Purified Bik1 and Bim1 Form Homodimers That Associate to Form a Tetrameric Complex
To investigate the biochemical activities of Bik1 and Bim1, we expressed 6xHis-tagged versions of each protein in SF9 cells using a baculovirus system and purified the proteins on Ni-NTA resin. After removal of the 6xHis tag, both proteins migrated on SDS-PAGE at their expected molecular weights (38 kDa for Bim1 and 51 kDa for Bik1) and were >90% pure (A).
Figure 1. Bik1 and Bim1 form homodimers that associate to form a tetrameric complex. (A) Coomassie-stained SDS-PAGE gels of ~5 μg of Bik1 (left lane) and Bim1 (right lane) purified from insect cells. (B) Diagram of Bim1 (top) and Bik1 (bottom) protein (more ...)
We have previously shown that Bim1 and Bik1 self-associate in vivo and that their coiled-coil regions are necessary for mediating this self-interaction (B; Wolyniak et al., 2006
). To determine the quaternary structure of the recombinant Bik1 and Bim1 proteins, we calculated the molecular weight of each from its Stokes radius and sedimentation coefficient (S; Siegel and Monty, 1966
). The Stokes radius was obtained by gel filtration using a Superose 6 column and the S value was obtained by centrifugation through a 5–20% sucrose gradient (, C and D; ). Purified Bim1 was calculated to have a molecular weight of 70 kDa, which is close to the predicted molecular weight of 77 kDa for a Bim1 homodimer. This finding agrees with the results of a recent study by Zimniak et al., (2009)
, which also concludes that Bim1 is a homodimer. Purified Bik1 was calculated to have a molecular weight of 106 kDa, close to the predicted molecular weight of 102 kDa for a Bik1 homodimer. Thus, purified Bim1 and Bik1 appear to exist as homodimers in solution. The early elution of Bim1 and Bik1 and their large calculated Stokes radii determined from size-exclusion chromatography indicate these proteins have elongated rod-like shapes. We determined the Perrin shape parameter for each protein (Bloom et al., 1988
), and used the polynomial inversion procedure to calculate an axial ratio for each protein, assuming a prolate ellipsoid (Harding and Colfen, 1995
). Bim1was found to have an axial ratio of 13.4, whereas Bik1 had an axial ratio of 23.2.
Physical properties of Bim1 and Bik1
To compare the physical properties of recombinant Bim1 and Bik1 with endogenous Bim1 and Bik1, yeast extracts were also subjected to gel filtration and sucrose gradient sedimentation, and the fractions analyzed by Western blotting (, C and D). We calculated molecular weights of 85 kDa for Bim1 and 113 kDa for Bik1, consistent with these proteins forming homodimers in vivo (). Previous work found Bim1 in a ~250-kDa complex in yeast extracts, although it may have existed as a homodimer within this larger complex (Lee et al., 2000
Bim1 and Bik1 have been shown to interact in vivo and in vitro (Ito et al., 2001
; Wolyniak et al., 2006
), so we assayed for the formation of a Bim1-Bik1 complex in vitro. Bim1 and Bik1 were mixed in equal molar amounts and then analyzed by gel filtration and sucrose gradient sedimentation. In both conditions, the majority of Bim1 and Bik1 comigrated as a larger complex than either Bim1 or Bik1 alone (C and D). The molecular weight of the complex was calculated to be 170 kDa, close to the predicted molecular weight of 179 kDa for two molecules of both Bim1 and Bik1 (). Thus, Bim1 and Bik1 homodimers associate in solution to form a stable tetrameric complex.
Analysis of Bim1 and Bik1 Binding to Tubulin Subunits and Microtubules
We looked for the formation of Bim1-tubulin and Bik1-tubulin complexes by mixing tubulin dimers with equal molar amounts of Bim1 and Bik1, respectively. The mixtures were then analyzed by gel filtration. In the Bim1-tubulin mixture, each protein eluted at the same position as it did alone, indicating that Bim1 does not bind tubulin dimers (A). In the Bik1-tubulin mixture, the majority of Bik1 eluted at the same position as it did alone, although a minor amount of Bik1 migrated as a larger species (B). Interestingly, a substantial fraction of the tubulin eluted as a broad peak that overlapped the Bik1 peak. It is unlikely that much of this tubulin is forming a stable complex with Bik1, because the Bik1 peak does not shift and tubulin in the broad peak is not concentrated in the Bik1-containing fractions. Similarly, in sucrose gradient analysis, tubulin in the presence of Bik1 sediments further into the gradient than tubulin alone (C). The molecular weight of the largest tubulin species is ~570 kDa, equivalent to approximately six tubulin subunits. Thus, we conclude that Bik1 binds only weakly, at best, to tubulin. In addition, Bik1 appears to stimulate the polymerization or aggregation of tubulin subunits.
Figure 2. Analysis of Bim1 and Bik1 binding to tubulin heterodimers. Size-exclusion chromatography of (A) Bim1 (blue), tubulin (green), and an equimolar mixture of Bim1 and tubulin (turquoise), and (B) Bik1 (red), tubulin (green), and an equimolar mixture of Bik1 (more ...)
We also measured the abilities of Bim1 or Bik1 to bind assembled microtubules by incubating each with various amounts of taxol-stabilized microtubules. Microtubule bound protein was separated from unbound protein by centrifugation and the supernatant and pellet analyzed by Western blotting. Bim1 bound to microtubules with an apparent dissociation constant, Kd, of 0.5 μM (A). In contrast, only small amounts of Bik1 bound microtubules even at much higher tubulin concentrations (B).
Figure 3. Microtubule binding and plus-end localization of Bim1 and Bik1. (A) Bim1 at 40 nM was incubated with various concentrations of taxol-stabilized microtubules. Left, microtubules were pelleted by centrifugation, and the pellet fraction (P) and the supernatant (more ...)
To examine the localization of Bim1 and Bik1 along microtubules, we purified Bim1-GFP and Bik1-GFP. Each of these was incubated with dynamic microtubules assembled from axonemes and then visualized by fluorescence microscopy. Bim1-GFP appeared as dots along microtubules with an average of 2.9 dots per microtubule (C). The positions of these dots were analyzed in two ways. First, we divided microtubules into 10 equal-length sections (van Breugel et al., 2003
) to determine the relative locations of the Bim1-GFP dots along the microtubule. Second, we measured the absolute distance from the plus end for each dot within 5 μm of the microtubule end. Both calculations showed that 73% of the microtubules had dots at their plus ends, whereas only ~20% of microtubules had dots in any other region along their length. These results show that Bim1 is an autonomous microtubule end-binding protein, in agreement with Zimniak et al., (2009)
. In contrast, we observed only faint Bik1-GFP straining along microtubules and axonemes (D).
Given that Bim1, but not Bik1, could localize to plus ends, we assayed whether Bik1 could be directed to the microtubule plus end through its interaction with Bim1. When Bik1-GFP was mixed with unlabeled Bim1, Bik1-GFP dots were observed along the microtubule and at the plus-end in a pattern similar to the Bim1-GFP localization (E). The average number of Bik1-GFP dots per microtubule was 1.6, and these were enriched at the plus ends. Therefore, Bim1 is able to target Bik1 to microtubule plus ends in vitro. Mixing Bik1 with Bim1-GFP did not substantially alter the plus-end localization of Bim1-GFP (unpublished data).
Bim1 Promotes and Bik1 Inhibits Microtubule Assembly In Vitro
We next examined the effects of Bim1 and Bik1 on the assembly of microtubules in vitro. Microtubules were nucleated from sea urchin axonemes in 11.5 μM tubulin alone or in the presence of 0.1, 0.5, and 1.0 μM Bim1 or Bik1. Microtubules were visualized using VE-DIC microscopy. Because axonemes contain parallel bundles of uniformly oriented microtubules, microtubules extending from one end are plus-ended, whereas microtubules extending from the other end are minus-ended. Thus, it was possible to examine the assembly of plus- and minus-ended microtubules independently.
Bim1 increased the length and number of both plus- and minus-ended microtubules (A). To quantify this effect, we calculated the total microtubule length per axoneme end by multiplying the average number of microtubules per end by their average length. Axonemes in 11.5 μM tubulin alone contained 11.9 ± 9.6 μm of total microtubule length at their plus ends and 4.4 ± 3.6 μm of total microtubule length at their minus ends (B and Supplementary Table 1). Addition of 0.1 μM Bim1 increased total microtubule length 2.8-fold at plus ends and 3.5-fold at minus ends; addition of 1 μM Bim1 increased total microtubule length by 12.8-fold at plus ends and 13.9-fold minus ends (B and Supplementary Table 1). Thus, Bim1 promotes microtubule assembly. In contrast to Bim1, Bik1 decreased the length and number of both plus- and minus-ended microtubules (, A and B). Addition of 0.1 μM Bik1 decreased total microtubule length by 8% at plus ends and 52% at minus ends; addition of 1 μM Bik1 decreased total microtubule length by 4.0-fold at plus ends and 14.2-fold at minus ends (B and Supplementary Table 2). Thus, Bik1 inhibits microtubule assembly.
Figure 4. Bim1 promotes and Bik1 inhibits microtubule assembly in vitro. (A) Sea urchin axonemes were incubated with 11.5 μM tubulin alone (left) and in the presence of 1 μM Bim1 (center) or 1 μM Bik1 (right). Microtubules were visualized (more ...)
Individual Effects of Bim1 and Bik1 on Microtubule Dynamics
To determine the mechanisms by which Bim1 and Bik1 influence microtubule assembly, we examined the effects of these proteins on microtubule dynamics. Microtubules dynamics are defined by four parameters: the rates of microtubule growth and shrinkage, and the frequencies of catastrophes (transitions from growing to shrinking) and rescues (transitions from shrinking to growing). To determine how Bim1 and Bik1 affect these parameters, we observed microtubules over time using VE-DIC microscopy.
The most substantial effect of Bim1 is on catastrophe frequency. The addition of 0.1 μM Bim1 decreased the frequency of catastrophes 2.5-fold at plus ends and 5.6-fold at minus ends (A and Supplementary Table 1). We did not observe any catastrophes at higher Bim1 concentrations, indicating that catastrophe frequencies were reduced at both plus and minus ends >20-fold in the presence of 0.5 μM Bim1 and >40-fold in the presence of 1 μM Bim1. Under the assembly conditions used, rescues are rare events with 11.5 μM tubulin alone; no rescue events were observed at plus ends and only two rescue events (1.96 events/min) were seen at minus ends (Supplementary Table 1). We did observe a higher frequency of rescues in 0.1 μM Bim1 with 0.51 events/min at plus ends, and 3.33 events/min at minus ends. We could not calculate a rescue frequency at higher Bim1 concentrations because of the absence of shrinking microtubules.
Figure 5. Effects of Bim1 and Bik1 on microtubule dynamics. (A–F) Sea urchin axonemes were incubated with 11.5 μM tubulin in the presence of varying concentrations of Bim1 or Bik1. Individual microtubules were visualized using VE-DIC microscopy (more ...)
In addition, Bim1 significantly increased microtubule growth rates and lowered shrinkage rates (, B and C, Supplementary Table 1). Bim1 at 1 μM increased plus- and minus-end growth rates by 57 and 83%, respectively. Bim1 at 0.1 μM lowered plus- and minus-end shrinkage rates by 2.5- and 3.5-fold, respectively. We could not calculate shrinkage rates at higher Bim1 concentrations because shrinking microtubules were not observed. Overall, these results demonstrate that Bim1 decreases the catastrophe frequency and shrinkage rate and increases the growth rate and rescue frequency.
Bik1 also had a substantial effect on catastrophe frequency but in the opposite direction from Bim1. In the presence of 1 μM Bik1, catastrophe frequencies rose by 38% at plus ends and 2.4-fold at minus ends (D and Supplementary Table 2). Growth rates decreased by 18% at plus ends and 43% at minus ends, but there was no significant change in shrinkage rates (, E and F, Supplementary Table 2). We did not observe a significant increase in rescue events in the presence of Bik1 (Supplementary Table 2). Because Bik1 stimulated catastrophes in 11.5 μM tubulin, we also examined its effect in 14.4 μM tubulin, a tubulin concentration at which catastrophes are rarely observed (Supplementary Table 3). The addition of 0.1 μM Bik1 to 14.4 μM tubulin increased the catastrophe frequency >8-fold at plus ends and >4-fold at minus ends, resulting in frequencies similar to those observed in 11.5 μM tubulin alone. In summary, these results indicate that Bik1 decreases growth rates and increases catastrophe frequency.
The Effect of Bim1, But Not Bik1, on Catastrophe Frequency Is Independent of Its Effect on Growth Rates
Catastrophe frequency is inversely related to growth rate (Drechsel et al., 1992
; van Breugel et al., 2003
), so the effects of Bim1 and Bik1 on catastrophe frequencies could be an indirect effect of their abilities to increase and decrease growth rates, respectively. To test this possibility, we examined the relationship between microtubule growth rate and catastrophe frequency for three conditions: tubulin alone, tubulin plus Bim1, and tubulin plus Bik1. For each condition, we binned microtubules according to their growth rates and calculated the corresponding catastrophe frequencies. We then plotted average catastrophe frequency versus average growth rate for each binned group. As expected for tubulin alone, catastrophe frequency decreases as growth rate increases for both plus and minus ends (, G and H). In the presence of Bik1, the catastrophe frequency at each growth rate is nearly the same as for tubulin alone, indicating that Bik1 likely promotes catastrophe frequency by inhibiting growth rate. However, in the presence of Bim1, the catastrophe frequency at each growth rate is lower than for tubulin alone. Thus, the reduction in catastrophe frequencies by Bim1 is not due solely to its effects on growth rates.
The Combined Effects of Bim1 and Bik1 on Microtubule Dynamics
To determine the effects of the Bim1-Bik1 complex on microtubule dynamics, we combined Bim1 and Bik1 in equimolar amounts (0.1 and 1 μM each). Overall, this complex affected microtubule assembly in nearly the same way as Bim1 alone ( and Supplementary Table 4). This result is more apparent at the higher concentrations of protein (1 μM) because the differences between the effects of the individual proteins at 1 μM are greater. The addition of both proteins at 1 μM increased total microtubule length 12.0-fold at plus ends and 12.1-fold at minus ends compared with tubulin alone (, A and E). We did not observe any catastrophes at 1 μM Bim1 and Bik1, indicating that catastrophe frequencies were reduced >48-fold at plus ends and >29-fold at minus ends (, B and F). Growth rates increased 50% at plus ends and 70% at minus ends (, C and G). All of these parameters are very close to those obtained with 1 μM Bim1 alone and substantially different from those produced by 1 μM Bik1. Lack of shrinking microtubules prevented measuring shrinkage rates at 1 μM (, D and H). However, at 0.1 μM Bim1-Bik1 complex, shrinkage rate at plus ends is the same as for 0.1 μM Bim1 alone.
Figure 6. Combined effects of Bim1 and Bik1 on microtubule dynamics. (A–H) Sea urchin axonemes were incubated with 11.5 μM tubulin in the presence of varying concentrations of both Bim1 and Bik1. Parameters of microtubule dynamic instability were (more ...)
To see whether Bim1 maintained this dominant effect at higher Bik1 to Bim1 ratios, we kept the Bik1 concentration at 1 μM and decreased the concentration of Bim1 to 0.5 or 0.1 μM. At 0.1 μM Bim1 and 1.0 μM Bik1, the assay should contain 0.1 μM Bim1-Bik1 complex and 0.9 μM Bik1, or a 9:1 ratio of free Bik1 to Bim1-Bik1 complex. This excess Bik1 did lead to decreases in total microtubule length and microtubule growth rate relative to 0.1 μM Bim1-Bik1 complex alone; the values for these parameters were approximately midway between those for 1.0 μM Bik1 and 0.1 μM Bim1. However, catastrophe frequency changed very little, remaining close the value for 0.1 μM Bim1 alone. Thus, even though it is out-numbered by Bik1 10:1, Bim1 is still the major influence on microtubule catastrophe frequency.