Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein

LETTERS

Reconstitution of a hierarchical +TIP interactionnetwork controlling microtubule end tracking of dyneinChristian Duellberg1,2, Martina Trokter1,2,4, Rupam Jha1, Indrani Sen3, Michel O. Steinmetz3and Thomas Surrey1,2,5

Growing microtubule end regions recruit a variety of proteinscollectively termed +TIPs, which confer local functions to themicrotubule cytoskeleton1. +TIPs form dynamic interactionnetworks whose behaviour depends on a number of potentiallycompetitive and hierarchical interaction modes2. The rules thatdetermine which of the various +TIPs are recruited to thelimited number of available binding sites at microtubule endsremain poorly understood. Here we examined how the humandynein complex, the main minus-end-directed motor and animportant +TIP (refs 2–4), is targeted to growing microtubuleends in the presence of different +TIP competitors. Using atotal internal reflection fluorescence microscopy-basedreconstitution assay, we found that a hierarchical recruitmentmode targets the large dynactin subunit p150Glued to growingmicrotubule ends via EB1 and CLIP-170 in the presence ofcompeting SxIP-motif-containing peptides. We further showthat the human dynein complex is targeted to growingmicrotubule ends through an interaction of the tail domain ofdynein with p150Glued. Our results highlight how theconnectivity and hierarchy within dynamic +TIP networksare orchestrated.

Important microtubule functions are mediated by proteins thatlocalize to growing microtubule plus ends and are therefore called+TIPs. Most +TIPs are recruited by proteins of the EB1 family1,2

(EBs). The amino-terminal calponin homology (CH) domain ofEB1 binds an extended region at growing microtubule ends that isconformationally different from the main body of the microtubule5,6.The carboxy-terminal domain of EB1, which contains the EBhomology domain (EBH) and a C-terminal EEY/F sequence motif,is responsible for the dimerization of EB1 monomers and representsa hub for interactions with other +TIPs (ref. 7). EB-recruitedproteins contain either a cytoskeleton-associated protein glycine-rich

(CAP-Gly) domain or the sequence motif SxIP (refs 7,8). CAP-Glydomains and SxIP motifs can interact with different parts of theC-terminal domain of EB1 (Table 1; ref. 7). As many proteins arerecruited by EB1, especially in higher eukaryotes where SxIP motifsare abundant7,9, the question arises of whether EB-dependent +TIPscompete with each other1,8. Furthermore, several +TIPs interact witheach other2, giving rise tomultiple possibilities for generating complexinteraction networks (Table 1). The logic underlying the connectivityand hierarchy of these networks is only poorly understood, raising thequestion of how +TIP networks act as a system.

One essential protein being part of +TIP networks is themicrotubule motor dynein4,10–12. Dynein is a multisubunit complexand the main minus-end-directed motor protein in highereukaryotes3. Dynein microtubule plus-end tracking has importantfunctions. In budding yeast, it is required for cortical anchoringand correct positioning of the nucleus during mitosis13. In neuronsand filamentous fungi, microtubule plus-end tracking of dynein iscritical for correct initiation of retrograde cargo transport4,14,15. Themechanism by which dynein is recruited to growing microtubule plusends is, however, not well understood.

Several proteins have been implicated in dynein end tracking,including canonical +TIPs (EB1, CLIP-170; refs 4,14), directdynein regulators (dynactin, LIS1; ref. 11) and plus-end-directedmotors13,16. It is unknown whether all of these proteins are requiredtogether to recruit dynein to microtubule ends or whetheralternative/complementary pathways for dynein targeting exist.One involved protein is the dynactin subunit p150Glued (refs 4,11;from here on referred to as p150). p150 mediates the interactionbetween dynein and dynactin via direct binding to the dyneinintermediate chain17 (Table 1) and regulates dynein processivity invitro18,19. It contains a CAP-Gly domain followed by two coiled-coilsequence regions18 (Table 1 and Supplementary Fig. 1). Mutations inthe CAP-Gly domain of human p150 are linked to Perry syndrome

1London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK. 2European Molecular Biology Laboratory, Meyerhofstrasse 1,69117 Heidelberg, Germany. 3Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.4Present address: Institute of Structural and Molecular Biology, University College London and Birkbeck, Malet Street, London WC1E 7HX, UK.5Correspondence should be addressed to T.S. (e-mail: [email protected])

Received 13 February 2014; accepted 29 May 2014; published online 6 July 2014; DOI: 10.1038/ncb2999

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1

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LETTERS

Table 1 Brief summary of known domains and motifs mediating interactions within the +TIP network.

Domain/motif Found in Interacts with

CH domain N-terminal part of EBs Nucleotide-dependent conformation at growing microtubule end regions

EBH domain Part of C-terminal region of EBs SxIP motifs, CAP-Gly domains of p150∗

EEY/F motif C-terminus of EBs, CLIP-170 and α-tubulin CAP-Gly domains

Zinc knuckle CLIP-170 CAP-Gly domains of p150 and CLIP-170

SxIP motif Several unrelated EB-dependent +TIPs EBH domains

CAP-Gly domain CLIP-170, CLIP-115 and p150 EEY/F motifs and EBH domains∗

p150 coiled coil 1 (CC1) p150 subunit of dynactin complex Intermediate chain subunit of the dynein complex

p150 coiled coil 2 (CC2) p150 subunit of dynactin complex Other dynactin components (Arp1, and potentially others)

CH: calponin homology. EBs: end-binding proteins. EBH: EB homology. CAP-Gly: cytoskeleton-associated protein glycine-rich. ∗p150 can bind composites of EEY/F motifs and EBH domains,or EEY/F motifs and zinc knuckles (bipartite interaction), respectively. In contrast, the CAP-Gly domain of CLIP-170 is not expected to interact with the EBH domain.

where plus-end tracking of p150 is impaired and initiation of dynein-dependent retrograde cargo transport in neurons is defective4,14,20,21.The isolated CAP-Gly domain of p150 interacts in vitro directly withboth the EBH domain and the EEY/F sequence motif of EB1 (Table 1;ref. 22). However, in living cells, EB1 is necessary but not sufficientto mediate microtubule end tracking of p150, which additionallyrequires CLIP-170 (refs 23,24).

CLIP-170 is a dimeric protein that contains two CAP-Gly domainsin its N-terminal part. These CAP-Gly domains interact with theC-terminal EEY/F motifs of both EB1 and α-tubulin25,26. Theseinteractions recruit CLIP-170 to growing microtubule ends, and areexpected to compete with p150 binding to EB1 (refs 25,27). Theisolated CAP-Gly domain of p150 interacts also with the isolatedC-terminal part of CLIP-170, more specifically with the distal zincknuckle and the C-terminal EEY/Fmotif of CLIP-170 (Table 1; refs 23,27,28). The presence of this additional p150 binding site suggests theexistence of a chain of interactions in living cells from EB1 to CLIP-170 to p150 (ref. 23). However, why p150 cannot localize to growingmicrotubule ends in cells23,24 or in vitro4 in the absence of CLIP-170,although its isolated CAP-Gly domains robustly interact with EB1in vitro22,29, is unclear. Therefore, several aspects of the molecularmechanisms that target dynactin and dynein to growing microtubuleends are not well understood.

Here, we examined the hypothesis that a hierarchical modeof recruitment, realized by a particular +TIP network module, isnecessary for the accumulation of human dynein/dynactin at growingmicrotubule ends specifically under conditions where other +TIPscompete for binding to EB1. This is probably the situation in humancells where many SxIP-motif-containing +TIPs are known7,9. To whatextent they compete with CAP-Gly-domain-containing proteins suchas p150 or CLIP-170 remains, however, unclear.

First, we examined whether p150 can be recruited to growingmicrotubule ends directly by EB1. We purified human EB1 andan mCherry-tagged version of a human tissue isoform30,31 of p150comprising the first 527 amino acids (mCherry–p150). The constructincludes the CAP-Gly domain and the first coiled-coil domain ofp150 (Fig. 1a). Using dual-colour total internal reflection fluorescence(TIRF) microscopy and an in vitro reconstitution assay6, we observedthat mCherry–p150 tracked the ends of Cy5-labelled microtubules inthe presence (Fig. 1b left and Supplementary Video 1) but not in theabsence (Fig. 1b right) of unlabelled EB1. We also purified mCherry-tagged constructs of the neuronal p150 isoform that containsan additional positively charged sequence encoded by exons 5–7

(Supplementary Fig. 1a; refs 30,31). This isoform boundmore stronglyto microtubules30,31 (Supplementary Fig. 1c) and, in contrast to aprevious report4, also tracked growing microtubule ends in an EB1-dependent manner (Supplementary Fig. 1c). In control experiments,p150 constructs very similar to those used previously4 were alsorecruited to growing microtubule ends by EB1 (SupplementaryFig. 1d). Possible reasons for the different observations4 couldbe differing buffer compositions (potentially affecting proteinsolubility—in particular, the p150 construct that is most similar tothe construct used in ref. 4 was found not to be very soluble) ordifferent EB1 labelling strategies. An mCherry-tagged version of theEB1 homologue EB3 was also sufficient to target both p150 isoformsto growing microtubule ends (Supplementary Fig. 1e and 3b right).We conclude that in vitro EBs are necessary and sufficient to recruitboth isoforms of p150 to growing microtubule ends, in agreementwith the established interaction between the isolated p150 CAP-Glydomain and the C-terminal part of EB1 (refs 22,29).

In living cells, EB1 is necessary but not sufficient for p150 endtracking23,24.We therefore reasoned that competitionwith other +TIPsmight displace p150 from EB1, suggesting an alternative mechanismof recruitment. Competing +TIPs might be SxIP-motif-containingproteins, because like the p150 CAP-Gly domain they also bind tothe EBH domain of EB1 (Table 1; refs 8,22). To mimic the action ofcompeting SxIP-motif-containing+TIPs in cells, we added to our assaya synthetic peptide comprising the sequence of the well-studied SxIPmotif of humanMACF2 (refs 8,32). As expected, a fluorescein-labelledversion of this peptide (Fluo–SxIP) was recruited to growing Cy5–microtubule ends by unlabelled EB1 (Fig. 1c). Adding 6 µMunlabelledSxIP peptide to 75 nMmCherry–p150 in the presence of 150 nM EB1abolished EB1-dependent end tracking of p150 (Fig. 1d right). Thiseffect was SxIP-sequence-specific, because an inactive control peptidein which the Ile-Pro dipeptide was replaced by Asn-Asn (denotedSxNN; ref. 32) did not displace p150 from EB1 at growingmicrotubuleends (Fig. 1d left). We conclude that the SxIP peptide and the p150CAP-Gly domain compete with each other for binding to EB1 atgrowing microtubule ends (Fig. 1e), consistent with structural datashowing a partial overlap of the two different binding modes8,22.

Next, we investigated whether EB1-mediated microtubule endtracking of the CAP-Gly domain containing CLIP-170 (Fig. 2a) issimilarly affected by SxIP peptides. A concentration of 6 µM SxIPpeptide was not sufficient to abolish EB1-dependent end trackingof 75 nM purified GFP-tagged CLIP-170 (Fig. 2b left and middle),but an increased concentration of 15 µM SxIP peptide displaced

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mCherry–p150 mCherry–p150

6 µM SxIP

mCherry–p150Fluo–SxIP Fluo–SxIPEB1+ EB16 µM SxNN

KymographsmCherry Cy5–tubulin mCherry Cy5–tubulin

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SxNN

EEY/F

Fluorescein mCherry

Cy5–tubulin Cy5–tubulin

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p150 SxIP

FP

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F

FPCoiled coilCH Fluorescent proteinEEY/F

CAP-Gly Serine-rich EBH Fluorescein tag

Figure 1 SxIP-motif-containing peptides disrupt EB1-mediated targeting ofp150 to growing microtubule ends. (a) Scheme of the proteins and peptidesused in the experiments shown in this figure: full-length EB1, mCherry-tagged p150 fragment consisting of the first 527 amino acids (tissue isoform;see Methods and Supplementary Fig. 1), fluorescein-labelled and unlabelledSxIP and an inactive SxNN control peptide. Note that EBs, CLIPs andp150 form dimers in solution but are depicted as monomers throughout,for simplicity. (b) Dual-colour TIRF microscopy images (top) of 125nMmCherry–p150 (green) binding to dynamic Cy5–microtubules (red) in thepresence (left) or absence (right) of 150nM unlabelled EB1. Single- anddual-colour kymographs (bottom) showing the time history of a selectedrepresentative microtubule for each condition, as indicated. For an illustration

of kymograph generation, see Supplementary Fig. 1b. (c) Single- and dual-colour kymographs of TIRF microscopy experiments, as indicated, showingthat 150nM unlabelled EB1 targets Fluo–SxIP (green in merge; present at150nM) to growing ends of Cy5–microtubules (red in merge). (d) Kymographsshowing that unlabelled SxIP peptide (right), but not inactive control peptideSxNN (left), impairs EB1-mediated end tracking of 75nM mCherry–p150(green in merge). (e) Scheme illustrating competition between SxIP peptidesand p150 for EB1 binding. Unlabelled EB1 was present at 150nM. TheCy5–tubulin concentration was 20 µM. In all experiments, the labelling ratiowas 0.08 Cy5 molecules per tubulin dimer. Experiments were performed inStandard TIRFM buffer (Methods). Horizontal and vertical scale bars are 3 µmand 20 s, respectively.

most CLIP-170 from microtubule ends in the presence of EB1(Fig. 2b right). The difference between the ability of p150 andCLIP-170 to withstand competition by the SxIP peptide may bedue to the tandem arrangement of CAP-Gly domains in CLIP-170.This duplication is probably, at least in part, the reason for thetighter interaction between EB1 and the N-terminal part of CLIP-170 compared with the interaction between EB1 and p150, as judgedby size-exclusion chromatography (Fig. 2d). Competition betweenSxIP peptide and CLIP-170 could also be demonstrated by displacing

150 nM Fluo–SxIP from growing microtubule ends in the presence of150 nM EB1 by the addition of 660 nM unlabelled CLIP-170 (Fig. 2c).In conclusion, binding of SxIP motifs and CAP-Gly domains to theC-terminal domain of EB1 are mutually exclusive (Figs 1d and 2b,cand Supplementary Fig. 2).

CLIP-170 is a relatively efficient competitor for EB1 binding (Fig. 2)and its C terminus offers an additional binding site for the p150CAP-Gly domain27,28. We therefore investigated in triple-colour TIRFmicroscopy experiments whether CLIP-170 can directly restore p150



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GFP–CLIP-170 GFP–CLIP-170

EB1 EB115 μM SxIP

GFP–CLIP-170

6 μM SxIPEB1

GFP

Merge

Fluo–SxIP Fluo–SxIP

Fluorescein

EB1

CLIP-170

EB1

Merge

EB1

GFP–CLIP-170ΔC

Complex

EB1/p150 (ratio 1:1) EB1/p150 (ratio 1:3)

EB1

mCherry–p150547-N

Complex

EB1

mCherry–p150547-N

Complex

EB1/CLIP-170ΔC (ratio 1:1)

(RIU

)

Competitivebinding

EB1

SxIP


CLIP-170

1.5 × 10–5

1.0 × 10–5

5.0 × 10–6

0.0

1.5 × 10–5 4.0 × 10–5

3.0 × 10–5

2.0 × 10–5

1.0 × 10–5

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5.0 × 10–6

0.0

5 10 15 5 10 15 5 10 15

(ml) (ml) (ml)

a

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FP

FP FPEEY/FEEY/F

GFP–CLIP-170 GFP–CLIP-170ΔCCLIP-170

Coiled coil Serine-rich Zinc knuckle CAP-Gly Fluorescent proteinEEY/F

Figure 2 SxIP motif and CAP-Gly domain binding to EB1 is mutuallyexclusive. (a) Scheme of the CLIP-170 constructs that are used: GFP-taggedand untagged full-length CLIP-170 and GFP-tagged CLIP-170 fragmentlacking the last 946 amino acids (GFP–CLIP-1701C). (b) Single- anddual-colour kymographs depicting the localization of 75nM GFP–CLIP-170(green in merge) in the presence of 150nM EB1 and SxIP peptides atthe indicated concentrations on dynamic Cy5–microtubules (red in merge).(c) Kymographs showing that 150nM EB1 targets Fluo–SxIP (green inmerge; present at 150nM) to the ends of growing Cy5–microtubules(red in merge; left), which is abolished by the presence of additional660nM unlabelled CLIP-170 (right). Cy5–tubulin concentration was 20 µM.Experiments were performed in Standard TIRFM buffer without EGTA(Methods). (d) Analytical gel filtration elution profiles of mixtures of EB1

and GFP–CLIP-1701C and of EB1 and mCherry–p150547-N (SupplementaryFig. 1a) at the indicated molar ratios. EB1 was always present at 12 µM.Whereas a 1:1 mixing ratio of EB1/CLIP-1701C is sufficient to shift theentire EB1 peak into the EB1–CLIP-1701C complex peak (at ∼10mlin the blue profile in the left panel), a 1:1 ratio of EB1/p150 is notsufficient to completely shift the EB1 into the EB1–p150 complex peak(in the middle panel where part of the EB1 still elutes at ∼13ml).Only at a 1:3 ratio of EB1/p150, does the EB1 become entirely partof the complex (blue profile, right panel). These experiments showtighter binding between EB1 and CLIP-170 than between EB1 andp150. (e) Scheme illustrating competition between CLIP-170 and SxIPpeptides for EB1 binding. Horizontal and vertical scale bars are 3 µmand 20 s, respectively.

end tracking in the presence of an excess of the SxIP peptide. Thiswas indeed the case: addition of 75 nM GFP–CLIP-170 to the assaywith 150 nM EB1, 75 nM mCherry–p150 and 6 µM SxIP competitorsrestored end tracking of mCherry–p150 (Fig. 3a third column, 3b bluecurve and Supplementary Video 2). This result is in contrast to thesame situation without CLIP-170 where the excess of SxIP peptidedisplaces mCherry–p150 from EB1 (Figs 1d right, 3a second columnand 3b red curve). End tracking of p150 was restored by CLIP-170 tosimilar levels as in experiments without CLIP-170 in the presence of anexcess of the inactive SxNN peptide variant (Fig. 3a first column and3b black curve). Therefore, EB1 and CLIP-170 are not only necessary

but also sufficient for p150 end tracking in the presence of SxIPpeptide competitors.

Adding a GFP-tagged truncated construct of CLIP-170 that lacksthe C-terminal part of the protein (GFP–CLIP-170-1C) did notrestore p150 end tracking (Fig. 3a right and 3b pink curve), althoughGFP–CLIP-170-1C clearly localized to growing microtubule endsin a manner similar to full-length GFP–CLIP-170 (compare Fig. 3asecond row, third and fourth column). Likewise, unfolding of theCLIP-170 zinc knuckles by addition of metal chelators33 stronglydecreased its binding to p150 (Supplementary Fig. 3). Therefore, anintact C terminus of CLIP-170 is necessary for the restoration of



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EB1

mCherry–p150

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EB1 EB1 EB1

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GFP–CLIP-170 GFP–CLIP-170ΔC mCherry

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c

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GFP

Cy5–tubulin

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SxIP

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Peak intensities of mCherry–p150

–500 5000Distance (nm)

No

Fluo

resc

ence

(a.u

.)

Fluo

resc

ence

(a.u

.)

competition

Quantification of mCherry–p150 signals

Time-averaged microtubule signal

Time-averaged mCherry–p150 signal

SxNNSxIPSxIP+ CLIP-170SxIP+ CLIP-170ΔC

SxNNSxIPSxIP+ CLIP-170SxIP+ CLIP-170ΔC

EB1

SxIP


CLIP-170

p150

200

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0

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Distance (nm)1,000

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Figure 3 CLIP-170 restores EB1-dependent end tracking of p150 in thepresence of SxIP peptides. (a) Representative single- and triple-colourkymographs showing the localization of 75nM mCherry–p150 (red inmerge) on dynamic Cy5–microtubules (blue in merge) in the presence of150nM unlabelled EB1 with 6 µM SxNN peptide or 6 µM SxIP peptidealone or together with 75 nM GFP–CLIP-170 or 75nM GFP–CLIP-1701C(green in merge), as indicated. Cy5–tubulin concentration was 20 µM.Experiments were performed in Standard TIRFM buffer without EGTA(Methods). Horizontal and vertical scale bars are 3 µm and 20 s, respectively.(b,c) Averaged mCherry–p150 fluorescence intensity profiles along themicrotubule end region for the conditions shown in a (b), and theirpeak intensities (c). In brief, mCherry–p150 images were cropped around

automatically detected growing Cy5–microtubule ends and averaged. One-dimensional mCherry intensity profiles along the microtubule axis weregenerated from these time-averaged images. The ‘zero’ position indicatesthe microtubule end position. Time-averaged images for one representativemicrotubule and the corresponding mCherry–p150 signal (correspondingto the black curve; ‘SxNN’) are depicted to visualize the orientationof the profiles. Error bars are standard errors of the mean (s.e.m.)and are derived from at least 250 individual frames per conditionfrom 3 independent experiments. For details, see Methods and ref. 42.(d) Scheme illustrating how the CLIP-170-mediated hierarchical recruitmentmode can rescue p150 end tracking in the presence of competition forEB1 binding.

microtubule end tracking of p150 by CLIP-170 in the presence of SxIPpeptide competitors, consistent with structural information27,28. Theobservation that CLIP-170-1C clearly acted as a competitor of p150end tracking (Supplementary Fig. 3c) provides an explanation why incells a knockout of CLIP-115, a close homologue of CLIP-170 lackingthe C-terminal zinc knuckles, resulted in increased p150 signals at

microtubule ends34. Binding of full-length CLIP-170 to EB1-loadedmicrotubule ends competes with direct binding of p150 to EB1, butat the same time introduces an additional binding site that is specificfor p150 but not for SxIP-motif-containing proteins. Consequently, inthe presence of competitors, p150 binds to microtubule ends usinga different interaction partner than in the absence of competition



LETTERS

GFP

mCherry

Cy5–tubulin

LIC2

IC1Tctex1

a

c

b

LC8RB1

CDHC

FP

FP

cc

Motor

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+TIP hierarchy controlling microtubule plus-end tracking of the human dynein complex

EEY/F

SxIP

EEY/

F

EB1

CLIP-170

p150

DynactinDyneincomplex

Dynein(artificial dimer)

Dynein(complex)

EB1 EB1 EB1


mGFP–Dynein mGFP–Dynein mGFP–Dynein (Artificial dimer)(Complex) (Complex)


EEY/FCH Coiled coil Zinc knuckle

CAP-Gly Serine-rich EBH

Figure 4 EB1 and p150 target the human dynein complex to growingmicrotubule ends. (a) Schematic representation of the human dynein complex(left) and a dimer of the two motor domains as used for experiments inthis figure. Abbreviations used as in ref. 36. (b) Representative single-and triple-colour kymographs depicting the localization of 14nM mGFP-labelled human dynein complex (green in merge) in the presence of200nM unlabelled EB1 and in either the additional presence (left) orabsence (middle) of 125nM mCherry–p150 (red in merge) on dynamicmicrotubules (blue in merge). 14 nM mGFP-tagged dimerized dynein motorsdomains (green in merge) do not localize to microtubule ends in thepresence of unlabelled EB1 and mCherry–p150 at concentrations as

above (right). Cy5–tubulin concentration was 20 µM. Experiments wereperformed in Dynein imaging buffer (Methods). Horizontal and verticalscale bars are 3 µm and 20 s, respectively. (c) Scheme of the +TIPnetwork directing the dynein complex to microtubule ends via thehierarchical dynactin/CLIP-170/EB1 interaction module in the presenceof SxIP competitors. SxIP-motif-containing proteins and CAP-Gly-domain-containing proteins compete for a limited amount of EB1-binding sites.CLIP-170 binds relatively efficiently to EB1 and provides new binding sitesfor p150 which SxIP motifs do not bind to. p150 in turn recruits the dyneincomplex essential for correct initiation of cargo transport from microtubuleplus ends.

(Supplementary Fig. 4). In this hierarchical mode of microtubule endtracking of p150, EB1 recruits CLIP-170 and in turn CLIP-170 recruitsp150 (Fig. 3d). This explains why in living cells, that is, in the presenceof multiple competitors, end tracking of p150 requires both EB1 andCLIP-170 (refs 23,24).

One key function of microtubule end tracking of the dynactincomponent p150 is to recruit the dynein complex to growingmicrotubule ends, which adds the next layer to the recruitmenthierarchy. The p150 construct used here contains the CAP-Glydomain required for microtubule end tracking35 and the first coiled-coil domain of p150 that interacts with the intermediate chainof the dynein complex17,18 (Table 1). To investigate whether thesetwo p150 domains are sufficient to mediate dynein recruitment,we combined purified EB1, mCherry–p150 and the reconstituted,mGFP-tagged human dynein complex consisting of 6 subunits36

(Fig. 4a). Triple-colour TIRF microscopy showed that a combinationof EB1 and mCherry–p150 was indeed sufficient to recruit thedynein complex to growing microtubule ends (Fig. 4b left andSupplementary Video 3). In contrast, EB1 alone did not recruitthe dynein complex to microtubule ends (Fig. 4b middle andSupplementary Video 3). To investigate the role of the dynein tailthat contains the intermediate chain, we used a tail-less mGFP-tagged dimer of the dynein motor domain36 (Fig. 4a). This constructwas not recruited to microtubule ends by EB1 and mCherry–p150(Fig. 4b right and Supplementary Video 3), in agreement with thenotion that the intermediate chain of dynein mediates binding top150 (ref. 18). These experiments define a minimal system for humandynein end tracking in the absence of competitors, consisting ofEB1, the dynactin component p150 and the dynein complex. Inthe presence of competitors, an alternative pathway using CLIP-



LETTERS

170 can target dynein to growing microtubule ends. Together, ourresults define the molecular mechanism underlying the hierarchicaldynactin-dependent mode of human dynein recruitment to growingmicrotubule ends (Fig. 4c).

We found that all tested EB1-dependent +TIPs compete with eachother. From previous structural studies with isolated domains, it wasexpected that the CAP-Gly domains of p150 and CLIP-170 com-pete for EB1 binding, because both bind to the EEY/F motif of EB1(refs 22,26,27,29). Similarly, the p150 CAP-Gly domain was expectedto compete with SxIP motif peptides because both form contacts withthe EBH domain8,22. Notably, in our experiments the CAP-Gly do-mains of CLIP-170 and SxIP peptides did also compete for EB1 bind-ing (Fig. 2b,c and Supplementary Fig. 2). Structural considerationssuggest that the CAP-Gly domains of CLIP-170 are not expected tosignificantly engage in contacts with the EBH domain but to bind theC-terminal EEY/F motif of EB1 (refs 2,22,26,27); CLIP-170 CAP-Glydomains and SxIPmotifs were thus not anticipated to directly competefor EB1 binding. However, the disordered C-terminal tail of EB1 getsstructured on binding of SxIP peptides8. This structuring could hinderaccess of the EEY/F motif for CLIP-170 CAP-Gly domain binding,thus explaining the observed competition between both molecules.As all EB1-dependent +TIPs tested here compete with each other forEB1 binding, this interaction is a bottleneck for building up +TIPinteraction networks. This provides amechanistic explanation for whyin living cells plus-end tracking of many proteins can be competitivelyinhibited by overexpression of SxIP peptides37.

Differences in the concentrations of competitors for EB1 binding,or the regulation of their ability to interact with EB1, for example byphosphorylation32,38, are expected to control whether p150 can be end-recruited directly byEB1 alone orwhether a hierarchical bindingmodeinvolving also CLIP-170 has to be used by the cell. In this hierarchicalrecruitment mode, CLIP-170 withstands competition by other +TIPsfor EB1 binding better than p150, offering an additional binding sitefor p150 for which no competition with SxIP motif peptides exists(Fig. 3). CLIP-170 can thus form a ‘bridge’ between EB1 and p150.p150 binds either composites of the EBH domain and the EEY/Fmotifof EB1 (ref. 22) or composites of the distal zinc knuckle domain ofCLIP-170 and its EEY/Fmotif27,28. Either of the two interactionmodescan mediate efficient end tracking of p150, in agreement with struc-tural information22,27,28. These results explain how p150 gets recruitedto growingmicrotubule ends in the presence of a large number of SxIP-motif-containing proteins, as it is the case in higher eukaryotes7,9.

CLIP-170 can be in an autoinhibited state when its N-terminalCAP-Gly domains engage in intramolecular interactions with its zincknuckle domains at its C terminus23,27. Therefore, the cell has severaloptions to regulate dynein/dynactin loading onto microtubule plusends by either controlling the level of direct competition by other+TIPs for EB1 binding (at the top of the hierarchy of interactions) orby controlling the degree of CLIP-170 autoinhibition, which can beregulated by several events: by EB1 binding27, by phosphorylation ofCLIP-170 (refs 39,40), and/or by binding of small ligands41.

The dynactin-dependent pathway of dynein recruitment dependsultimately on EB family members recognizing a transiently existingstructural feature at growing microtubule ends5. This pathwayseems to be prominent in all mammalian cells studied so far4,11.In addition to dynactin-dependent dynein recruitment, as studied

here, alternative and dynactin-independent pathways exist thatcontribute to dynein enrichment at microtubule plus ends viatransport by plus-end-directed kinesinmotors13,15,16. These alternativepathways often require the conserved dynein regulator LIS1 (refs 13,16). Dynein transport along microtubules has been demonstrated inneurons and several fungal systems15,16. Depending on the cell type,kinesin-dependent transport and EB1-dependent plus-end-trackingmechanisms of dynein can be intertwined or work in parallel. Therecombinant dynein complex used here is naturally non-processive36,which indicates that processivity is not needed for its accumulationat growing microtubule ends. In the future, it will be interesting toalso reconstitute kinesin- and LIS1-dependent pathways of dyneinend localization. Such studies should clarify how the accumulationof dynein at microtubule plus ends and its own minus-end-directedmotility for cargo transport away from microtubule plus ends areregulated and coordinated.

In conclusion, this study showed how general principles ofdomain–domain and domain–motif interactions organize a defined,dynamic protein network. These principles include competition for alimited amount of binding sites, system plasticity due to alternativerecruitment routes, and an overall hierarchical organization, whichallows for multiple layers of regulation. These features define thefunctional order in the microtubule–EB1–CLIP-170–p150–dyneinintermediate chain interaction network that ultimately controlsthe targeting of the multisubunit dynein motor complex. Ourreconstitution system provides the basis for the future investigationof additional layers of +TIP network regulation such as, for example,phosphorylation reactions. It will be useful to study different steadystates of +TIP recruitment pathways that depend on local, cell-cycle-dependent and/or species-specific regulation modes within +TIPnetworks that are required for specific cellular functions. �

METHODSMethods and any associated references are available in the onlineversion of the paper.

Note: Supplementary Information is available in the online version of the paper

ACKNOWLEDGEMENTSWe thank R. M. Buey for cloning of the p150–GCN4 construct, I. Lüke for help withinsect cell culture and protein expressions, the Peptide Chemistry facility LRI forpeptide synthesis, J. Roostalu for Atto488-labelled tubulin, N. Cade for microscopysupport and critical reading of the manuscript, and H. Walden for useful advice onprotein purification. C.D. andT.S. acknowledge financial support from the EuropeanResearch Council (ERC project ID 323042) and the German Research Foundation(DFG SU 175/7-1). M.O.S. is supported by a grant from the Swiss National ScienceFoundation (310030B_138659).

AUTHOR CONTRIBUTIONSC.D. performed experiments, C.D., M.T., R.J. and I.S. prepared reagents, and C.D.,M.O.S. and T.S. analysed data, designed research and wrote the manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at www.nature.com/doifinder/10.1038/ncb2999Reprints and permissions information is available online at www.nature.com/reprints

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2. Akhmanova, A. & Steinmetz, M. O. Tracking the ends: a dynamic protein networkcontrols the fate of microtubule tips. Nat. Rev. Mol. Cell Biol. 9, 309–322 (2008).



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8. Honnappa, S. et al. An EB1-binding motif acts as a microtubule tip localizationsignal. Cell 138, 366–376 (2009).

9. Jiang, K. et al. A proteome-wide screen for mammalian SxIP motif-containingmicrotubule plus-end tracking proteins. Curr. Biol. 22, 1800–1807 (2012).

10. Kobayashi, T. & Murayama, T. Cell cycle-dependent microtubule-baseddynamic transport of cytoplasmic dynein in mammalian cells. PLoS ONE 4,e7827 (2009).

11. Splinter, D. et al. BICD2, dynactin, and LIS1 cooperate in regulating dyneinrecruitment to cellular structures. Mol. Biol. Cell 23, 4226–4241 (2012).

12. Vaughan, K. T., Tynan, S. H., Faulkner, N. E., Echeverri, C. J. & Vallee, R. B.Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubuledistal ends. J. Cell Sci. 112, 1437–1447 (1999).

13. Moore, J. K., Stuchell-Brereton, M. D. & Cooper, J. A. Function of dynein in buddingyeast: mitotic spindle positioning in a polarized cell. Cell Motil. Cytoskeleton 66,546–555 (2009).

14. Moughamian, A. J. & Holzbaur, E. L. Dynactin is required for transport initiation fromthe distal axon. Neuron 74, 331–343 (2012).

15. Zhang, J. et al. The microtubule plus-end localization of Aspergillus dynein isimportant for dynein-early-endosome interaction but not for dynein ATPase activation.J. Cell Sci. 123, 3596–3604 (2010).

16. Yamada, M. et al. LIS1 and NDEL1 coordinate the plus-end-directed transport ofcytoplasmic dynein. EMBO J. 27, 2471–2483 (2008).

17. Vaughan, K. T. & Vallee, R. B. Cytoplasmic dynein binds dynactin through a directinteraction between the intermediate chains and p150Glued. J. Cell Biol. 131,1507–1516 (1995).

18. Schroer, T. A. Dynactin. Annu. Rev. Cell Dev. Biol. 20, 759–779 (2004).19. King, S. J. & Schroer, T. A. Dynactin increases the processivity of the cytoplasmic

dynein motor. Nat. Cell Biol. 2, 20–24 (2000).20. Ahmed, S., Sun, S., Siglin, A. E., Polenova, T. & Williams, J. C. Disease-associated

mutations in the p150(Glued) subunit destabilize the CAP-gly domain. Biochemistry49, 5083–5085 (2010).

21. Farrer, M. J. et al. DCTN1 mutations in Perry syndrome. Nat. Genet. 41,163–165 (2009).

22. Honnappa, S. et al. Key interaction modes of dynamic +TIP networks. Mol. Cell 23,663–671 (2006).

23. Lansbergen, G. Conformational changes in CLIP-170 regulate its binding tomicrotubules and dynactin localization. J. Cell Biol. 166, 1003–1014 (2004).

24. Watson, P. Microtubule plus-end loading of p150Glued is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells. J. CellSci. 119, 2758–2767 (2006).

25. Bieling, P. et al. CLIP-170 tracks growing microtubule ends by dynamicallyrecognizing composite EB1/tubulin-binding sites. J. Cell Biol. 183,1223–1233 (2008).

26. Mishima, M. et al. Structural basis for tubulin recognition by cytoplasmiclinker protein 170 and its autoinhibition. Proc. Natl Acad. Sci. USA 104,10346–10351 (2007).

27. Weisbrich, A. et al. Structure–function relationship of CAP-Gly domains. Nat. Struct.Mol. Biol. 14, 959–967 (2007).

28. Hayashi, I., Plevin, M. J. & Ikura, M. CLIP170 autoinhibition mimicsintermolecular interactions with p150Glued or EB1. Nat. Struct. Mol. Biol. 14,980–981 (2007).

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30. Zhapparova, O. N. et al. Dynactin subunit p150Glued isoforms notable fordifferential interaction with microtubules. Traffic 10, 1635–1646 (2009).

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39. Li, H. et al. Phosphorylation of CLIP-170 by Plk1 and CK2 promotes timely formationof kinetochore-microtubule attachments. EMBO J. 29, 2953–2965 (2010).

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DOI: 10.1038/ncb2999 METHODS

METHODSCloning and protein biochemistry. Untagged full-length human EB1 wasexpressed from a published expression plasmid43 in BL21 RIL cells at 18 ◦C.Collected cells were lysed in 20mM Bis/Tris buffer at pH 6.0, supplemented withDNAse I (Roche) and Complete protease inhibitors (Roche) using a microfluidizer.Clarified lysates were loaded onto a Mono Q ion exchange column (GE Healthcare)equilibrated with 20mM Bis/Tris buffer at pH 6.0 and a linear NaCl gradient from0 to 0.5 M was applied over 160ml and fractions were collected, using an AEKTApurifier (GE Healthcare). EB1-containing fractions were pooled and gel filteredusing a Superdex 200 column (GE Healthcare) in 20mM Tris/HCl at pH 7.4 and350mM NaCl and 1mM 2-mercaptoethanol. EB1-containing peak fractions werepooled (Supplementary Fig. 5) and snap frozen in liquid N2. mCherry–EB3 waspurified as described previously44 (Supplementary Fig. 5).

Humanp150DNAwas PCR amplified from a cDNAclone containing the humanDCN1 gene (GenBank: BX640799.1, which represents the neuronal splice variant ofp150 including the sequence of exons 5–7) and was cloned into pETM11 vector. Theconstruct codes for an N-terminal hexa-histidine tag, followed by a TEV cleavagesite, then mCherry or SNAP and finally the first 547 amino acids of the neuronalp150 isoform. mCherry and p150 were separated by a short GGGGTS linker.

A shorter artificially dimerized p150 construct (GFP–p150218GNC4N) wascloned into the pET28a vector (Novagen). An N-terminal 6× histidine tag wasfollowed by GFP, the N-terminal 218 amino acids of p150 and the GCN4 sequencefor dimerization4.

Site-directed mutagenesis was applied to delete exons 5–7 to generate thecorresponding construct of the tissue form of p150 (refs 30,31). All p150 constructswere expressed in BL21 RIL cells at 18 ◦C. For purification, cells were lysed in30mMHEPES at pH 7.4, 350mMKCl, 5mMMgCl2 and 1mM 2-mercaptoethanol,DNAse I and complete protease inhibitors, using a microfluidizer. Protein inclarified lysates was bound to Proteino Ni-TED beads (Macherey-Nagel 745200.5)in batch format. Then the beads were transferred to a column and washed with∼100ml wash buffer (30mM HEPES at pH 7.4, 350mM KCl, 1mM MgCl2 and1mM 2-mercaptoethanol). Proteins were eluted using wash buffer supplementedwith 400mM imidazole. Peak fractions were concentrated (VIVASPIN4; SartoriusStedim) and gel filtered using a Superose 6 column (GE Healthcare) in washbuffer. SNAP-tagged protein was labelled using SNAP-Surface Alexa Fluor 647(New England Biolabs) according to the manufactures recommendation. Purefractions were pooled (Supplementary Fig. 5), aliquoted and snap frozen inliquid N2.

Human full-length CLIP-170 and GFP–CLIP-170 were expressed from existingexpression constructs25 using baculovirus-infected SF21 cells. The growth mediumwas supplemented with 0.1mM ZnCl2 (Sigma, 429430). Cells were lysed in 30mMHEPES, pH 7.4, 400mM KCl, 20mM L-arginine, 20mM K-glutamate, 0.01% Brij35 (Thermo), 10mM 2-mercaptoethanol, 3mM MgCl2, DNAse I and Completeprotease inhibitors, using a glass homogenizer (1234F36, Fischer). Protein inclarified lysates was bound to Proteino Ni-TED beads in a batch format, beadswere transferred to a column and washed with ∼100ml CLIP-170 wash buffer(30mMHEPES, pH 7.4, 400mM KCl, 20mM arginine, 20mM K-glutamate, 0.01%Brij 35, 10mM 2-mercaptoethanol, 3 mM MgCl2). Then 200 µl (1mgml−1) ofpurified GST-tagged TEV protease was added for 1 h at room temperature. TEVcleavage released the CLIP-170 constructs from the beads and fractions could beeluted using CLIP-170 wash buffer. CLIP-170-containing fractions were pooledand concentrated (VIVASPIN 4) and gel filtered using a Superose 6 column (GEHealthcare) in CLIP-170 wash buffer. Pure fractions were pooled (SupplementaryFig. 5) and directly used in TIRF microscopy assays to avoid damage due tofreeze–thaw cycles.

Corresponding constructs lacking the 946 final C-terminal amino acids of CLIP-170 (CLIP-170-1C and GFP–CLIP-170-1C), previously also called H2 (ref. 45)were expressed and purified as described25 (Supplementary Fig. 5). GFP–CLIP-170-1CSxNN was generated by site-directed mutagenesis using the original GFP–CLIP-170-1C as a template at the position as specified previously32. Purification wasperformed as described for full-length CLIP-170.

A fusion protein between EB1 without its last 4 amino acids and the last40 C-terminal amino acids of CLIP-170, here called EB11EEY–CLIP-C40, wasconstructed in a pETG-20A expression vector. This construct codes for an N-terminal thioredoxin-solubility tag, followed by a hexa-histidine purification tag27,then human EB1 (from the cDNA clone as used in ref. 43) without its last 4 aminoacids, directly followed by the last 40 amino acids of human CLIP-170 (from thecDNA clone used in ref. 27). Protein expressionwas performed in BL21 RIL cells andgrowth medium was supplemented with 0.1mM ZnCl2. Cells were lysed in 30mMHEPES, pH 7.4, 350mM KCl, 5mM MgCl2, 5mM 2-mercaptoethanol, DNAse Iand Complete protease inhibitors, using a microfluidizer. Clarified lysates werebound to Proteino Ni-TED beads in a batch format, the beads were transferredto a column and washed with ∼100ml wash buffer. Protein was eluted with washbuffer supplemented with 400mM imidazole. Directly after elution, imidazole was

removed using PD10 columns (GEHealthcare). Finally, gel filtration was performedin wash buffer using a Superdex 200 column (GE Healthcare). After gel filtrationpeak fractions were pooled (Supplementary Fig. 5) and directly used for TIRFmicroscopy experiments without freezing.

A construct consisting of the 40 C-terminal amino acids of CLIP-170, calledCLIP-C40, was expressed from an existing expression plasmid27 as describedpreviously, except that the N-terminal solubility tag was not cleaved to enhancesolubility. Pure fractions were pooled (Supplementary Fig. 5) and directly used inTIRF microscopy assays without freezing.

The recombinant human dynein complex and the artificial dynein dimer wereprepared as previously described36.

Porcine brain tubulin was purified using standard procedures. To generatebiotinylated, Atto488-labelled and Cy5-labelled tubulin, NHS-biotin (Pierce),NHS-Atto488 (Sigma) or NHS-Cy5 (Lumiprobe), respectively, was covalentlybound to purified tubulin using standard procedures. For all experiments thatinvolved zinc-binding domains of CLIP-170, EGTA was removed from tubulinthrough an additional polymerization/depolymerization cycle in the absenceof EGTA.

During all purifications the temperature was always kept at ∼4 ◦C. Proteinconcentrations were measured using their absorbance at 280 nm. The extinctioncoefficient was calculated on the basis of their primary amino-acid composition.

Peptide synthesis. A human MACF2-derived peptide (HRPTPRAGSRPSTAKPSKIP TPQRKSPASK LDKSSKRW) without or with an N-terminalfluorescein label (SxIP and Fluo–SxIP, respectively) was synthesized using standardprocedures32. As a negative control, the same peptides with a SKIP → SXNNreplacement were synthesized (SxNN, Fluo-SxNN). Peptides were dissolved in10mM Tris/HCl pH 7.0 and 50mM NaCl aliquoted and snap frozen in liquid N2and stored at−80 ◦C.

+TIP reconstitution assay. In vitro +TIP reconstitution assays were performed aspreviously described6.

Briefly, GMPCPP-stabilized biotinylated and fluorescently labelled seeds wereattached to PEG–biotin-functionalized glass coverslips, assembled into flowchambers using double-sided sticky tape. Free tubulin (fluorophore/tubulin dimerlabelling ratio was always 0.8) assembled from these seeds in the presence of GTPinto microtubules exhibiting dynamic instability behaviour. Fluorescently labelled+TIPs andmicrotubules were visualized bymulti-colour TIRFmicroscopy. StandardTIRFM buffer was 80mM K-PIPES at pH 6.85, 60mM KCl, 1mM EGTA, 4mMMgCl2, 5mM 2-mercaptoethanol, 50 µgml−1 β-casein (Sigma), 2mMGTP (Sigma),0.1% methylcellulose (Sigma), 20mM glucose, 1.3mgml−1 glucose oxidase (Serva)and 0.66mgml−1 catalase (Sigma), unless stated otherwise in the figure legend.For experiments that involved zinc-binding domains of CLIP-170, EGTA wasusually omitted (as stated in the legends), because zinc chelation can damage theintegrity of the zinc knuckle domain. Experiments with the neuronal isoform ofGFP–p150218GCN4 were performed in Standard TIRFM buffer supplemented withadditional 25mMKCl and 85mMK-acetate (as stated in the legends) to compensatefor stronger microtubule binding of this construct. For all experiments with dynein,the concentrations of K-PIPES and KCl were lowered to 60mM and 45mM,respectively, and ATP was present at 1mM. All other components of Standardimaging buffer were unchanged (= Dynein imaging buffer). All experimentswere performed at 30 ◦C and three independent experiments were performed foreach condition.

TIRF microscopy. Images were acquired on a total internal reflection fluorescence(TIRF) microscope (iMIC, FEI Munich) equipped with 3 cooled electron-multiplying CCD (charge-coupled device) cameras (Evolve, Photometrics) usinga 1.49 N.A. ×100 oil immersion objective (Olympus) with additional ×1.33optical magnification, giving an image pixel size of 120 nm. The microscope wasequipped with 488 nm, 561 nm and 640 nm diode lasers (Toptica). A quadbanddichroic mirror (405/488/561/64, Semrock), and long-pass dichroic mirrors (T565LPXR, T660 LPXR, Chroma) were used to split the different wavelength emissionchannels to separate cameras, with additional GFP (525/50, Semrock), RFP (600/37,Semrock), and Cy5 (700/75, Semrock) emission filters. Images were acquired intwo or three cameras consecutively (non-simultaneously) at a rate of 1 frameper second for each fluorescence channel. Exposure times were 100 ms and laserintensities were kept constant, whenever two or more conditions were directlycompared. TIRF excitation was performed in annual excitation mode to averageout illumination inhomogeneities. Images of a calibration grid (Compugraphics)were used to align images recorded in different fluorescence channels, aspreviously described42.

TIRF microscopy image analysis. Image analysis was performed using ImageJand MATLAB. ImageJ was used to create merged images taken in different

NATURE CELL BIOLOGY


METHODS DOI: 10.1038/ncb2999

fluorescence channels (single frames or kymographs) to visualize the localizationsof proteins or peptides on microtubules. For a more clear presentation of acquiredimages, ImageJ was used for background subtraction (30 pixel radius rolling ballsubtraction and 2 frame running average using the ‘running z projector’ plug in).The calculation of averaged fluorescence intensity profiles of mCherry–p150 in themicrotubule end region (Fig. 3b) was performed as previously described42. In brief,growing Cy5–microtubule end regions were detected by automated tracking and thecorresponding fluorescence intensity profiles of mCherry–p150 were extracted andaveraged, resulting in mCherry–p150 profiles with enhanced signal-to-noise ratio.Error bars are standard errors of the mean (s.e.m.) and are derived from at least 250individual frames per condition from 3 independent experiments. For the bar graphin Fig. 3c, intensities at microtubule end regions (0 nm–150 nm) were averaged.Averaged intensities from a region without microtubule (−700 nm to −1,000 nmaway from the microtubule) were averaged and subtracted from the previous valueto correct for background. Similarly, in Supplementary Fig. 2c the peak intensitiesfor GFP–CLIP-170 constructs (150 nm away from the plus end) and themicrotubulelattice signal (2 µmaway from the plus end) were averaged and plotted as a bar graphas indicated.

Analytical gel filtration. Analytical gel filtrations were performed at roomtemperature and all proteins were dialysed into gel filtration buffer (10mM NaPiat pH 7.4, 150mM NaCl, 5mM 2-mercaptoethanol) and were loaded eitherindividually or asmixtures as indicated onto a Sephadex 75 gel filtration column (GEHealthcare) at a flow rate of 0.5mlmin−1. Elution profiles were monitored using thechange in refractive index (Wyatt). All experiments were performed in duplicates.

SDS–PAGE. SDS–PAGE was performed under denaturating conditions usingstandard procedures. As a size marker, ‘Precision Plus Protein Standards’ (BioRad,161-0377) were used. Gels were stained using Coomassie brilliant blue.

43. Honnappa, S., John, C. M., Kostrewa, D., Winkler, F. K. & Steinmetz, M. O. Structuralinsights into the EB1-APC interaction. EMBO J. 24, 261–269 (2005).

44. Montenegro Gouveia, S. et al. In vitro reconstitution of the functionalinterplay between MCAK and EB3 at microtubule plus ends. Curr. Biol. 20,1717–1722 (2010).

45. Scheel, J. et al. Purification and analysis of authentic CLIP-170 and recombinantfragments. J. Biol. Chem. 274, 25883–25891 (1999).

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Supplementary Figure 1 EB1 targets the neuronal splice variant of p150 to growing microtubule ends in vitro. (a) Top: Schematics illustrating the difference between the tissue and neuronal p150 isoforms: insertion of a positively charged stretch of 20 amino acids, encoded by exons 5-7. Note: In the main figures, the addition ‘-T’ is omitted, because only experiments with the tissue form of p150 (except in Fig. 2d) are shown there. Bottom: Schematics illustrating the composition of the p150 constructs used for the experiments shown in this figure: mCherry labelled neuronal and tissue p150 isoforms containing the first 547 and 527 amino acids of p150 (mCherry-p150547-N and mCherry-p150527-T, ending at corresponding amino acid positions), respectively; GFP-labelled neuronal p150 isoform containing the first 218 amino acids (with the CAP-Gly domain and the basic domain) followed by the artificial dimerization domain GCN4 (p150218GCN4-N). This latter construct was made for comparison with previously published work 4. (b) Illustration of the conversion of a time series (top) to a kymograph (“space-time plot”, bottom). Example images and corresponding kymographs of a time series (corresponding to Fig. 1b) are shown for the individual fluorescent channels (middle and right) as well as their merge (left, with mCherry-p150 in green and Cy5-microtubules in red). (c) Left column: Dual (top) and single colour overview images (middle) and representative kymographs (bottom) of 125 nM mCherry-p150547-N (green) tracking growing Cy5-microtubule (red) ends in the presence of 150 nM unlabelled EB1. Remaining panels: Comparison of the microtubule binding strength of the two p150 isoforms in the absence of EB1: dual (top) and single (middle)

colour overview images and a single colour time-averaged images (from 60 consecutive frames; bottom) showing 125 nM mCherry-p150547-N (green in merge, left pair of columns) or 125 nM mCherry-p150527-T (green in merge, right pair of columns) binding along Cy5-microtubules (red in merge) in Standard TIRFM buffer or standard TIRFM buffer without KCl, as indicated. (d) An overview dual colour TIRF microscopy image (left) and single and dual colour kymographs (right) of 10 nM GFP-p150218GCN4-N (green in merge) tracking growing ends of Cy5-microtubules (red in merge) in the presence of 150 nM unlabelled EB1. Standard TIRFM buffer was supplemented with additional 25 mM KCl and 85 mM K-acetate, which was necessary to keep this construct soluble and to reduce its overall microtubule binding strength. (e) Three colour experiment showing that mCherry-EB3 recruits Alexa647-p150547-N to growing microtubule ends. Left: overview images of 150 nM mCherry-EB3 (top, blue) and 75 nM Alexa647-p150547-N (bottom, green) on Cy5-microtubules (red) in Standard TIRFM buffer (the same image frames are shown). Right: Single colour and merged triple colour kymographs of a representative microtubule in the same experiment (same colour code in merge). Cy5-tubulin concentration was always 20 µM. Horizontal and vertical scale bars are 3 µm and 20 s, respectively. Taken together, these results show that despite the stronger binding of the neuronal p150 isoform to microtubules, both the neuronal and the tissue isoform can track growing microtubule ends in an EB dependent manner (irrespective of the construct of the neuronal isoform used and irrespective of whether EB1 or EB3 is used).


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SxIP

Supplementary Figure 2 CLIP-170 without a putative SxIP motif competes with SxIP peptides for EB1 binding at growing microtubule ends. (a) Schematic of a mutated version of GFP-CLIP-170ΔC with a putative SxIP motif between the CAP-Gly domains changed to SxNN (GFP-CLIP-170ΔCSxNN). (b) Representative dual-colour images (top) and kymographs (bottom) of 75 nM GFP- CLIP-170ΔC (green, left) or 75 nM GFP-CLIP-170ΔCSxNN (green, right) in the presence of 150 nM unlabelled EB1 and either no additional SxIP peptide or with 15 µM unlabelled SxIP peptide, as indicated. Cy5-microtubules (red) elongated from immobilised seeds in the presence of 20 µM Cy5-tubulin in Standard TIRFM buffer. Horizontal and vertical scale bars are 3 µm and 20 s, respectively. (c) Quantification of fluorescence intensities of the GFP-CLIP-170ΔC and GFP-CLIP-170ΔCSxNN at microtubule end regions and on the microtubule lattice in the presence of unlabelled EB1 and in the presence or absence of competing

unlabelled SxIP peptides. Error bars are standard errors of the mean (s.e.m) and are derived from at least 250 individual frames per condition from 3 independent experiments. These experiments show that GFP-CLIP-170ΔC and GFP-CLIP-170ΔCSxNN are both recruited to growing microtubule ends by EB1 without a significant difference. GFP-CLIP-170ΔCSxNN is also displaced from EB1 at growing microtubule ends by an excess of SxIP peptides, similar to wild type GFP-CLIP-170ΔC. This demonstrates that the putative SxIP motif of CLIP-170 does not contribute significantly to binding of CLIP-170 to EB1, even in the context of the native flanking CLIP-170 domains. This confirms previous results with a short CLIP-170 sequence peptide 32 and demonstrates that the CAP-Gly domains, and not the putative SxIP sequence in CLIP-170 are responsible for the competition between CLIP-170 and SxIP peptides for EB binding.




a bmCherry-EB3

CLIPc40plus EGTAminus EGTA

mCherry

Merge

GFP

GFP-p150198GCN4-T

no CLIP-170ΔC + CLIP-170ΔCEB1 + GFP-p150218GCN4-N

c

Kymograph

competitive binding

p150

EB1

CLIP-170ΔC


EEY/FS

EEY/F

GFP-p150198GCN4-T

mCherry-EB3

CLIPc40

CAP-Gly Serin rich

Coiled coil

Fluorescent proteinFP

S Solubility tag

Zinc knuckle

EBH

CH

EEY/F

FP

FPGCN4

Supplementary Figure 3 An intact distal zinc knuckle of CLIP-170 is required for efficient interaction with the CAP-Gly domain of p150. (a) Schematics of mCherry-EB3, of the GFP-labelled tissue p150 isoform containing the first 198 amino acids of p150 followed by the artificial dimerisation domain GCN4 (GFP-p150198GCN4-T), and of a construct comprising the final 40 amino acids of CLIP-170 containing the distal zinc knuckle domain and the C-terminal EEY/F motif (CLIP-C40). A thioredoxin tag on CLIP-C40 was added to improve solubility (solubility tag). (b) Single and triple colour kymographs showing the localisation of 75 nM mCherry-EB3 (red in merge) and 20 nM GFP-p150198GCN4-T (green in merge) on a Cy5-microtubule (blue in merge) in the presence of 660 nM unlabelled CLIP-C40 (C-terminal 40 amino acids of CLIP-170), used here as a sequestering agent, in the absence (left) and presence (right) of 5 mM EGTA. EGTA chelates zinc ions33 and is therefore expected to unfold the zinc knuckle domain of CLIP-170. Standard TIRFM buffer without or with 5 mM EGTA was used as indicated. We observe that the localisation of mCherry-EB3 (used here instead of EB1, because N-terminal fusions with fluorescent proteins tend to impair EB1, but not EB3 activity) to growing microtubule ends is unaffected by EGTA, but that GFP-p150198GCN4-T

localises to microtubule ends only in the presence of EGTA. This demonstrates that CLIP-C40 efficiently sequesters GFP-p150218GCN4-T in solution only in the absence of EGTA when the distal zinc knuckle domain of CLIP-170 is properly folded. This supports the conclusion from Figs. 1-3 that the CAP-Gly domain of p150 can either interact at microtubule ends with the C-terminal part of EBs or with the C-terminal part of EB-recruited CLIP-170. (c) Left: Representative overview images (top) and kymographs (bottom) showing 150 nM unlabelled EB1 targeting 10 nM p150218GCN4-N (green) to the ends of growing Cy5-microtubules (red) only in the absence (left), but not in the presence (right) of unlabelled 660 nM CLIP-170ΔC. Standard TIRFM buffer was supplemented with additional 25 mM KCl and 85 mM K-acetate, which was necessary to keep this construct soluble and to reduce its overall microtubule binding strength. This experiment provides evidence that the N-terminal part of CLIP-170 competes with p150 for EB1 binding at growing microtubule ends. Cy5-tubulin concentration was always 20 µM. Horizontal and vertical scale bars are 3 µm and 20 s, respectively. Right: The scheme illustrates the competition between the N-terminal part of CLIP-170 and p150 for EB1 binding at growing microtubule ends.




a bEB1 EB1ΔEEY-CLIPc40

GFP-p150198GCN4-T

EB1ΔEEY-CLIPc40

GFP-p150198GCN4-T+ SxIP

EB1ΔEEY-CLIPc40

Fluo-SxIP488

Tubulin

Merge

EB1ΔEEY-CLIPc40

c

EB1

SxIP


p150

c40 no competitive binding

S EEY/F

from EB1 from CLIP-170

Coiled coil

S Solubility tagZinc knuckle

EBH

CH

EEY/F

Supplementary Figure 4 CLIP-170-mediated recruitment of p150 does not require a direct interaction between EB1 and p150. (a) Scheme of a fusion protein consisting of EB1 lacking its 4 most C-terminal amino acids followed by the 40 most C-terminal amino acids of CLIP-170 (EB1ΔEEY-CLIP-C40). This fusion protein contains the EBH domain that interacts with both SxIP peptides and with the CAP-Gly domain of p150, but lacks the terminal EEY sequence of EB1 known to also interact with the p150 CAP-Gly domain (see Table 1); instead of the terminal EEY sequence of EB1 the construct contains the distal zinc knuckle and the EEY/F tail motif of CLIP-170 that interact with the CAP-Gly domain of p150, but not with SxIP motif peptides. A thioredoxin tag was added to improve solubility. (b) Single and dual colour kymographs showing that 150 nM unlabelled EB1 (left column of panels) as well as 150 nM unlabelled EB1ΔEEY-CLIP-C40 (second column) recruits 20 nM GFP-p150198GCN4-T (top kymograph, green in bottom merge) to the ends of growing Cy5-microtubules (middle kymograph, and red in bottom merge). This interaction cannot be strongly suppressed by the additional presence of a large excess of 60 µM unlabelled SxIP peptide (far right),

although SxIP peptides are recruited by the EB1ΔEEY-CLIP-C40 fusion protein (third column of panels) as demonstrated by end tracking of 150 nM Fluo-SxIP (top kymograph, green in bottom merge). This means that the interaction between the zinc knuckle domain of the EB1ΔEEY-CLIP-C40 fusion protein and the p150 CAP-Gly domain is sufficient to rescue recruitment of p150 in the presence of competing SxIP motif peptides (see Fig. 3) that occupy the EBH domain of EB1. The EEY/F motif of EB1 that is absent in the EB1ΔEEY-CLIP-C40 fusion is not required for this rescue. Representative two or single-colour kymographs for each condition are depicted. ‘488’ indicates excitation at 488 nm which excites either GFP fused to p150 constructs or fluorescein covalently linked to SxIP peptide. Cy5-tubulin concentration was 20 µM. Experiments were performed in TIRFM buffer without EGTA (Methods). Horizontal and vertical scale bars are 3 µm and 20 s, respectively. (c) Scheme illustrating non-competitive binding of SxIP peptides and p150 to the EB1ΔEEY-CLIP-C40 fusion protein: SxIP peptides bind to the EBH domain and the CAP-Gly domain of p150 binds to the sequence corresponding to the C-terminal part of CLIP-170.




1 2 3 4 5 6 7 8

9 10 11

Purity of recombinant proteins

2501501007550

37

2520

250150100755037

2520

12 13

1 EB12 mCherry-EB33 GFP-p150218GCN4-N4 GFP-p150198GCN4-T5 mCherry p150527-T6 mCherry-p150547-N7 mGFP-Dynein (artificial dimer)8 mGFP-Dynein (complex)9 EB1ΔEEYCLIPc40

10 GFP-CLIP-17011 CLIP-17012 CLIPc40

13 CLIP-170ΔC14 GFP-CLIP-170ΔC15 GFP-CLIP-170ΔCSxNN

16 SNAP-Alexa647-p150547-N14

a

c

b

a: Heavy chainb: Intermediate chain 1c: Light intermediate chain 2

15 16

Supplementary Figure 5 Purity of recombinant proteins. Coomassie-stained SDS-PAGE gels of the purified proteins, as indicated. The double band seen for mCherry constructs is likely due to different maturation forms of mCherry

as reported previously. Mass spectrometry analysis supported this by showing that both bands correspond to the expected construct and not to a potential contaminant or truncated protein (data not shown).




Supplementary Videos

Supplementary Video 1 EB1 targets p150 to microtubule ends. Time-lapse TIRF microscopy movie depicting the localisation of 125 nM mCherry-p150 (green) on dynamic Cy5-labelled microtubules (red) in the presence of 150 nM unlabelled EB1. This video corresponds to Fig. 1b. The time stamp (upper left corner) is in seconds. Scale bar: 5 µm. Supplementary Video 2 CLIP-170 restores EB1-dependent end tracking of p150 in the presence of SxIP peptides. Time-lapse TIRF microscopy movies depicting the localisation of 75 nM mCherry-p150 (green) on dynamic Cy5-labelled microtubules (red) in the presence of 150 nM unlabelled EB1 and 6 µM SxNN control peptide (left), 6 µM SxIP peptide (middle) or 6 µM SxIP peptide with additional 75 nM GFP-CLIP-170 (right). Note: GFP-CLIP-170 is not shown here. This video corresponds to Fig. 3a. The time stamp (upper left corner) is in seconds. Scale bar: 5 µm.

Supplementary Video 3 EB1 and p150 target the dynein complex to microtubule ends. Time-lapse TIRF microscopy movies depicting the localisation of 14 nM mGFP-tagged human dynein complex (green) on dynamic Cy5-labelled microtubules (red) in the presence of 200 nM EB1 and 125 nM mCherry-p150 (A; left) or in the presence of 200 nM EB1 but without mCherry-p150 (B; middle). EB1 and mCherry-p150 fail to target a tail-less mGFP-tagged dimer of the dynein motor domain (green) to microtubule ends (C; right). Note: mCherry-p150 is not shown here. This video corresponds to Fig. 4b. The time stamp (upper left corner) is in seconds. Scale bar: 5 µm.


Documents

Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein