, D) Confocal images of ANF::GFP loaded DCVs in motor axons of live larvae. (C) Single frames showing large swellings that contain DCVs in Milton RNAi motor axons (scale bar = 5 μm). (D) Kymographs of ANF::GFP in swelling-free portions of distal motor axons (A5–A6) show that Milton knockdown had little effect on DCV motion (x-axis bar = 5 μm; y-axis t = 0 s at top and 100 s at bottom; two frames/s). (E) Quantification of ANF::GFP DCV flux in control (n = 6) and Milton RNAi (n = 8) larvae (1 nerve per animal) in segments A5–A6. Bars show mean ± SE. Retrograde DCV flux was somewhat reduced, but anterograde flux was not affected (*p ≤ 0.05).

FIGURE 3:

Unc104 and kinesin-1 colocalize with DCVs in neurons. Primary neurons were cultured from larvae that expressed ANF::GFP in motor neurons (OK³⁷¹ driver). (A) A maximum projection of a 3D SIM superresolution Z-series of a motor neuron with ANF::GFP/DCVs (green) stained with anti-Unc104 for kinesin-3 (blue) and anti-Khc for kinesin-1(red). The neuron had neurites (n) growing out of the cell body (CB). The nucleus (nc) was identified by DAPI staining (not shown). (B) An expanded view of the box marked in A. Arrowheads (1–3) point to DCVs whose centroids are within 180 nm of anti-Khc and anti-Unc-104 centroids. Scale bars in A and B = 2 μm. (B1–B3) Line scans across the three marked DCVs and motor punctae show fluorescence intensities as a function of position. (C) The mean percentage (±SE) of DCV punctae that were within 180 nm of anti-Unc104, anti-Khc, or both as measured in three dimensions from Z-stacks of entire cells (n = 6 cells from three different experiments). Note that the fraction of DCVs that colocalize with both motors (15.2%) is nearly twofold greater than predicted by chance (8.3%). (D) To assess differences between observed and chance colocalization over a range of threshold distances, Khc and Unc104 locations were randomly and uniformly distributed across a volume region of interest (ROIs) within each of the six cells. This was repeated 100 times for each ROI. A mean colocalization (±SE) of real DCV punctae with the random motor punctae was then determined for each iteration at a variety of distance thresholds for each of the six ROIs (random). Colocalization of DCVs with real motor signals in the same ROIs was determined over the same range of threshold distances (observed). At 400 nm and less, the observed vs. random values were significantly different (Chitest, p value ≤ 0.001) (E) Plot showing the mean (±SE) of the fold differences between observed and random colocalization across the six ROI analyzed for D as a function of threshold distance.

FIGURE 4:

Kinesin-1 influences on DCV runs. Mobile DCVs were individually tracked, and their run behaviors were quantified in control and Khc mutant larval axons. The mutant data displayed were collected from Khc⁶/Khc²⁷ animals. (A) Frequency distributions for run velocities (bin size = 0.1 μm/s). Arrows indicate means for each distribution. Note the single mode distribution of anterograde run velocities and the shift toward slower velocities in Khc mutants (right panel). (B) Frequency distributions for run lengths (bin size = 1 μm). Arrows indicate means for each distribution. Note the shift toward shorter anterograde run lengths in Khc mutants (right panel). ***p < 0.001.

FIGURE 5:

Kinesin-1 and kinesin-3 have different effects on DCV distribution in neurons. CCAP GAL4, which is expressed in four neurons per segment in A1–A4 of the ventral ganglion, was used to coexpress ANF::GFP (green) with either Khc RNAi or Unc-104 RNAi. Anti-elav was used to stain all neurons (red). (A) Diagram illustrating that the primary neurites of the two CCAP neurons (arrows) in each hemisegment extend toward the midline (dashed line) where they arborize (*). The axon of the motor neuron (white circle, black line) then proceeds across the contralateral longitudinal tract (LT) and leaves the ganglion in a segmental nerve, eventually synapsing with body wall muscles 12 and 13 (Hodge et al., 2005; Vomel and Wegener, 2007). The neurite of the interneuron (black circle, gray line) projects to the contralateral tract and then bifurcates to project in both directions along it (LT) (Santos et al., 2007; Vomel and Wegener, 2007; Karsai et al., 2013). (B–D) Large-depth-of-field maximum projections of confocal Z-stacks of ANF::GFP DCVs (green) in CCAP neurons and of anti-Elav (red) in ventral ganglia of fixed larvae. The images in each column were collected with identical confocal and camera settings, so the differences in fluorescence intensities for different genotypes can be compared directly. Examples of CCAP neuron cell bodies are marked by arrows in B. The blue and yellow boxes mark areas with expanded views in E–G and H–J, respectively. Scale bar = 50 μm. (E–G) Expanded view of the blue boxes from B to D showing CCAP cell bodies (arrows). Scale bar = 10 μm. (E) Note the fairly even distribution of ANF-GFP punctae in the control cell body. (F) Unc-104 RNAi caused overaccumulation of ANF-GFP signal in cell bodies. This is seen in the cell body on the right, which was entirely within the Z-stack. The cell body on the left, which was only partially in the Z-stack, shows that the bright signal reflects crowding of punctae. DCVs in Unc-104 RNAi neurites were rare. (G) Khc RNAi caused some clumped accumulation of ANF-GFP in cell bodies. (H–J) Images of the central regions of the ventral ganglia (yellow boxes in B–D) giving views of neurites and longitudinal tracts. Insets show expanded views of the neurite commissures. Primary neurites (white arrowheads) project in commissures toward the midline (dashed line) where they arborize. Note that Unc-104 RNAi (I) greatly reduced DCV signal in neurites (inset), at the midline arborization zone, and in longitudinal tracts (LT). In contrast, Khc RNAi (J) showed strong DCV signal in longitudinal tracks and commissures, while causing excessive DCV signal in the arborization zone. Scale bar = 10 μm. (K–M) Images of DCVs in CCAP axons within segmental nerves as they leave the ventral ganglia. Note that DCVs are rarely seen in Unc-104 RNAi axons (L). They are present in the Khc RNAi axon (M) but are clustered and often appear in focal accumulations (**). Scale bars = 10 μm. These images are representative of five larvae analyzed for each genotype.