Ndel1 structure prediction and peptide selection
Short folded peptides derived from a protein are likely to reveal functional activities, which may be masked in full-length proteins due to changes in conformation. We chose our peptides based on their structure within the full-length protein. The target of our study, the Ndel1 protein was predicted to be unstructured using the FoldIndex prediction algorithm (http://bip.weizmann.ac.il/fldbin/findex
) (Prilusky et al., 2005
), with the exception of approximately fifty amino acids surrounding amino acid 250 (). This domain is predicted to form a beta-sheet (http://www.ics.uci.edu/~baldig/betasheet.html
). In addition, it contains the cysteine (C273) that can undergo lipid modification (Shmueli et al., 2010
). We have demonstrated that palmitoylation of C273 negatively regulates the interaction between Ndel1 and cytoplasmic dynein (Shmueli et al., 2010
). Other programs such as COILS (http://www.ch.embnet.org/software/COILS_form.html
), and more importantly direct structural data for residues 6–166, indicate that the N-terminal part of Ndel1 forms a coiled-coil structure divided into three regions () (Derewenda et al., 2007
). The dimerization domain of Ndel1 is composed of residues 8–99 (Derewenda et al., 2007
). Residues 103–153 contain the minimal LIS1-binding domain within the N-terminal part of Ndell; deletion of twenty amino acids (114–133) was sufficient to abrogate the interaction with LIS1 (Yan et al., 2003
). In this study we were interested in investigating LIS1-independent activities of Ndel1. Therefore, based on the above information we generated two peptides from Ndel1: DID, consisting of amino acids 4–103 and pep3, consisting of amino acids 238–284 (schematically shown in ). Based on the limited amino acid similarity between Nde1 and Ndel1 within the DID domain (supplementary material Fig. S1), we also generated one corresponding peptide from Nde1 DID amino acids 3–102 (Nde1-DID). All peptides were fused to gluthathione S-transferase (GST) for easy purification and solubility. GST was used as a control peptide in all experiments.
Ndel1 and Nde1 derived peptides.
Protein interactions of these peptides were examined by pull-down experiments (). Our results indicate that Ndel1 and the three peptides pulled down a relatively small amount of conventional kinesin in brain extract, nonetheless no interaction was observed with the control GST peptide (), suggesting specificity. Ndel1, DID (derived from Ndel1) and the Nde1-DID peptide pulled down dynein intermediate chain (DIC), whereas no signal was noticed with either pep3 or the control GST peptide. Similar results were obtained using extracts of HEK293 or COS-7 cells. The interaction between Ndel1 with pep3 in a cell lysate was enhanced when Ndel1 was palmitoylated (). Ndel1 and pep3 can interact directly as demonstrated by a GST pull down experiment, whereas GST-pep3 pulled down 6×His Ndel1 but the GST protein did not (). Furthermore, pep3 can self-interact since GST-pep3 pulled down 6-His-pep3, but GST did not (). The DID peptide pulled down detectable amounts of dynein heavy chain and dynein intermediate chain identified by mass-spectrometry sequencing of the indicated bands (). Our results indicate that the DID peptide interacts with dynein (therefore named Dynein Interacting Domain) and pep3 is an additional Ndel1 self-association domain, which is sensitive to the palmitoylation status of Ndel1.
The squid genome is not yet available, however, it has been shown to contain multiple molecular motors including cytoplasmic dynein and members of the kinesin superfamily of proteins, which exhibit high degree of similarity to the mammalian ones (Degiorgis et al., 2011
). Although the Ndel1 ortholog has not been detected in squid so far, the squid genome is likely to contain one based on Western blot analysis (). Using polyclonal anti-Ndel1 antibodies we detected a cross-reactive band in a protein lysate from the squid axoplasm (). This band has the same molecular weight as Ndel1 detected in a mouse brain protein lysate (). Furthermore, our previous bioinformatic analysis revealed that Ndel1 and Nde1 are highly conserved proteins (Shmueli et al., 2010
). Nevertheless, our interpretation of the results will include the possibility that the squid genome does not contain an ortholog of Ndel1.
We further examined whether Ndel1 and its related peptides may interact with conventional kinesin using far-Western analysis (). The different tested proteins of kinesin included α2β2 that is the recombinant wild-type kinesin consisting of two heavy chains and two light chains, α2 is kinesin heavy chain homodimer and ΔTail is kinesin heavy chain deleted after residue M559. Our results indicate that the tested Ndel1/Nde1-derived peptides interacted with all the tested kinesin proteins whereas the GST control did not. We are not sure what are the functional implications of these interactions since NDEL1 did not activate kinesin in single-molecule assays.
To evaluate the role of Ndel1 on transport in vivo, we injected either GST, GST-Ndel1 (Ndel1), GST-Ndel1-DID (DID), the related peptide from Nde1 (Nde1-DID) and GST-pep3 (Pep3) mixed with 100 nm diameter carboxylated fluorescent beads into the giant axon of the squid (). Transport of the fluorescent beads was monitored using confocal microscopy (supplementary Movies 1–3) (). Movements of the fluorescent beads were analyzed using the automatic spots-tracking module of the Imaris software package (Bitplane, Inc.) to generate a set of position lists. The generated lists consisted of a track index, followed by x and y coordinates and a time stamp. The deduced tracks enabled to resolve the direction and velocity of each bead during each captured time frame. The anterograde direction was given a positive sign while the retrograde direction has a negative sign.
Injection of peptides mixed with carboxylated beads into the squid giant axon.
Each experiment was conducted in 4–6 different axons, recorded at 4 sec intervals for 100 frames. The motile beads were tracked resulting in 1194–4046 tracks. The tracks were separated into segments that were defined by continuous movements of a bead in one direction (4765–12324 segments per treatment). The movements from one frame to the next were used to calculate instantaneous movements (20,304–49,640 per treatment). The summary of the data is shown in . A few representative tracks are shown in . Beads appear and disappear as they enter and leave the focal plane. Some beads moved in one direction only, along with pauses of zero velocity (the green, turquoise and purple traces, ). Other beads changed directions, either with or without intervening pauses (the red and blue traces, ). Thus several tracks include both anterograde and retrograde segments. We noticed a large variability of the velocity within the individual segments; beads were often accelerating or slowing down even while moving in one direction. To distinguish the effects of the various peptides on motor-induced bead motion a number of statistical tests to the data were applied. First, we compared the individual, instantaneous movements at the measurement interval from the four treatments in comparison to GST (). The histograms have been normalized to the same areas, so that heights of the bars can be interpreted as probabilities. The horizontal axes show velocities in units of microns per second. The GST control appears in turquoise in all panels for comparison. The first and most notable feature is that there are no peaks at the canonical free motor velocities, which we would expect to be approximately 0.5~1 µm/sec based on in vitro measurements (Howard et al., 1989
; King and Schroer, 2000
; Toba et al., 2006
; Vale et al., 1996
). Instead there is a tall central peak at zero, surrounded by a broad shoulder to the anterograde side and a slim shoulder to the retrograde side. These shoulders decay to a maximum velocity of approximately 1 µm/sec in both retrograde and anterograde directions, just where we might have expected to see peaks due to single motor activity. The shoulders are not symmetric, but show a skew toward the anterograde direction. This is consistent with the visual observation that the cloud of injected beads moves and spreads mainly toward the axon tip, away from the cell body. Overall, the instantaneous velocities are much smaller than expected from in vitro motility assays. This might indicate a drag against the motion, or the effect of competing motors. Subtle differences appear in the shoulders of the distributions with the various peptides. Specifically, the full-length Ndel1 protein increased instantaneous velocities slightly in the anterograde direction (), while the DID and Nde1-DID peptides increased the instantaneous velocities in both directions with respect to the GST control (). Pep3 had a more pronounced effect in comparison with that of full-length Ndel1, namely it increased the velocity of anterograde steps without an effect on the retrograde steps (). Furthermore, addition of the peptides, but not of the full-length Ndel1, significantly reduced the number of immobile beads ( and ). DID had the most pronounced effect in this regard and its addition decreased the percentage of paused beads from 58% to 38%. Addition of Pep3 or Nde1-DID resulted in 45% of paused beads in comparison with 58% with the GST control. As a quick test of significance we split each of the datasets into six random sub-groups and confirmed that the visual differences remained qualitatively unchanged among them. In order to confirm quantitatively that GST and treatment step velocity distributions are distinct, we applied the Kolmogorov-Smirnov test, which yielded extraordinarily small p values (<10−21
Table 1: Summary of data derived from the experiments conducted in the giant axon of the squid. Tracks were defined when individual beads could be followed for at least three time frames. Segments were derived from the tracks that were split when a bead stopped (more ...)
Fig. 3. (A) Directed velocity of representative tracks along time (x axis time in seconds, y axis velocity in µm/second). Example of five individual long tracks over time, each track is shown in a different color. Note that there are fast and slow movements (more ...)
Percentage of paused beads. The percentage of paused beads (two bins located at –0.02 and 0.02 µm/sec shown in ) was derived.
The recorded tracks were further divided into retrograde and anterograde segments; when the movement of the bead stopped or changed direction a new segment was defined. To demonstrate the differences in the distributions of the segments more effectively we turned to a two-dimensional, color-based presentation (). For each treatment the segment duration (in seconds) is plotted along the x-axis, and the directional run length (in microns) along the y-axis. These plots show the correlation between persistence in length and temporal duration. In particular, each point in 2D shows the total length and duration of the segments it represents and the coloring represents the percentage of the segments with the particular duration and velocity. This presentation contains more information than conventional bar histograms as it shows how the segment lengths depend on their duration. We see clearly that anterograde segments are more numerous than retrograde in the GST control and in all treatments (about 2/3 of the total segments following exclusion of the very short segments, ). In order to emphasize the differences, thin lines were drawn over the 2D plots representing bounds of 85% in the distributions of both length and duration of continuous runs (s). A shift of the vertical line to the left indicates that a persistent motion is shorter on average; while the vertical position of the two horizontal lines indicates represents the segment lengths in either direction. The intersections of the horizontal and vertical lines with the y and the x axes indicate the duration of segments and the displacement of the segments and their confidence interval are shown in . The duration of segments in the anterograde direction was slightly affected by the treatments. It shortens in the presence of full length Ndel1 and extended a little with pep3 addition. All treatments decreased the duration of the retrograde segments, mainly Nde1-DID and DID. The displacement of the retrograde tracks was increased in all the treatments, where the most evident effects were noted following addition of DID and Nde1-DID.
Table 3: Summary of time and displacement intercepts and interval from 85% cutoff presented in left column as well as average velocities (µm/sec) of 95 or 50 percentile of anterograde or retrograde segments presented in as (more ...)
In addition, we examined the average velocity per segment. The peak follows the lightest-colored ridges in (right panels). The diagonal bounding lines are drawn to include 95% of the distributions (turquoise) and the median, 50% (grey). The median shows us the typical or most probable value, while the limit shows the average velocity of the fastest segments. All points that lie along a diagonal line starting from the origin have the same ratio of length to duration, so the slope represents a similar average velocity within the segments. The average velocities and their confidence interval were calculated at the 95% and 50% criteria (), and were normalized in relation to the retrograde or anterograde average velocities of control GST treated segments (). Overall, the effect of the peptides was more pronounced when the median velocities are inspected in comparison to the 95 percentile. All of the treatments increased the median velocities of the retrograde segments where DID and Nde1-DID exhibited a 4.5 fold increase in comparison with GST whereas the duration decreased (). In addition, all of the treatments resulted in increased velocities of the anterograde segments. A modest increase was noted when Ndel1 was added (13%), Pep3 and Nde1-DID each increased the anterograde velocities by 20% and DID contributed additional 40%.