KIF17 motors are in an inactive state when expressed in mammalian cells
To study the regulation of mammalian KIF17 in its native environment, tagged versions of human KIF17 were expressed in COS cells whose flat morphology is particularly amenable to live cell imaging. This approach has been used successfully to study kinesin-1 and kinesin-3 motors produced and labeled under native conditions, and avoids potential problems associated with in vitro purification and/or labeling (Cai et al., 2007a
When expressed in cells, KIF17 motors tagged at the N or C termini with small epitope tags (Flag-KIF17 and KIF17-Myc) localized diffusely throughout the cytoplasm ( and not depicted). As steady-state expression patterns do not reveal whether the motor is actively binding to and/or moving on microtubules, we took advantage of the fact that the nonhydrolyzable ATP analogue 5′-adenylylimididiphosphate (AMPPNP) locks kinesin motors in a microtubule-bound state. Although many mechanistic and thermodynamic factors contribute to microtubule binding by kinesin motors, the AMPPNP assay can be used to assess the inhibited state of kinesin motors in live cells (Cai et al., 2007a
; Hammond et al., 2009
). The cells were permeabilized with the bacterial toxin streptolysin O (SLO), then AMPPNP was added, and the cells were fixed and stained with antibodies to the epitope tags. Upon addition of AMPPNP, Flag-KIF17 and KIF17-Myc remained cytosolic and did not become locked in a microtubule-bound state (; and not depicted). This suggests that KIF17 motors exist in an inhibited state when expressed in mammalian cells.
Figure 1. The kinesin-2 motor KIF17 is inactive for microtubule binding in mammalian cells. (A) Microtubule binding assay in fixed cells. COS cells expressing KIF17-Myc were untreated or treated with AMPPNP, then fixed and immunostained with anti-Myc and anti-tubulin (more ...)
To study KIF17 motors in live cells, the motor was tagged at the N or C termini with a monomeric version of Citrine (Cit, a variant of yellow fluorescent protein [FP]). The monomeric citrine (mCit)-KIF17 and KIF17-mCit motors localized diffusely throughout the cytoplasm at steady state but became locked in a microtubule-bound state upon addition of AMPPNP (). This indicates that the addition of larger, e.g., FP tags, to either end of the molecule interferes with the inhibitory mechanisms that prevent AMPPNP-induced microtubule localization. We reasoned that the FP tags may sterically hinder motor–tail interactions that contribute to autoinhibition. To test this possibility, we created additional tagged versions of KIF17 that contained spacer sequences (18-aa random sequence or a 20-aa sequence containing a Flag tag) between the mCit tag and the KIF17 polypeptide. Interestingly, the addition of spacer residues between the FP tag and KIF17 resulted in motors (mCit-18aa-KIF17 and mCit-Flag-KIF17) that remained largely cytosolic upon addition of AMPPNP (; and not depicted), which suggests that moving the FP further from the motor domain allows a return to the autoinhibited state. Our interpretation of these results is that KIF17 is regulated by an autoinhibition mechanism that prevents AMPPNP-induced microtubule binding in the absence of cargo. However, unlike other motors tested to date (Cai et al., 2007a
; Hammond et al., 2009
), attachment of a bulky epitope tag via short linkers to the N or C termini interferes with autoinhibition.
Truncated KIF17 motors retain a dimeric state
To identify the domains of KIF17 involved in autoinhibition, we created truncated versions by removing successive segments from the C-terminus (). Analysis of the human KIF17 sequence () shows four regions of potential coil following the N-terminal motor domain—the neck coil (NC) and coiled-coil segments 1–3 (CC1-CC3)—followed by a C-terminal tail domain that can bind to cargo proteins (Setou et al., 2000
; Takano et al., 2007
). When compared with CC predictions of OSM-3 (), the overall domain organization appears similar, with the exception of the region between CC1 and CC2. In OSM-3, this region contains a short hinge segment that is required for the folded conformation of the autoinhibited motor (Imanishi et al., 2006
). In KIF17, this central region contains an ~300 residue segment of undefined structure, which suggests that the mechanisms of autoinhibition of OSM-3 and KIF17 may be different.
Because truncation of the OSM-3 tail domain destabilized the dimeric state of the molecule (Imanishi et al., 2006
), we analyzed the dimeric state of our truncated KIF17 motors. We first used coimmunoprecipitation of Flag/Myc-KIF17 and mCit-KIF17 proteins coexpressed in COS cells. As expected, mCit-KIF17 coprecipitated with Flag-KIF17 in the presence of anti-Flag antibodies but not control (IgG) antibodies (, left). Likewise, when Myc- and mCit-tagged versions of the KIF17 truncations 1–846, 1–738, 1–490, and 1–369 were coexpressed, the mCit-tagged truncations coprecipitated with their Myc-tagged counterparts upon addition of anti-Myc but not control antibodies (, right). These results demonstrate that FL and truncated versions of KIF17 exist in an oligomeric state.
We then used the step-wise photobleaching of mCit to confirm the dimerization state of FL and truncated KIF17 motors at the single-molecule level. The KIF17 constructs were tagged with three tandem copies of mCit (3xmCit) and expressed in COS cells, then the lysates were analyzed by total internal reflection fluorescence (TIRF) microscopy. For each construct, the fluorescence intensity of ~100 individual fluorescent molecules was recorded over time. The number of bleaching steps was determined for each molecule (Cai et al., 2007b
; Hammond et al., 2009
) and then plotted in a histogram to show the population distribution (). In control experiments, although we can detect at least 10 bleaching steps for 3xmCit-tagged proteins by this method, a maximum of six bleaching events were observed for dimeric 3xmCit-tagged kinesin-1 motors, whereas a maximum of three bleaching events were observed for monomeric 3xmCit-tagged kinesin-3 motors (Cai et al., 2007b
; Hammond et al., 2009
; unpublished data). For all FL and truncated KIF17 constructs, no more than six bleaching steps were observed (). These data indicate that FL and truncated KIF17 motors exist in a dimeric state and that the NC segment is sufficient for dimerization.
The C-terminal tail domain of KIF17 is required for inhibition of microtubule binding
We then used the truncated KIF17 constructs to identify regions involved in autoinhibition of microtubule binding. Deletion of the KIF17 tail domain resulted in a motor, 1–846, that was diffuse and cytosolic at steady state but became trapped in a microtubule-bound state upon addition of AMPPNP, regardless of whether 1–846 was tagged with mCit () or Myc tags (Fig. S1
). These results indicate that removal of the C-terminal tail region results in a motor that is no longer inhibited for AMPPNP-induced microtubule binding.
Figure 3. The C-terminal tail domain is required for inhibition of microtubule binding. Live cell microtubule binding assay. (left) COS cells expressing the indicated truncated KIF17 proteins (C-terminal mCit tag) were imaged, transiently permeabilized with SLO, (more ...)
Further truncations of KIF17 that removed the CC3 segment ([1–795]-mCit], the CC2 segment ([1–738]-mCit), the central region of unknown structure ([1–490]-mCit), or the CC1 segment ([1–369])-mCit) resulted in no change in microtubule binding behavior. That is, all truncations showed diffuse cytosolic localization before addition of AMPPNP and redistributed to a microtubule-trapped localization upon addition of AMPPNP (). The percentage of cells showing microtubule-bound motors in the presence of AMPPNP was lower for these truncations than for 1–846 motors (), which indicates that the CC3 segment may play a role in facilitating microtubule binding, either by interacting directly with the microtubule as has been reported for kinesin-1’s kinesin heavy chain (KHC) tail domain (Dietrich et al., 2008
), or by promoting a conformation of the KIF17 motor that facilitates microtubule binding and/or the retention of AMPPNP in the nucleotide pocket.
It should also be noted that in the presence of AMPPNP, active KIF17 motors still maintain a pool of motors that is localized diffusely throughout the cytoplasm ( and ; and unpublished data). This is in contrast to active versions of kinesin-1, where addition of AMPPNP traps virtually all expressed motors on microtubules, effectively clearing the cytoplasm (Cai et al., 2007a
; Hammond et al., 2009
). Whether this indicates a weaker binding of KIF17 than KHC to microtubules in the presence of AMPPNP, or whether these KIF17 motors are still partially inhibited for microtubule binding, is presently unclear.
The CC2 segment inhibits the processive motility of KIF17
At steady-state, expressed (1–738)-mCit and (1–490)-mCit motors showed an accumulation at the periphery of the cell, presumably at the plus ends of the microtubules, in addition to their diffuse cytosolic localization (, arrowheads). This suggests that, unlike the FL and other truncated motors, 1–738 and 1–490 may have the ability to move processively toward the plus ends of microtubules. To test this directly, we performed two assays that measure the ability of kinesin motors to undergo processive motility.
As a first test of the ability of FL and truncated KIF17 motors to move processively along microtubules, we used neuronal cells whose microtubule minus and plus ends are spatially segregated to the cell body and neurite tips, respectively. In this assay, active motors accumulate at neurite tips, and this localization generally correlates with their processive motility in vitro (Nakata and Hirokawa, 2003
; Lee et al., 2004
; Jacobson et al., 2006
; Hammond et al., 2009
). When expressed in differentiated CAD cells, mCit- and Myc-tagged versions of FL KIF17 were primarily localized in the cell body and did not concentrate in neurite tips (; and Fig. S2
), which suggests that FL KIF17 motors are inactive for processive motility when expressed in mammalian cells. Similar results were obtained for the truncated constructs 1–846 and 1–795 (), even though these motors can bind to microtubules in the presence of AMPPNP (). These results indicate that microtubule binding and processive motility are two separate events that are regulated by different segments of the KIF17 protein.
Figure 4. The CC2 segment is required for inhibition of processive motility. (A) Processive motility in vivo. CAD cells expressing FL or truncated KIF17 constructs (C-terminal mCit tag) were imaged. Shown are mCit fluorescence (top) and phase contrast (bottom) (more ...)
Further truncation to remove the CC2 segment of KIF17 resulted in a motor, 1–738, that accumulated at the plus ends of microtubules in the neurite tips (). This suggests that the CC2 segment is critical for preventing the processive movement of KIF17 in mammalian cells. Similar results were obtained upon further truncation to remove the central region ([1–490]-mCit, ; and [1–490]-Myc, Fig. S2). However, removal of the CC1 segment resulted in a decrease in processive motility, as (1–369)-mCit motors maintained a pool of motor in the cell body in addition to accumulation at neurite tips ().
To verify the processive motility of these KIF17 motors and to obtain quantitative information about their motile properties, we performed in vitro single-molecule motility assays. For this assay, KIF17 motors were tagged with three tandem copies of mCit for increased signal and decreased photoblinking and photobleaching (Cai et al., 2007b
). Lysates from COS cells expressing KIF17-3xmCit motors were added to a flow chamber containing polymerized microtubules and assayed by time-lapse imaging on a TIRF microscope. Only motility events that lasted at least six frames (300 ms) were included in the data analysis. To compare motility properties between KIF17 constructs, the amount of expressed protein was normalized by Western blotting with an antibody to mCit (unpublished data), and each construct was analyzed for the same total amount of time.
Few in vitro motility events were detected for FL KIF17-3xmCit motors (n = 21; ; and ), which is consistent with their inability to accumulate at the plus ends of microtubules in neuronal cells (). Analysis of these motility events showed that FL KIF17-3xmCit motors moved for only short distances (mean run length 0.36 ± 0.04 µm; and ). A limited ability to undergo processive motility in vitro was also seen upon removal of the tail domain ([1–846]-3xmCit) and the CC3 segment ([1–795]-3xmCit; not depicted), which is consistent with their in vivo behavior (). However, further truncations that removed the CC2 segment resulted in a dramatic increase in the number of motility events observed for (1–738)-3xmCit motors (n = 112; ; and ) and (1–490)-3xmCit motors (see and ). (1–738)-3xmCit motors moved with a mean speed of 0.77 ± 0.02 µm/s ( and ), and moved in runs that were as long as 6–7 µm but averaged 1.13 ± 0.10 µm ( and ). Truncations that remove the CC1 segment ([1–369]-3xmCit) resulted in a reduction in the number of motility events observed (n = 40) and the mean run length (0.45 ± 0.06 µm; and ), which is consistent with the in vivo analysis (), which suggests that the CC1 segment may facilitate processive motility. We conclude that the CC2 segment plays an important role in autoinhibition of KIF17 by preventing processive motility.
Motile properties of truncated KIF17 constructs
Figure 8. The CC2 and tail domains interact directly with the KIF17 motor. (A) Schematic of GST-tagged constructs. (B) Coomassie-stained gel showing recombinant, purified GST-(1–490)-Myc motor, WT and mutant GST-CC2, and WT and mutant GST-tail proteins. (more ...)
Effects of the CC2 and tail domains on the motility of KIF17 motors
Mutation of the CC2 segment relieves autoinhibition of processive motility
The results presented thus far suggest that two regions of KIF17 are involved in autoinhibition of motor activity: the tail domain interferes with microtubule binding (), whereas the CC2 segment blocks processive motility (). We hypothesized that KIF17 exists in a folded state that allows these segments to inhibit motor activity. If so, then KIF17 should undergo conformational changes that correlate with its activation, as observed for OSM-3 (Imanishi et al., 2006
), and the inhibitory CC2 and tail segments should directly interact with and inhibit the activity of the motor domain.
To test the relationship between KIF17 conformation and activity, we performed sucrose gradient sedimentation assays. However, we first had to generate mutations in FL KIF17 that result in activation of processive motility. Deletion of the entire CC2 segment in a manner that preserves the predicted CC architecture of KIF17 (not depicted) resulted in an active motor, ΔCC2, that accumulated at neurite tips in differentiated CAD cells (), which confirms that the CC2 segment plays a critical role in autoinhibition of KIF17 motility. To generate more subtle activating mutations, we created a point mutation based on the OSM-3 allele sa125
in which mutation of Gly444 to Glu (G444E) results in activation of OSM-3 ATPase activity, processive motility in vitro, and chemosensory defects in worms (Imanishi et al., 2006
). Because Gly444 and surrounding residues in OSM-3 align best with Gly754 and adjacent residues of KIF17 (unpublished data), we mutated Gly754 to Glu (G754E) in FL KIF17. This mutation resulted in a dramatic accumulation of motors at neurite tips in differentiated CAD cells (), which demonstrates that a single point mutation in CC2 is sufficient to relieve autoinhibition of FL KIF17.
Figure 5. Deletion or mutation of CC2 is sufficient to relieve autoinhibition of processive motility. (A–E, left) Schematics of FL KIF17 (A), constructs in which the CC2 domain was deleted (B, ΔCC2), or constructs in which mutations were introduced (more ...)
Because ionic interactions are critical for inhibition of the kinesin-1 motor by its tail domain (Stock et al., 1999
), we looked for charged regions in the tail and CC2 segments of KIF17 that could be involved in inhibition of microtubule binding and processive motility, respectively. In both the tail domain and the CC2 segment, a stretch of basic residues was identified and mutated to alanines (1016–1019A and 764–772A, respectively; ). When expressed in CAD cells, 764-772A motors but not 1016-1019A motors showed a dramatic accumulation in neurite tips (), which indicates that charged residues in CC2 may be critical for interaction with the motor domain in the autoinhibited state.
To confirm that mutation of residues G754 and 764–772 in the CC2 domain results in activation of processive motility, we performed in vitro single-molecule motility assays. The motility of individual motors in lysates of COS cells expressing 3xmCit-tagged versions of FL KIF17, G754E, or 764–772A motors was determined by time-lapse TIRF microscopy. Again, few motility events were observed for FL KIF17 (n = 8; and ). However, mutation of either G754E or 764–772A resulted in a dramatic increase in the number of motility events observed as well as the overall speed and processivity of the motor (; and ). Notably, mutation of the basic residues 764–772 resulted in a larger increase in KIF17 activation than mutation of the single glycine residue. Collectively, these results demonstrate that the CC2 segment plays a critical role in autoinhibition of processive motility and that the ionic interactions between CC2 and the KIF17 motor domain may be critical for this regulation.
Motile properties of mutant FL KIF17 constructs
Release of inhibition correlates with a shift to an extended conformation
We then used sucrose gradient sedimentation analysis to test the relationship between KIF17 activity and conformation. Lysates of COS cells expressing Flag-tagged versions of inactive (FL) and active (G754E and 764–772A) KIF17 motors were loaded on the top of linear sucrose gradients and separated by sedimentation. The majority of FL KIF17 motor was found in fractions 6 and 7 (). Mutation of G754E or 764–772A resulted in a slower migration in the sucrose gradients (), which is consistent with the possibility that the active motors are in an extended conformation. A similar shift in sedimentation behavior was observed for the activated OSM-3(G444E) motor (Imanishi et al., 2006
). The change in sedimentation is consistent with the hypothesis that KIF17 undergoes a conformational transition between an inactive folded state and an active extended state.
Figure 6. Mutations in CC2 that relieve autoinhibition convert the motor to an extended conformation. Lysates of COS cells expressing Flag-tagged FL KIF17, or the G754E or 764-772A mutants were subjected to sucrose gradient sedimentation. Fractions were removed (more ...)
The CC2 and tail segments inhibit KIF17 motility via direct interactions with the motor domain
We tested whether the CC2 and tail constructs could act in trans to inhibit the processive motility of active KIF17 motors. In differentiated CAD cells, the ability of active (1–738)-mCit motors to accumulate in neurite tips () was not affected by expression of a Myc-tagged version of KIF17’s central region (aa 466–738; ), but was abolished upon coexpression of Myc-tagged CC2 + CC3 or CC2 segments (). Although expression of the Myc-tail domain reduced the accumulation of (1–738)-mCit motors in neurite tips, the result was not statistically significant, perhaps because of nuclear accumulation of the tail construct ().
Figure 7. The CC2 and tail domains inhibit KIF17 motility in trans. (A) Processive motility in vivo. Differentiated CAD cells expressing (1–738)-mCit motor alone (NT, nontransfected control) or together with the indicated Myc-tagged constructs were fixed (more ...)
To obtain quantitative information about the ability of the CC2 and tail segments to inhibit the activity of the KIF17 motor, we used single-molecule motility assays. Lysates of COS cells expressing active (1–490)-3xmCit motors were incubated alone or in the presence of recombinant purified GST-tagged CC2 or tail domains (), and the motility of individual motors was analyzed by TIRF microscopy. A large number of motility events were observed for (1–490)-3xmCit motors alone ( and ) and in the presence of GST ( and ). When added in trans, the CC2 segment completely blocked motor activity, as no motility events were observed for (1–490)-3xmCit motors in the presence of GST-CC2 ( and ). Addition of the GST-tail domain did not completely block motor activity but did result in a dramatic reduction in the number of motility events observed ( and ).
To test whether inhibition of KIF17 motility is caused by direct interactions between the motor domain and the CC2 or tail segments, we performed binding assays using recombinant GST-tagged motor, CC2, and tail constructs (). For these experiments, we define the KIF17 motor domain as a dimer of heads, including the NC and CC1 segments. We generated both wild-type (WT) and mutant (764–772A and 1016–1019A) versions of the CC2 and tail domains, and in both cases, mutation of the basic residues resulted in a faster mobility in SDS-PAGE gels (). The WT and mutant GST-tail constructs were highly susceptible to proteolysis ().
Direct binding assays were performed by loading GST-KIF17(1–490)-Myc motors in a column via anti-Myc antibodies. Purified GST-tagged WT and mutant (764–772A) versions of the CC2 segment, and WT and mutant (1016–1019A) versions of the tail domain were allowed to bind to the column. After extensive washing, the bound fraction was analyzed by Western blotting with an anti-GST antibody to detect all proteins. GST alone was used as a control and did not bind to the motor (, lane 1). The CC2 segment was able to interact directly with the motor as GST-CC2 protein bound to the column (, lane 2), but this interaction was dramatically reduced by mutation of the basic residues 764–772 (, lane 3). In contrast, both WT and mutant 1016-1019A forms of the tail domain bound to the motor (, lanes 4 and 5). These results indicate that both CC2 and tail segments interact directly with the KIF17 motor domain and that residues 764–772 are critical for the interaction of CC2 with the motor. We conclude that autoinhibition of KIF17 results from intramolecular interactions between the KIF17 motor domain and the CC2 and tail segments in the folded inactive state.