Establishing the properties of axonal APP vesicle movement in vivo
We probed the transport behavior of individual APP vesicles in vivo by using SG26.1 Gal4 (Gal4 gene enhancer trap strain) to drive the expression of UAS-APPYFP in Drosophila
. Because the expression of SG26.1 Gal4 is confined to a subset of ventral ganglion neurons, APPYFP movement could be tracked with single-axon resolution within segmental nerves. Analysis of green fluorescent protein–tagged EB1 (EB1-GFP) transport in these neurons confirmed that movement away from cell bodies is microtubule plus end–directed and anterograde (Supplemental Figure S1A), consistent with a previous report (Stone et al., 2008
). Using high spatial (0.126 micron) and temporal (10 Hz) resolution imaging, we characterized the movement of APP in an otherwise wild-type background (, and S1, A–F, and Supplemental Movie S1; see Materials and Methods
): 32.3% of the vesicles moved anterogradely and 18.2% moved retrogradely (), whereas 40.4% were stationary and 9.1% reversed direction with an average switch frequency of 6.09 switches/min ( and S1B). Switches generally did not involve pausing, consistent with a previous report of lipid droplet movement in Drosophila
embryos (Welte et al., 1998
). Thus directional switches are mediated by an apparently instantaneous change in the balance of active motors.
FIGURE 1: Characterization of the properties of APPYFP transport in Drosophila segmental nerve axons in vivo. (A) In vivo data were collected from an axonal region ~900 μm from the cell body (imaging field size: 88 μm in length). A standard (more ...)
Anterograde velocities of APP vesicles depend on the amount of kinesin-1
Considerable evidence demonstrates that APP movement is driven by kinesin-1 (Koo et al., 1990
; Ferreira et al., 1992
; Amaratunga et al., 1993
; Simons et al., 1995
; Tienari et al., 1996
; Kamal et al., 2000
; Szodorai et al., 2009
). We observed that a substantial fraction of APPYFP vesicles traveled at velocities faster than those observed for kinesin-1 motors in vitro, up to a maximum of 2.85 μm/s (), consistent with previous reports (Kaether et al., 2000
; Stamer et al., 2002
; Goldsbury et al., 2006
Kinesin-1-driven movement in vivo, which is substantially faster than in vitro, may be related to a different regulation of the ATPase cycle in vivo. Alternatively, multiple kinesin motors may coordinate to generate faster velocities, as speculated by Kural and colleagues (Kural et al., 2005
). The latter would be incompatible with the independence of velocity and motor number for kinesin-1 seen in vitro (Howard et al., 1989
; Block et al., 1990
; Spudich, 1990
). Additionally, it would be inconsistent with more recent in vivo data of lipid droplet movement in Drosophila
embryos, which suggest that neither velocity nor run length changes significantly with varying amounts of kinesin-1 (Shubeita et al., 2008
To test the hypothesis that the velocities and run lengths of APP vesicles may be determined by the amount of kinesin-1, we performed genetic, biochemical, and statistical analyses to correlate the number of active kinesin-1 molecules with vesicle behavior. We tested four predictions made by this hypothesis. A first prediction is that the anterograde velocity distribution should be nonnormal with multiple modal peaks reflecting differential occupancy of active motors. We tested this possibility by statistical mode analysis (Fraley and Raftery, 2002
) and found that the distribution was best fit by three modes (; see Materials and Methods
). Thus the first prediction is fulfilled. Interestingly, multimodal cargo velocity distributions have been reported previously in motility assays under substantial mechanical load of viscoelastic drag and in intracellular transport of PC12 cells and Xenopus
melanophores (Hill et al., 2004
; Levi et al., 2006
; Gagliano et al., 2010
A second prediction is that increased velocities should correlate with increased run lengths (or reduced pause frequency; Spudich, 1990
). Because our relatively short observation window of 15 s leads to truncation of run length for many vesicles (Figure S1C), we tested for a correlation of segmental velocity with pause frequency (see Supplemental Material). For controls, the anterograde correlation coefficient was −0.45, indicating that the faster the cargo, the lower the average probability of a pause. Thus the second prediction is also fulfilled.
A third prediction is that reducing the amount of kinesin-1 on vesicles should shift the distribution of velocities to lower modes without changing the mode centers. We tested this possibility by partially reducing kinesin-1 level, thereby circumventing the issues of organismal lethality and cellular defects reported in previous in vivo studies (Gindhart et al., 1998
; Hurd and Saxton, 1996
; Boylan and Hays, 2002
). We used two null point mutation alleles for kinesin heavy chain (KHC), khc8
(Saxton et al., 1991
; Brendza et al., 2000
), and one deletion allele for kinesin light chain (KLC), klc8ex94
(Gindhart et al., 1998
). Western blot analysis of kinesin-1 heterozygotes (see Materials and Methods
) confirmed that the removal of one functional khc
gene caused ~50% reduction in KHC or KLC proteins (). Interestingly, khc
reduction also resulted in KLC protein reduction, whereas klc
reduction did not affect KHC protein levels. Thus KLC protein levels appear to depend on KHC but not vice-versa, consistent with previous work in Drosophila
S2 cells (Ligon et al., 2004
FIGURE 2: Anterograde APPYFP velocities are relatively stable over time and depend on kinesin-1 amounts. (A–C) Western blot analysis in animals heterozygous for null mutations in khc or klc subunits of kinesin-1: khc8/+, khc20/+, and klc8ex94/+ (syntaxin (more ...)
To test whether kinesin-1 reduction translates to less motor protein assembly on vesicles, we used bottom-loaded sucrose flotation step gradients (see Materials and Methods
). In our experimental design, a fraction at the interface of the 8/35 sucrose step consists of floated membranes and vesicles. We found that 50% khc
gene reduction leads to ~50% reduction in both KHC and KLC proteins in the 8/35 fraction (), indicating that khc
reduction leads to less kinesin-1 associated with vesicles. Under these conditions, we observed substantial decreases in anterograde APPYFP velocities ( and S2A). Although the distributions were best fit by three modes (as in control), there were significant shifts in the relative contribution of each mode in kinesin-1 reductions (, and S5B and Supplemental Table S1). Moreover, either khc
reduction led to significantly shorter run lengths, increased pause frequency, increased pause duration, and a larger fraction of stationary vesicles (Figure S2, B–H). Together these biochemical and genetic findings are consistent with the notion that the number of functional kinesin-1 motors assembled per cargo is reduced with kinesin-1 reduction, causing a decrease in the anterograde velocity and a shift in the occupancy of modal velocities (unlike lipid droplet transport in Drosophila
embryos [ Shubeita et al., 2008
A fourth prediction of the hypothesis that the velocities of APP vesicles may be determined by the amount of kinesin-1 is that the correlation of anterograde velocity and pause frequency should be preserved in kinesin-1 reduction. Indeed, the magnitude of the correlation coefficients for all kinesin-1 reduction genotypes was in the range of −0.45 to −0.55, which is statistically highly significant (; see Supplemental Material). Thus all four predictions are fulfilled.
A potential concern is whether kinesin-1 reduction causes nonspecific global toxicity to axonal transport, including swellings, “organelle jams,” and blockage in transport (Hurd and Saxton, 1996
; Martin et al., 1999
; Gunawardena and Goldstein, 2001
; however, see Pilling et al., 2006
). Several observations suggest nonspecific global toxicity is an unlikely explanation for our data. First, there was no organismal phenotype characteristic of axonal transport defects, such as a tail-flip phenotype or animal inviability. Second, while axonal swellings were present in our kinesin-1 reduction genotypes, they were generally found in distal axonal regions, rather than in the more proximal region we analyzed. Third, nonselective poisoning would likely shift modal velocity values, whereas we observed fractional shifts in mode. Fourth, both kinesin-1–dependent and kinesin-1–independent transport would be expected to show impairment. To test the latter possibility, we analyzed the movement of synaptotagmin (SYT)—a kinesin-3 cargo (Yonekawa et al., 1998
; Pack-Chung et al., 2007
) tagged with GFP (SYTGFP), in khc8/+
animals. As expected, we observed that kinesin-1 reduction does not significantly change the total number of SYTGFP moving vesicles (see later in the paper) or the population percentages (Figure S6, G–J; see also Table S2). Furthermore, kinesin-1 reduction has no significant effect on retrograde velocity (Figure S6, B and D) or anterograde run length (Figure S6E). Although we observed a mildly significant decrease in anterograde segmental velocity for khc8/+
, this was not seen in klc8ex94/+
(Figure S6, A and C). Interestingly, a significant increase in retrograde run length was noted (Figure S6F), which may relate to the overall decrease in kinesin-1 related transport. Taken together, the SYTGFP data suggest that nonspecific global toxicity is an unlikely mechanism underlying the effects of kinesin-1 reduction on APP movement.
FIGURE 4: Kinesin-1 overexpression and reduction experiments suggest that the impairment of APPYFP retrograde transport seen in kinesin-1 reduction may result from a sampling bias. (A–C) Western blot analysis of kinesin-1 proteins in khc and klc overexpression (more ...)
Kinesin-1–driven anterograde velocity modes are relatively stable
Current models of vesicle movement make different predictions about the stability of movement behavior during axonal transport. For example, models in which motor proteins actively associate and dissociate with vesicles to determine directionality predict that certain parameters will vary over time (e.g., before and after pauses). Alternatively, models that invoke stable loading of vesicles in neuronal cell bodies predict substantial stability of movement before or after pauses and during runs. In fact, a prediction of the hypothesis that varying kinesin-1 motor amounts on APP vesicles control their behavior is that while the distribution of velocities is broad, the velocity of any given vesicle should remain relatively stable if motor number does not change over time. Hence, we analyzed movement behaviors relative to pauses and run lengths.
Our measurements of pause frequency indicate that APP vesicles pause infrequently during movement (Figure S1, D–E). Consistent with this, the observed average run length for APPYFP vesicles was 7.63 μm (Figure S2B), severalfold higher than in vitro (Howard et al., 1989
; Block et al., 1990
). Because our cargo trajectories are truncated spatially and temporally, even this value is too low. Our calculated estimated bulk anterograde run length is 11.7 μm (see Supplemental Material). Indeed, longer-duration movies (2 min of continuous imaging) generated an average run length of 21.85 μm (SEM = 2.3 μm; n = 47 segments; unpublished data).
We also probed the stability of APP movement by calculating the probability that a vesicle changes velocity mode during runs and after pauses and comparing it with random changes (see Supplemental Material). Consistent with the idea of stable motor loading for APP axonal transport, we found that APP velocity modes are relatively stable.
Kinesin-1 reduction impairs retrograde transport
To examine the effects of kinesin-1 reduction on the retrograde transport of APP, we first determined the retrograde behavior of APPYFP vesicles under control conditions. The observed mean retrograde segmental velocity was 0.87 μm/s (), similar to values reported for cytoplasmic dynein in vitro (King and Schroer, 2000
; Reck-Peterson et al., 2006
). However, retrograde segmental velocity distribution showed three modes, with velocities as high as 3.14 μm/s ( and Table S1). As with anterograde transport, retrograde velocities showed high modal stability (). The observed retrograde run length was 7.08 μm (Figure S2J), and the calculated bulk retrograde run length was 13.2 μm. Longer-duration recordings confirmed the longer calculated retrograde run length (mean = 14.18 μm; SEM = 2.7 μm; n = 26 segments; 2 min of imaging; unpublished data).
FIGURE 3: Kinesin-1 reduction leads to impairment in retrograde APPYFP transport. (A) Distribution of retrograde segmental velocities. (B) Retrograde unweighted segmental velocity mode analysis for control shows three modes (cyan); see Table S1 for a definition (more ...)
Reduction in khc
gene dosage led to substantial impairment in retrograde transport. We found significant decreases in APPYFP segmental velocities ( and S2I) and a major shift in occupancy (but not value) of velocity modes (, and S5F and Table S1). In addition, there was a significant decrease in retrograde run lengths and significant increases in retrograde pause frequencies and durations (Figure S2, J–L). Thus kinesin-1 plays an important role in bidirectional transport, as proposed previously (Brady et al., 1990
; Martin et al., 1999
; Pilling et al., 2006
; Kim et al., 2007
; Colin et al., 2008
). Although khc
reductions produced more pronounced effects compared with klc
, we observed that both subunits play a similar role, unlike previous work in which only KHC had a significant contribution (Ling et al., 2004
We considered three explanations for the impairment in retrograde transport by kinesin-1 reduction: dynein vesicle loading, motor activation, and sampling bias.
If loss of kinesin-1 leads to reduced loading of cytoplasmic dynein on APP vesicles, as proposed previously (Ally et al., 2009
), we should see evidence for this in our flotation assay. However, we noted that dynein heavy chain (DHC) protein levels in khc8
/+ remained unchanged in the postnuclear supernatant (PNS) and exhibited only a minor (15%) decrease in the 8/35 fraction (). Thus loss of kinesin-1 does not appear to significantly lower the amounts of cytoplasmic dynein on floated vesicles.
The hypothesis of dynein activation by kinesin (Ally et al., 2009
) cannot be tested directly in our system. However, a prediction of this hypothesis is that kinesin overexpression will lead to increased dynein activation. Thus we overexpressed khc
, or both together (see Materials and Methods
) and analyzed APPYFP movement. Western blot analysis confirmed the expected increase in KHC and KLC proteins in kinesin-1 overexpression genotypes (). Movement analysis in these backgrounds showed a significant impairment in retrograde transport, with decreases in segmental velocities and run lengths, as well as increases in pause frequency and duration ( and S2, I–L). These findings do not support the hypothesis that kinesin-1 activates cytoplasmic dynein. Intriguingly, we also observed a significant impairment in anterograde transport ( and S2, A–D), even though there was a significant increase in the anterograde population fraction (Figure S2F).
Sampling bias attributes the observed impairment in retrograde transport to an overall reduction in APP movement. The bias would result from fewer axonal retrograde vesicles within the observation window, resulting in an apparent inhibition of retrograde transport (). Indeed, we observed a significant decrease in the total number of APPYFP vesicles in kinesin-1 reductions, an effect we observed for no other genotype (). Thus sampling bias alone may explain the apparent impairment of retrograde transport upon reduction of kinesin-1.
Reduction of cytoplasmic dynein and dynactin subunits confirms modal stability is a generic feature of kinesin-1–driven APP transport
Previous work in Drosophila
embryos showed that impairment to DHC or the p150 subunit of dynactin disrupts anterograde movement (Gross et al., 2002
). From this, dynactin emerged as a potential coordinating factor. Other work has proposed that dynactin promotes dynein attachment to vesicles and enhances retrograde motor processivity through p150 binding to microtubules (Karki and Holzbaur, 1999
; King and Schroer, 2000
; Schroer, 2004
). However, more recent work demonstrated that cargo motility in vivo does not require p150 microtubule binding (Kim et al., 2007
). In addition, loss of dynactin does not prevent dynein association with vesicles (Haghnia et al., 2007
). Although disruption of the dynactin complex via arp1
deletion or p50
overexpression led to bidirectional impairment in axonal transport (Haghnia et al., 2007
; Kwinter et al., 2009
), the severity of the effects raised concerns about the specificity of the inhibition. Thus we further probed models of motor protein coordination and attachment by evaluating the effects of reduction in cytoplasmic dynein and dynactin on APP transport (see Materials and Methods
). For cytoplasmic dynein, we studied two null point mutation alleles for DHC, dhc64cp4163
(see Materials and Methods
), two null point mutations for dynein IC (DIC), dic1
(Boylan and Hays, 2002
), and one deletion mutation of the dynein light chain (DLC), roblk
(Bowman et al., 1999
). Western blot analysis in dynein and dynactin heterozygotes showed the expected ~50% reduction in protein levels (DHC: ; DIC: Figure S3A; DLC: Figure S3B; dynactin: Figure S4, A and B).
FIGURE 5: Reduction of cytoplasmic dynein and dynactin subunits confirms that modal stability is a generic feature of kinesin-1–driven APP transport. (A) Generation of dhc null alleles for evaluation of APP transport. Protein extracts were either treated (more ...)
For dynein, dhc reduction resulted in an expected overall impairment of many features of retrograde transport but enhancement of a number of anterograde parameters (, S3, C–J, and S5C, and Table S1), including significant increases in anterograde run length (Figure S3E) and average segmental velocity (Figure S3C), as well as shifts in the relative contribution of higher modes to the anterograde velocity distribution ( and S5C and Table S1). This suggests that DHC acts as an inhibitor of anterograde transport. DLC behaved in similar manner, although the overall effect on anterograde was milder (e.g., significant increase in anterograde run length [Figure S3E], but not segmental velocity [ ]). In contrast, dic reduction not only led to impairment in retrograde transport but also to strong impairment in anterograde transport (, and S3, C–J). As with kinesin, velocity modal stability was maintained in dynein reductions (). Thus DHC/DLC appear to act as inhibitors of anterograde movement, while DIC acts as a promoter.
In general, dynactin reduction led to bidirectional impairment of APP transport (, and S4, C–F, and Table S1), although some variability was observed among the subunits. For dmn (p50/dynamitin), there was a mild decrease in mean retrograde segmental velocity but no significant change in anterograde velocity (). However, there was a significant increase in both pause frequency and duration (Table S1) and in the fractions of the population of vesicles with anterograde movement (Figure S4, G–J). Perturbation of Gl (p150Glued) and grid (Arp1) caused stronger transport impairment in terms of reduced segmental velocities and run lengths (, and S4, C–F). However, the impairment was less pronounced in terms of increased pause frequencies and durations (Table S1). As with kinesin and dynein, velocity modes were maintained in dynactin reductions (). Overall, these data confirm that dynactin promotes anterograde and retrograde transport, although the transport impairment associated with perturbation in these dynactin subunits is milder than is observed for kinesin-1 and cytoplasmic dynein. In addition, these data support the notion that modal stability is a generic feature of APP transport.
Stimulation of anterograde transport by overexpression of DIC
Cytoplasmic dynein has been proposed as an activator of kinesin-1 (Martin et al., 1999
; Ling et al., 2004
; Pilling et al., 2006
; Kim et al., 2007
). Previous biochemical data established that the only detectable interactions between kinesin-1, cytoplasmic dynein, and dynactin protein complexes are mediated by DIC. Interactions were reported between DIC:p150Glued
(Vaughan and Vallee, 1995
) and DIC:KLC (Ligon et al., 2004
) and appear to involve similar regions of DIC (Vaughan and Vallee, 1995
; Ligon et al., 2004
; Schroer, 2004
). Previous work, however, did not distinguish whether KLC, dynactin, or both mediate the interaction between endogenous kinesin and dynein, since the in vitro work demonstrating an interaction between KLC and DIC used recombinant protein for the affinity chromatography assay (Ligon et al., 2004
). Thus we performed RNA interference (RNAi) experiments in S2 cells to test whether KLC binds to DIC in vivo (see Materials and Methods
). We observed that KLC mediates binding of the KHC/KLC kinesin-1 tetramer to DIC (). Hence, it is possible that motor protein activity on APP vesicles is controlled by a competition between p150Glued
and KLC for binding to DIC. Consistent with this idea, we noted that among the dynein reduction subunits, DIC reduction caused by far the strongest and most consistent impairment to bidirectional transport.
FIGURE 6: DIC stimulates anterograde APPYFP transport. (A) RNAi pulldown experiments in S2 cells demonstrate that KLC mediates binding of the KHC/KLC kinesin-1 tetramer to DIC in vivo. (B) DIC overexpression has no significant effect on mean anterograde duration-weighted (more ...)
If DIC controls the balance between anterograde and retrograde transport by activating kinesin-1, overexpression of DIC would stimulate anterograde transport. To test this, we overexpressed DIC protein (see Materials and Methods; Figure S3K). This led to significant increases in both anterograde vesicle fraction and run length (Figure S3, E and H), as well as significant decreases in the fraction of stationary and reversing vesicles (Figure S3, G and J). In addition, the mean anterograde velocity increased significantly (Figure S3C), due to a systematic shift of each of the three velocity distribution modes toward higher center values ( and S5D and Table S1). It should be noted that this was the only genetic condition for which we observed significant shifts in the mode centers. Taken together, these findings indicate that DIC acts as a promoter of kinesin-1 activity.