A transient helix in the disordered region of dynein light intermediate chain links the motor to structurally diverse adaptors for cargo transport

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.


Introduction
Microtubule-based cargo transport and force production are critical for a wide range of cellular and developmental processes. In animal cells, the 1.4-MDa complex cytoplasmic dynein 1 (dynein) is the major molecular motor with motility directed toward microtubule minus ends. Dynein-driven cargo transport is particularly crucial in highly polarized cells such as neurons. In axons, whose microtubule plus ends are uniformly oriented toward the axonal tip, dynein is responsible for the retrograde transport of diverse vesicle and organelle cargo toward the cell body. Mutations in dynein that alter axonal transport kinetics have been linked to a variety of nervous system disorders, including spinal muscular atrophy, motor neuron disease, Perry syndrome, and Charcot-Marie-Tooth 2 disease [1,2]. In addition to transporting cargo, dynein can exert pulling forces on microtubules when the motor is stably anchored at subcellular sites such as the cell cortex. A striking example of dynein-dependent force production occurs during mitosis, when cortically localized dynein pulls on astral microtubules to position and orient the bipolar spindle, which in turn defines the axis along which the cell will divide. Dynein's functional versatility implies tight regulation of localization and motor activity, the molecular basis of which has only recently begun to be understood.
Dynein is a 12-subunit complex consisting of a dimerized heavy chain (HC) with a C-terminal motor domain and two copies each of five accessory chains that bind along the HC Nterminal tail: dynein intermediate chain (IC), light intermediate chain (LIC), and three types of light chain (LC). In vivo, dynein function requires the cofactor dynactin, which is itself a 1.1-MDa complex built around a short filament of actin-related protein 1 (ARP1) [3]. In recent years, a number of coiled-coil proteins, referred to as activating adaptors [4], have been shown to recruit dynein to cargo while simultaneously promoting the association of dynein with dynactin [5,6]. Dynein, dynactin, and the N-terminal coiled-coil region of activating adaptors form a stable three-way assembly capable of highly processive motility in vitro [7,8], whereas the C-terminal region of adaptors links to cargo [4]. Cryo-electron microscopy (EM) studies with the adaptors bicaudal D homolog 2 (BICD2), BICD-related protein 1 (BICDR1), and Hook homolog 3 (HOOK3) have revealed the molecular arrangement within the dyneindynactin-adaptor assembly [3,[9][10][11]: the adaptor coiled-coil region binds along the length of

The C-terminal region of LIC1 is intrinsically disordered and contains two short segments with α-helical propensity
To gain mechanistic insight into how LIC-C interacts with dynein adaptors that are diverse in structure and function (Fig 1A and 1B, S1 Fig), we first characterized LIC1-C (residues 388-523) by NMR spectroscopy. The 15 N-1 H heteronuclear single quantum coherence (HSQC) spectrum of LIC1-C at 25˚C showed a narrow 7.8 to 8.6 parts per million (ppm) amide-proton chemical shift range, indicating a predominance of structural disorder (Fig 1C). Near-complete backbone resonance assignment was achieved (104 out of the 117 nonproline residues; Fig 1C, S2 Fig). An overlay of 15 15 N-1 H HSQC spectrum of human LIC1(388-523) with backbone NH assignments. The spectra were acquired at 25˚C and 900 MHz. Labels marked with an asterisk indicate peaks with heights below the cutoff contour level. (D) SSP scores for LIC1(388-523) calculated using 1 H N , 15 N, CO, CA, and CB chemical shifts for each assigned residue. "+1" indicates a fully formed α helix, "-1" indicates a fully formed β sheet, and "0" indicates disorder. GOR4 helix prediction scores are also shown. Values from 0 to 1,000 reflect absence of a fully formed α-helical structure. Scores obtained for residues 442-453 and 493-502 indicate transient α helix formation. (E) 15 N relaxation measurements for LIC1 (388-523). R 1 and R 2 relaxation rate constants at 700 MHz are shown for each assigned residue. (F) 15 N-1 H heteronuclear NOEs of LIC1(388-To assess residual secondary structure in LIC1-C, we computed the secondary structure propensity (SSP) score developed by Forman-Kay and colleagues [24]. The method combines different backbone chemical shifts into a single residue-specific SSP score. The results closely matched those obtained from the GOR4 secondary structure prediction program [25] (Fig 1D,  S1 Data). An SSP score of +1 and −1 indicates a fully formed α helix and β sheet, respectively. The SSP scores for LIC1-C residues with predicted α-helical propensity (residues 444-454 and 497-504) were positive values up to 0.35. However, assuming a linear relationship between the score and the population, helical structure was only transiently sampled at about 20%. These two segments of LIC1-C, which are highly conserved (S1A Fig), will be referred to as helix 1 and helix 2, respectively. The remaining segments scored mostly slightly negative SSP values and GOR4 values around zero, indicating complete structural disorder (note that β sheet and random coil have both extended secondary structure with similar scores in practice).
To characterize the dynamic properties of LIC1-C, we performed 15 N NMR relaxation measurements ( Fig 1E, S3 Fig). The relaxation rates are sensitive probes of the amplitudes and timescales of residue-specific structural fluctuations. The longitudinal relaxation rates R 1 , which report on motion faster than nanoseconds, were 1.2 s −1 on average. Although such a value is not compatible with a globular protein, it is indicative of an intrinsically disordered protein that undergoes extensive local reorientation. Residues 441-456 and 492-508 had larger R 1 values, which are suggestive of increased structural order of the residues with α-helical propensity. The residues between helices 1 and 2 had consistently lower-than-average R 1 values, indicating a highly flexible interhelical linker.
The fast dynamics inferred from R 1 measurements reduce the transverse relaxation rates R 2 typically observed for folded proteins, but in addition, potential motion slower than nanoseconds might increase them. Whereas the measured R 2 average of 4.7 s −1 is again only reconcilable with high structural disorder, the helical regions showed larger values in line with partial helix formation ( Fig 1E). Since the R 1 values suggest less flexibility in the helices on the fast timescale, it is likely that the increased R 2 values originate also exclusively from reduced fastmotion amplitudes. In support of this, relaxation dispersion experiments did not indicate any slow micro-millisecond dynamics. The segment with the smallest values was again the interhelical linker. Since slow motion is absent throughout the protein, the ratio R 2 /R 1 allowed extraction of an effective residue-specific reorientational tumbling time that is independent of the motional amplitude [26]. The average tumbling time was 4.5 ns, which is much smaller than a typical value expected for a folded protein consisting of approximately 140 residues.
The 1 H-15 N nuclear Overhauser enhancement (NOE) is another probe of the amplitudes of motions taking place on the sub-nanosecond timescale (Fig 1F). The values of nearly all residues fall below 0.5, which is again indicative of structural disorder with large amplitudes. In support of the findings described above, the largest values were typically found for the helix 1 and 2 segments, which suggests higher structural order than the other regions.
In conclusion, the relaxation data show that LIC1-C is intrinsically disordered with large amplitudes of motions faster than nanoseconds. In helix 1 and 2, the amplitudes are smaller and the local reorientation slower but still on the fast timescale. These results support the highly transient character of the helices derived from the SSP score. 523) probing amide bond vector motion. Relaxation rate constants (E) and NOEs (F) are indicative of intrinsic structural disorder, and larger  values indicate more restricted sub-nanosecond motion, which are observed for the helical regions. Underlying data for Fig 1 can be found in S1  Data. BICD2, bicaudal D homolog 2; dynein, cytoplasmic dynein 1; HC, heavy chain; HOOK3, Hook homolog 3; HSQC, heteronuclear single  quantum coherence; LIC, light intermediate chain; MIRO, mitochondrial Rho; NIN, ninein; NOE, nuclear Overhauser enhancement; ppm,  parts per million; RAB11FIP3, RAB11 family-interacting protein 3; RILP, RAB-interacting lysosomal protein; RZZ, ROD-ZW10-ZWILCH;  SPDL1, Spindly; SSP, secondary structure propensity; TRAK1, trafficking kinesin-binding  For independent confirmation of the residual helical propensity in LIC1-C, we collected circular dichroism (CD) spectra of LIC1-C (S4 Fig). Whereas fully formed α-helical proteins show prominent negative bands at 222 and 208 nm and a positive band at 193 nm, disordered proteins have very low ellipticity above 210 nm and negative bands near 195 nm [27]. The spectrum of LIC1-C corresponded largely to structural disorder. However, there were negative values at 208 nm and a negative shoulder at 222 nm. These features are indicative of residual helical structure, in agreement with our findings from NMR spectroscopy. The helical content was maintained over a large temperature range (25, 37, 50, 60, and 70˚C), similar to previous reports on other intrinsically disordered proteins [28].
The C-terminal helix 1 in LIC1 is the main binding site for SPDL1, BICD2, and HOOK3 To identify adaptor binding sites in LIC1-C, we recorded 15 N-1 H HSQC spectra of LIC1-C bound to SPDL1(2-359), BICD2(2-422), and HOOK3(2-239) using molar ratios of 1:0, 1:0.5, and 1:1 (Fig 2A, 2B, 2C and 2D). The spectra obtained from each titration series revealed similar modes of interaction for the three adaptors. Rather than chemical shifts being perturbed, many peaks were significantly attenuated because of binding. This indicates decrease in the local motion of the bound residues; increase of the hydrodynamic radius of the entire complex, resulting in increase in the effective tumbling time; and/or contributions from conformational and chemical exchange [29]. Inspection of the peak height ratios between bound and free LIC1-C showed that the residues affected were mainly located in helix 1 and to a lesser extent in helix 2 ( Fig 2B, 2C and 2D). In addition, we observed intensity reduction for the peaks of residues 418-421, which also tend to be conserved (S1A Fig). The quenching of segment 418-421 and helix 2 was more pronounced for SPDL1(2-359) than for BICD2  or HOOK3 (2-239) (Fig 2B, 2C and 2D).
NMR spectroscopy suggested that, in addition to helix 1, residues N-terminal to helix 1, as well as residues in helix 2, also make contact with adaptors. To investigate the contribution of these sites to adaptor binding, we first truncated LIC1(388-471) from its N terminus to generate progressively smaller fragments (residues 402-471, 414-471, 424-471, and 440-471), all of which retained helix 1 ( Fig 3A). SPDL1(2-359), BICD2(2-422), HOOK3(2-552), FIP3(2-756), and NIN(1-693) showed reduced binding to LIC1-C with decreasing fragment size, whereas RILP bound equally well to all LIC1-C fragments ( Fig 3E). Thus, for most adaptors, efficient binding to LIC1-C requires residues located N-terminally of helix 1. To assess the contribution of helix 2, we generated a fragment from the beginning of helix 1 until the end of LIC1-C (residues 440-523). Although SPDL1(2-359), BICD2(2-422), and HOOK3(2-552) were not detected in pull-downs using LIC1(440-455), they bound weakly to LIC1(440-523), potentially reflecting a modest contribution of helix 2 to the interaction ( Fig 3E). Overall, our binding experiments with purified components demonstrate that helix 1 of LIC-C is essential for the interaction with adaptors but suggest that most adaptors also need to make additional contacts with the flexible LIC-C scaffold for efficient binding. Specifically, residues preceding helix 1 appear to make a significant contribution. The exception is RILP, for which LIC-C helix 1 on its own is sufficient for binding.

Point mutations in the C-terminal helix 1 of C. elegans LIC cause severe developmental defects, impair locomotion, and shorten life span
To determine whether helix 1 of LIC-C contributes to dynein function in vivo, we turned to the animal model C. elegans, which expresses a single dynein LIC, dli-1 [30]. Just as in other LIC orthologs, the C-terminal helix 1 is highly conserved in DLI-1 ( Fig 5A). We used clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 (CRISPR/Cas9)-mediated genome editing to mutate either the two phenylalanines or the two leucines in helix 1 to alanine (F392A/F393A and L396A/L397A), in analogy to the human LIC-C mutations we characterized in vitro ( Fig 5B). For comparison, we also deleted the entire DLI-1 C-terminal region (Δ369-443). For all three dli-1 mutants, homozygous offspring from heterozygous mothers developed to adulthood but were sterile, suggesting that the C-terminal region of DLI-1, and helix 1 in particular, is essential for dynein function in vivo ( Fig 5B). Because RNA interference (RNAi)-mediated depletion of DLI-1 showed that DLI-1 is required for the stability of dynein HC 1 (DHC-1; S7 Fig), we addressed the possibility that the phenotype of our dli-1 mutants was a consequence of reduced DHC-1 levels. Immunoblotting of homozygous adults with an antibody against DHC-1 demonstrated that this was not the case. On the contrary, DHC-1 levels appeared slightly increased in all three dli-1 mutants (Fig 5C and S7B Fig). This indicates that the C-terminal DLI-1 mutants do not interfere with the binding of the Nterminal GTPase-like domain to DHC-1. Dynein light intermediate chain uses a conserved mechanism to bind structurally diverse cargo adaptors Differential interference contrast imaging revealed severe morphological defects in day 1 adults homozygous for the dli-1 mutations (Fig 5D, 5E and 5F). All three dli-1 mutants had a slightly dumpy body with a protruding vulva and a highly disorganized and underdeveloped gonad ( Fig 5D and 5E), which is indicative of problems with postembryonic cell divisions. Furthermore, the prevalence of vacuoles suggested widespread necrotic-like cell death ( Fig 5F). Consistent with this, dli-1 mutants exhibited severe defects in locomotion (Fig 5G), which was assessed by determining the body bending frequency in liquid medium (wild type, 1.50 ± 0.02 Hz; Δ369-443, 0.50 ± 0.03 Hz; F392A/F393A, 0.46 ± 0.04 Hz; L396A/L397A, 0.50 ± 0.03 Hz). Dli-1 mutants had a significantly shorter life span compared to wild-type animals (wild type, 18.3 ± 0.7 d; Δ369-443, 13.7 ± 0.4 d; F392A/F393A, 12.3 ± 0.5 d; L396A/L397A, 13.0 ± 0.5 d) ( Fig 5H). None of the dli-1 mutant animals lived beyond 24 d, whereas 27% of wild-type animals did. We conclude that deletion of the DLI-1 C-terminal disordered region and point mutations in the conserved helix 1 cause similar defects in development, locomotion, and life span. These phenotypes are reminiscent of those reported for other dynein mutants, including null mutants of dli-1 [30,31].

Point mutations in the C-terminal helix 1 of DLI-1 cause accumulation of membrane cargo at nerve endings of mechanosensory neurons and perturb retrograde axonal transport
Next, we sought to assess the impact of the C-terminal dli-1 mutations on dynein-dependent cargo transport. We chose to examine the distribution and transport kinetics of membrane cargo in axons of touch receptor neurons, which are mechanosensory neurons whose processes extend along the length of the animal just underneath its cuticle ( Fig 6A). Axonal microtubules in touch receptor neurons are polarized with their plus ends oriented toward the axonal tip, and dynein is therefore responsible for retrograde transport of cargo toward the cell body [31,32]. Using Mos transposase-mediated single-copy insertion [33], we constructed transgenic animals expressing mKate2::RAB-5 from the mec-7 promoter and crossed them with animals expressing soluble green fluorescent protein (GFP) from the mec-4 promoter (allele zdIs5). This allowed simultaneous visualization of early endosomes (mKate2), a cargo of dynein, and neuronal morphology (GFP) (Fig 6A). In contrast to control day 1 adults, all three dli-1 mutants exhibited a pronounced misaccumulation of mKate2::RAB-5 at the axonal tips and nerve ring synapses of anterior lateral mechanosensory (ALM) and anterior ventral mechanosensory (AVM) neurons ( Fig 6B, 6C, 6D and 6E). This defect resembled the previously described misaccumulation of synaptic vesicle proteins in touch receptor neurons of dynein mutants [31].
To determine how dli-1 mutants affect the kinetics of axonal transport, we imaged mKate2:: RAB-5-labeled early endosomes at high temporal resolution in an axonal segment close to the cell body of ALM neurons at the larval L4 stage (Fig 6A and 6F and S1 Movie). Quantitative analysis of kymographs revealed that the three dli-1 mutants had near-identical defects in axonal transport. The fraction of particles moving in the retrograde direction was reduced significantly, with a corresponding increase in the fraction of stationary particles ( Fig 6G). Analysis of individual runs showed that particles spent less time in retrograde motion and paused more frequently and for longer periods of time (S8A Fig). Particles also had a decreased run length and moved at reduced speed, especially in the retrograde direction (Fig 6H and 6I). Overall, this analysis revealed that all three dli-1 mutants strongly impaired retrograde axonal transport of early endosomes, consistent with compromised dynein function.
We also examined the axonal distribution of two additional types of dynein cargo, each labeled with a marker expressed from a single-copy transgene: synaptic vesicles (SNB-1  [31]. When examining the distribution of TOMM-20(1-54)::mKate2 particles (Fig 7A), we found that axons of ALM neurons contained an average of 21 ± 1 mitochondria in control day 1 adults, corresponding to a density of 5.6 mitochondria per 100 μm, and that mitochondria were evenly distributed along axons (Fig 7B, 7C, 7D and 7E). These findings are consistent with prior reports [34,35]. Mitochondria remained evenly distributed in ALM axons in dli-1 (L396A/L397A) animals ( Fig 7E). However, the average number of axonal mitochondria increased to 33 ± 1, corresponding to a density of 9 mitochondria per 100 μm (Fig 7C and 7D). Close inspection of mitochondrial morphology suggested that the increase was primarily due to an excess of small TOMM-20(1-54)::mKate2 puncta, which may represent fragmented mitochondria ( Fig 7B). Thus, the dli-1(L396A/L397A) mutation does not result in distribution bias of mitochondria along the axon but appears to promote mitochondrial fission. Similar results were obtained for the dli-1(Δ369-443) mutation (Fig 7C, 7D and 7E). Analysis of mitochondrial transport kinetics in dli-1(L396A/L397A) animals revealed a reduction in the speed of transport, with a more pronounced effect in the retrograde direction (Fig 7F and 7G, and S2 Movie). However, none of the other transport parameters were significantly altered (S8E and S8F Fig).
Given the modest impact of the dli-1(L396A/L397A) mutation on mitochondrial transport, we asked whether the mitochondrial adaptor TRAK1, which contains a CC1 box similar to SPDL1 and BICD2, binds LIC1. GST pull-downs with purified human proteins showed that maltose-binding protein (MBP)::TRAK1(103-187), which corresponds to the first coiled-coil segment, was able to bind GST::LIC1(388-523), albeit weakly and only when artificially dimerized using a general control nondepressible 4 (GCN4) sequence (Fig 7H and 7I). The interaction was abolished when the two leucines in LIC1-C helix 1 were mutated, suggesting that LIC1 binds TRAK1 through a similar mechanism as the other adaptors.

The C-terminal helix 2 of DLI-1 is largely dispensable for dynein function in vivo
Just like helix 1, helix 2 in LIC-C shows high sequence conservation, including in C. elegans (residues 420-428; Fig 8A and S1A Fig). To assess the role of helix 2 in vivo, we used genome editing to generate animals that express DLI-1 without its C-terminal 30 residues (Δ414-443). This truncated version of DLI-1 retains helix 1 (residues 388-400) (Fig 8A). In contrast to the helix 1 point mutants, homozygous dli-1(Δ414-443) animals were fully viable and fertile (embryonic viability 98.4% ± 0.6% and 97.3% ± 0.7% for wild type [>300 progeny, 12 mothers] and mutant [>300 progeny, 14 mothers], respectively). Furthermore, we observed no increase axons of ALM neurons, imaged at the L4 larval stage. Time-lapse sequences of confocal images were recorded every 200 ms in an axonal region approximately 50 μm away from the cell body, which is positioned to the right. Scale bar, 10 μm. (G-I) Quantification of early endosome motility, based on the analysis of kymographs as shown in (F). Graphs represent the mean ± SEM. For (G), n is the total number of axons. For (H) and (I), n is the total number of segments within tracks of moving particles framed by a pause or a reversal. Results are derived from 2-5 independent imaging sessions. Statistical significance was determined as described for (D, E). ���� P < 0.0001; ��� P < 0.001; ns indicates P > 0.05. Also see S8A Fig  and axonal length (D) in day 1 adults. Graphs represent the mean ± SEM. Statistical significance (mutant versus WT dli-1) was determined by one-way ANOVA on ranks (Kruskal-Wallis nonparametric test) followed by Dunn's multiple comparison test. ���� P < 0.0001; ns indicates P > 0.05. (E) Relative distribution of mitochondria along the ALM axon from the proximal (0%) to the distal (100%) end in day 1 adults. Graph corresponds to the mean, and the in mKate2::RAB-5 signal at axonal tips of touch receptor neurons, indicating that dyneindependent transport of early endosomes was not significantly affected in this mutant (Fig 8B,  8C and 8D). Because homozygous dli-1(Δ414-443) animals produced progeny, we were able to examine the first mitotic division of embryos coexpressing GFP::β-tubulin and mCherry:: histone H2B, which mark microtubules and chromosomes, respectively ( Fig 8E). During mitosis, dynein-dependent pulling on centrosome-nucleated microtubules is essential for the separation and positioning of centrosomes, which form the spindle poles after nuclear envelope breakdown (NEBD). At the time of NEBD in control animals, centrosomes were fully separated and the centrosome-centrosome axis was oriented approximately parallel to the anterior-posterior axis (Fig 8E and 8F). In dli-1(Δ414-443) embryos, centrosomes separated normally, but the centrosome-centrosome axis was frequently severely tilted relative to the anterior-posterior axis (angle > 45˚in 7 out of 21 mutant embryos versus 0 out of 13 controls), resulting in misoriented spindles. Despite initial misorientation, spindles ultimately correctly aligned with the anterior-posterior axis, and chromosome segregation completed without defects. The delay in spindle orientation indicates a subtle impairment of dyneindependent pulling forces [36,37]. To further probe the functional significance of helix 2, we combined the dli-1(Δ414-443) mutation with a null allele of the dynein cofactor nud-2, nud-2 (ok949), which represents a sensitized dynein background that also exhibits a spindle orientation delay and that we had previously exploited for an enhancer screen [36,38]. We observed neither an increase in lethality nor enhanced defects in spindle orientation in the double mutant relative to the single mutants (embryonic viability 85.9% ± 1.6% and 89.8% ± 1.4% for ok949 [>300 progeny, 15 mothers] and ok949; dli-1[Δ414-443] [>300 progeny, 19 mothers], respectively), consistent with the idea that dynein function in dli-1(Δ414-443) animals is relatively unperturbed.
We conclude that helix 2 of the DLI-1 C-terminal region makes a modest contribution to dynein-dependent pulling forces during mitosis but overall plays a minor role in vivo compared to helix 1, which is critical for dynein function. These results are consistent with our biochemical characterization of human LIC-C in vitro, which shows that helix 1, but not helix 2, is essential for the interaction between LIC-C and cargo adaptors.

Discussion
How dynein achieves specificity for cargo is a key question underlying the functional versatility of the motor. Studies over the past few years have revealed that the N-terminal region of cargo adaptor proteins helps bring together the motor with its essential cofactor dynactin and that multiple distinct protein-protein interactions participate in the assembly of a processive number n of axons imaged for each condition is indicated. (F) Kymographs of mitochondrial motility in axons of ALM neurons, imaged at the L4 larval stage. Time-lapse sequences of confocal images were recorded every 5 s in an axonal region approximately 50 μm away from the cell body, which is positioned to the right. Scale bar, 10 μm. Also see S2 Movie. (G) Quantification of mitochondrial transport velocity, based on the analysis of kymographs as shown in (F). Graphs represent the mean ± SEM. n is the total number of segments within tracks of moving particles framed by a pause or a reversal. Results are derived from 5-8 independent imaging sessions. Statistical significance was determined as described for (C, D). ���� P < 0.0001; ��� P < 0.001. Also see S8E and S8F Fig. (H) Coomassie Blue-stained protein gel of purified GST-tagged human LIC1-C and purified MBP::TRAK1 fragments containing a C-terminal Strep-tag II (also see S1B Fig dynein-dynactin-adaptor transport machine. Here, we dissected the structural determinants and the functional relevance of the interaction between the C-terminal region of the dynein LIC subunit and cargo adaptors. We present high-resolution molecular evidence that LIC-C is disordered and that a conserved segment with helical propensity, helix 1, is the main binding site for seven adaptors that differ in structure and cargo specificity. Our results agree with recent work that identified LIC1-C helix 1 as the critical structural element for the interaction Dynein light intermediate chain uses a conserved mechanism to bind structurally diverse cargo adaptors with HOOK1/3, SPDL1, and BICD2 [13], and we extend these findings to RILP, NIN, FIP3, and TRAK1. The highly conserved phenylalanines and leucines of the amphipathic helix 1 are essential for the interaction, which occurs with single-digit micromolar affinity for SPDL1, BICD2, HOOK3, and RILP, suggesting a similar binding mechanism. NMR spectroscopy analysis shows that helix 1 is transient. The presence of transiently sampled secondary elements in intrinsically disordered proteins is common and may facilitate the transition to more rigid states upon binding [39][40][41]. The observed signal quenching upon binding to adaptors is unlikely to be caused entirely by an increased local tumbling time. Instead, micro-millisecond conformational exchange between various states of the helical elements or binding kinetics contribute significantly to the quenching. Both mechanisms suggest residual structural disorder. Indeed, various examples of disordered proteins that maintain partial disorder during interaction with other proteins have recently been reported [40,[42][43][44]. A prior study used Xray crystallography to show that LIC1-C helix 1 inserts into a hydrophobic cleft on HOOK3 (1-160) [13]. An analogous hydrophobic cleft is likely formed by the CC1 box, the LIC1-C binding region in SPDL1 and BICD2 [13,16]. CC1 boxes are also present in other adaptors whose N-terminal regions are structurally similar to SPDL1 and BICD2, such as HAP1 and TRAK [45]. Consistent with this, we show that the first coiled-coil segment of TRAK1 interacts with LIC1-C in a helix 1-dependent manner. RILP, NIN, and FIP3 contain neither a Hook domain nor a CC1 box. The structural element that accommodates the LIC-C helix 1 in these adaptors remains to be identified.
LIC1-C helix 1 is essential for adaptor binding, but we find that helix 1 on its own-i.e., LIC1(440-455)-is not sufficient for efficient binding to 5 out of 6 adaptors tested, RILP being the exception. Pull-down assays with LIC1-C truncations show that segments N-terminal to helix 1 contribute to adaptor binding. LIC1-C binding to SPDL1, FIP3, and NIN appears to be particularly sensitive to N-terminal LIC1-C truncations, as we find that LIC1(424-471) has markedly reduced affinity relative to LIC1(388-471). This is in agreement with NMR analysis, which indicates that residues 418-421 participate in the interaction with SPDL1. NMR analysis also indicates that LIC1-C helix 2 participates in the interaction with adaptors, but in vitro binding experiments show that the contribution of helix 2 to the overall binding affinity is relatively minor.
Vertebrates possess two LIC paralogs that differ significantly in their C-terminal region. Prior studies in cultured cells suggest that there is functional redundancy among LIC1 and LIC2 but also hint at functional specialization in dynein-dependent cell division processes and membrane trafficking [46][47][48][49][50][51][52][53]. One attractive possibility is that the C-terminal regions of LIC1 and LIC2 discriminate between adaptors. Our results show that LIC1 and LIC2 use the same helix 1-based mechanism for adaptor binding, and we did not detect any striking differences in the ability of the two C-terminal regions to interact with our set of six adaptors. Nevertheless, it is plausible that LIC1 and LIC2 exhibit subtle preferences for specific adaptors. For example, we find that NIN binds slightly better to LIC2 than LIC1. Posttranslational modifications could possibly further modulate the affinity for adaptors in an isoform-specific manner.
Previous work in vitro showed that the dynein-dynactin-adaptor assembly fails to form in HOOK3 and SPDL1 mutants that cannot bind to LIC1 [16,17] and that addition of excess LIC1-C helix 1 inhibits processive movement mediated by HOOK3 or BICD2 fragments in motility assays [13]. Here, we present direct evidence that LIC-C helix 1 is important for dynein function in vivo. Like other invertebrates, the nematode C. elegans expresses only one LIC isoform, which facilitates the identification of functionally critical regions. In all assays, the outcome of mutating the two conserved phenylalanines or leucines in the C-terminal helix 1 of DLI-1 is identical to that of deleting the entire C-terminal region. Unlike DLI-1 depletions, the phenotype of the C-terminal DLI-1 mutations cannot be attributed to destabilization of DHC-1 and therefore likely reflects defective adaptor binding. On the animal level, sterility and other developmental defects suggest failure of postembryonic cell division, similar to what was described for dli-1 null mutants [30,31]. We show on the cellular level that the C-terminal DLI-1 mutations disrupt the distribution of early endosomes and presynaptic vesicles in axons of touch receptor neurons. Since we find that axonal length is normal in day 1 adults of dli-1 (L396A/L397A) and dli-1(Δ369-443) mutants, this is unlikely to be an indirect consequence of a developmental defect. Instead, the misaccumulation at the axonal tip is suggestive of impaired retrograde transport by dynein, as previously described for other dynein mutants, including the dli-1 null mutant js351 [31]. Consistent with this, we show that the frequency, velocity, and run length of early endosome movement is significantly decreased in our dli-1 mutants, with a predominant effect on retrograde movement. Neurodegeneration (i.e., axon beading) starts to become evident in dli-1 mutant day 1 adults and likely contributes to the animals' severe locomotory deficit and shortened life span.
The residual retrograde movement of early endosomes in axons of dli-1 mutants could indicate that dynein retains a limited ability to form processive dynein-dynactin-adaptor complexes without the interaction between DLI-1 and adaptors. This may also explain the modest effect of dli-1 mutants on mitochondrial transport. However, it is difficult to rule out that a small fraction of wild-type maternal DLI-1, passed on from the heterozygous mother to homozygous mutant progeny, could persist in touch receptor neurons at the L4 stage and promote residual dynein activity. Nevertheless, the observation that dli-1 mutants have a more pronounced effect on early endosomes and synaptic vesicles than on mitochondria indicates that DLI-1 binding to adaptors may not be equally important for all cargo transport.
In contrast to the point mutants in the DLI-1 C-terminal helix 1, there are no obvious developmental defects in animals expressing the DLI-1(Δ414-443) mutant that lacks the C-terminal helix 2, and mKate2::RAB-5 is not misaccumulated at the axonal tip of touch receptor neurons in this mutant. We have not examined neuronal cargo transport kinetics, but given that dli-1(Δ414-443) animals are healthy and propagate normally, any defects are unlikely to be substantial. We do, however, observe a delay in mitotic spindle positioning in one-cell dli-1 (ΔΔ414-443) embryos. The mild phenotype contrasts with the failure of spindle assembly observed in one-cell embryos after DLI-1 depletion [30]. Furthermore, the mitotic defects of dli-1(Δ414-443) embryos are not enhanced by a null allele of the dynein cofactor nud-2, which partially compromises dynein [36,38]. Thus, analysis in vivo and in vitro indicates that LIC-C helix 2, despite its high sequence conservation, makes a relatively modest contribution to dynein function compared to helix 1.
Together with the work of Lee and colleagues [13], our study establishes the molecular mechanism used by LIC to interact with structurally diverse cargo adaptors. An interesting open question is whether LIC-C could have additional binding partners besides adaptors. Two recent cryo-EM studies revealed that two dyneins can be recruited by a single dynactin-adaptor complex [10,11]. In one of the structures, an extra density, most likely corresponding to LIC-C of the first dynein, packs against the N-terminal coiled-coil of BICDR1 while simultaneously contacting one of the HCs of the second dynein [10]. It is tempting to speculate that this interaction between LIC-C and HC facilitates the incorporation of a second dynein into dynein-dynactin-adaptor assemblies.

Protein expression and purification from bacteria
All bacterial expression vectors were transformed into the Escherichia coli strain BL21, except for the NIN and HOOK3 constructs, which were transformed into the E. coli strain Rosetta. Expression was induced with 0.1 mM IPTG at 18˚C overnight at an OD 600 of 0.9, and cells were harvested by centrifugation for 20 min at 4,000g.
For Purification of LIC1::6xHis (residues 388-523, 388-471, and 472-523) for NMR spectroscopy, SPR, and MST experiments was carried out as described above with the following modifications: GST::LIC1::6xHis was captured using a GSTrap FF column (GE Healthcare) and eluted with elution buffer A. The GST moiety was cleaved off in solution with PreScission Protease, glutathione was removed by dialysis (50 mM HEPES, 150 mM NaCl [pH 8.0]), and the sample was applied again to a GSTrap FF column to remove GST and GST-tagged Prescission Protease. The flow-through containing LIC1::6His was subjected to size-exclusion chromatography using a Superdex 75 increase 10/300 GL column (GE Healthcare) in NMR buffer (50 mM sodium phosphate, 150 mM NaCl [pH 6.5]).
For purification of cargo adaptors, bacterial pellets were resuspended in lysis buffer B (50 mM HEPES, 250 mM NaCl, 10 mM imidazole, 0.1% Tween 20, 1 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, 1 mg/mL lysozyme [pH 8.0]), disrupted by sonication, and cleared by centrifugation at 34,000g for 45 min. The 6xHis::MBP::adaptor::Strep-tag II proteins were purified by tandem affinity chromatography using HisPur Ni-NTA resin followed by Strep-Tactin Sepharose resin (IBA). HisPur Ni-NTA resin was incubated in batch with the cleared lysate and then washed with wash buffer B, and proteins were eluted on a gravity column with elution buffer B. Fractions containing the recombinant proteins were pooled, incubated overnight with TEV protease to cleave off the 6xHis::MBP moiety (except for TRAK1 fragments, which were not cleaved), incubated in batch with Strep-Tactin Sepharose resin, and washed with wash buffer A. Proteins were eluted on a gravity column with elution buffer E (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin [IBA] [pH 8.0]). Fractions containing the proteins were pooled and dialyzed against storage buffer or further purified by size-exclusion chromatography using a Superose 6 10/300 column equilibrated with storage buffer. Glycerol and DTT were added to final concentrations of 10% and 1 mM, respectively, and aliquots were flash-frozen in liquid nitrogen and stored at −80˚C.

Protein expression and purification from insect cells
Bacmid recombination and virus production were performed as described previously [54]. A 500-mL culture (SFM4 medium; Hyclone) of Sf21 cells (0.8 × 10 6 cells/mL) was infected with RILP::Strep-tag II-encoding virus. Cells were harvested by centrifugation at 800g for 5 min.

NMR spectroscopy
For backbone resonance assignment of LIC1(388-523)::6xHis, 15 N-1 H HSQC, HNCACB, CBCA(co)NH, HNCO, HN(ca)CO, and HNN spectra [55] were recorded on a triple-resonance Varian 900 NMR cryoprobe spectrometer at 25˚C using a 13 C/ 15 N-labeled sample in 50 mM sodium phosphate and 150 mM NaCl at pH 6.5 and 375 μM protein concentration in a standard 5-mm Shigemi tube. The 3D spectra were acquired with a nonuniform sampling (NUS) scheme generated by the NUS@HMS scheme generator software [56] with 1,024 complex data points in the direct dimension and 30% sampling of the original 96 and 80 points in the indirect 13 C and 15 N dimension, respectively. The spectral widths were 14,045 Hz ( 1 H), 3,200 Hz ( 15 N), 3,770 Hz (C = O), and 15,835 Hz (Cα/Cβ); the interscan delay was 1.7 s; and the number of scans was 16 for all experiments. The NUS-acquired data were reconstructed using the hmsIST software [56]. Zero-filling was achieved by addition of 256 points in both indirect dimensions. A solvent subtraction function was applied in the direct dimension. Further data processing and visualization were performed using NMRPipe/NMRDraw [57] and NMRFAM Sparky [58]. Resonance assignment was performed using CCPNmr Analysis software [59].
Because of high sequence redundancy and extensive peak overlap, we used a "divide-andconquer" approach for chemical shift assignment. We measured and overlaid 15 N-1 H HSQC spectra for two smaller LIC1 constructs, 388-471 and 472-523, with 388-523 to facilitate the verification of assignment. The 15 N-1 H HSQC spectra of the small constructs were measured with 128 scans and 128 complex points in the indirect dimension.
We assessed residual secondary structure using the SSP score program developed by Forman-Kay and colleagues with the re-referencing algorithm for 13 CA and 13 CB shifts [24]. The method combines different chemical shifts into a single residue-specific SSP score. Our input shifts were those of 1 H N , 15 N, 13 CO, 13 CA, and 13 CB.
The 15 N R 1 and R 1ρ relaxation rate constants and 15 N-1 H heteronuclear NOEs of LIC1 (388-523)::6xHis were measured on a Bruker 700 MHz spectrometer equipped with a tripleresonance cryoprobe at 25˚C using a 15 N-labeled sample at 400 μM protein concentration in 50 mM sodium phosphate buffer, 50 mM NaCl, 0.05% (w/v) NaN 3 , and 5 mM DTT at pH 6.5. For the R 1 and R 1ρ experiments, the sampling time points were 40,88,136,192,288,392,592,688,792, and 992 ms and 30, 60, 120, 150, 180, and 210 ms, respectively. During the R 1ρ relaxation time, a 15 N spin-lock field of 1,433-Hz strength was applied. R 2 was calculated from R 1 and R 1ρ using the following equation: R 2 = R 1ρ + (R 1ρ -R 1 ) � tg 2 (θ), where θ = tan -1 (2πΔν/ γ N B 1 ), Δν is the resonance offset, |γ N B 1 |/2π is the strength of the spin-lock field B 1 , and γ N the gyromagnetic ratio of the 15 N spin. The 15 N-1 H heteronuclear NOEs were determined from two spectra recorded in presence and in the absence of 1 H saturation in an interleaved manner.
For NMR titration analysis, 15 N-1 H HSQC spectra with 128 complex points in the indirect dimension and 128 scans were recorded on a triple-resonance Varian 900 NMR cold-probe spectrometer at 25˚C of 40 μM samples of 15  :Strep-tag II in 50 mM sodium phosphate and 150 mM NaCl at pH 6.5. We used freshly prepared samples for each titration step to limit confusion with potential degradation peaks. Data processing and visualization were performed using NMRPipe/NMRDraw [57] and NMRFAM Sparky [58].
The 1 H, 13  SPR analysis with RILP::Strep-tag II was performed using a Biacore X100 system equipped with a CM5 sensor chip (GE Healthcare). Anti-GST antibody was immobilized using aminecoupling chemistry using the Amine Coupling Kit (GE Healthcare) and the GST Capture Kit Flow cell 1 was injected with GST::6xHis at a density of 575-430 RU to serve as a reference surface. RILP::Strep-tag II (analyte) was injected in running buffer over the two flow cells at concentrations of 50, 11, 4.5, 1.8, and 0.3 μM at a flow rate of 30 μL/min and a temperature of 25˚C. The complex was allowed to associate and dissociate for 120 and 600 s, respectively. Surfaces were regenerated with two 2-min injections of regeneration solution. Three independent runs were performed for each condition. The data were fit to a 1:1 interaction model using the evaluation module of the Biacore X100 software, version 2.0.1 (GE Healthcare).

CD
CD spectra of LIC1(388-523)::6xHis were collected on a J-815 CD spectrometer (Jasco) with a wavelength range from 190 to 250 nm, data pitch 1 nm, standard sensitivity, 1 nm bandwidth and 20 nm/min scanning speed, at temperatures of 25, 37, 50, 60, and 70˚C. The baseline of the spectrum was obtained from measurement of the buffer (50 mM sodium phosphate, 150 mM NaCl [pH 6.5]) and subtracted from the spectra of the samples to remove artificial CD signals that might originate from the optical system. Data measurement and analysis were performed using Spectra Manager version 2 (Jasco). Molar ellipticity was calculated as m°M/ (10LC), where m°is the degree value (mdeg), M is the molecular weight in g/mole, L is the path length in cm, and C is the concentration in g/L.

Life span assay
Animals were collected at the L4 stage (day 0) and transferred every 2 d to a new NGM plate with bacteria. Animals were scored as alive or dead every 1-3 d. Animals were considered dead if they did not respond when touched with a platinum wire and if there was no evidence of pharyngeal pumping. Animals that were found dead on the edge of the plate, escaped, or died because of internal hatching of progeny were excluded from the assay.

Imaging
Body bending assay. L4 hermaphrodites were transferred to a new NGM plate with bacteria 16 h before performing the assay. For imaging, animals were transferred to a slide containing a 2-μL drop of M9. Movements were tracked at 20˚C for 1 min at 40 frames per second using an SMZ 745T stereoscope (Nikon) mounted with a QIClic CCD camera (QImaging) and controlled by Micro-Manager software (Open Imaging). The wrMTrck plugin for Image J (http://www.phage.dk/plugins/wrmtrck.html) was used for automated counting of bodybends.
Differential interference contrast imaging of adults animals. L4 hermaphrodites were transferred to a new NGM plate with bacteria for 16 h, paralyzed with 50 mM of sodium azide for 5 min, mounted on a freshly prepared 2% (w/v) agarose pad, and covered with an 18 mm × 18 mm coverslip (No. 1.5H, Marienfeld). Imaging was performed on an Axio Observer microscope (Zeiss) equipped with an Orca Flash 4.0 camera (Hamamatsu) controlled by ZEN 2.3 software (Zeiss). Multiple images covering the entire animal were recorded at 1 × 1 binning with a 40x NA 1.3 Plan-Neofluar objective and assembled into one image using the tiles mode in ZEN.
For live imaging of mKate2::RAB-5 and TOMM-20(1-54)::mKate2, hermaphrodites at the L4 stage were paralyzed with 5 mM levamisole in M9 buffer for 10 min and mounted onto freshly prepared 2% or 5% agarose pads, respectively. ALM neurons were identified using the GFP signal, and only neurons with morphologically healthy axons were imaged. Time-lapse sequences of an axonal region approximately 50 μm away from the cell soma were recorded in a temperature-controlled room at 20˚C, using a Nikon Eclipse Ti microscope coupled to an Andor Revolution XD spinning disk confocal system, composed of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i, Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corporation) controlled by Andor iQ3 software (Andor Technology). For mKate2::RAB-5, a single image was acquired every 200 ms for a total of 30 s at 1 × 1 binning using a 100x NA 1.45 Plan-Apochromat objective. For TOMM-20(1-54)::mKate2, a 5 × 0.5 μm z-stack was acquired every 5 s for a total of 5 min at 1 × 1 binning using a 60x NA 1.4 Plan-Apochromat objective.

Image analysis
Image analysis was performed using Fiji software (Image J version 1.52d). mKate2::RAB-5 and SNB-1::mKate2 levels. For axonal tip measurements, the GFP signal was used as a reference to draw a region around the last 20 μm of the ALM or AVM axon, and the integrated intensity of mKate2 fluorescence within that region was measured. The region was then expanded by a few pixels along the length of the axonal segment. The difference in integrated fluorescence intensity between the outer and inner region was used to define the integrated background intensity after normalization to the area of the inner region. The final integrated intensity of mKate2 signal at the axonal tip was then calculated by subtracting the integrated background intensity from the integrated intensity of the inner region. The same approach was used to determine integrated mKate2 intensity in the nerve ring.
TOMM-20(1-54)::mKate2 distribution. To determine the number and position of mitochondria along the ALM axon (marked by GFP), line segments connecting adjacent TOMM-20(1-54)::mKate2 particles were drawn with the first segment starting at the cell body and the last segment terminating at the axonal tip. The sum of the lengths of individual line segments was equal to the total length of the axon. The distance of each TOMM-20(1-54)::mKate2 particle from the cell body was then normalized to axon length (i.e., 0% corresponds to the beginning of the axon at the cell body and 100% denotes the axonal tip).
Axonal transport parameters. Only time-lapse sequences during which there was no discernable movement of the animal and that had uniformly bright mKate2 signal along the axonal segment were considered. Kymograph generation and image analysis were performed with KymoAnalyzer, a semiautomated open-source ImageJ package of macros designed to quantify transport parameters of fluorescently labeled stationary or moving particles [62]. A track is defined as a single particle trajectory, and a segment corresponds to a portion within a track of an individual moving particle framed by a pause or a reversal. Values for run length and velocity reported in this study are for segments. To determine the fraction of particles moving in the anterograde or retrograde direction, the position of the start and end point of the track was considered (i.e., a net anterograde moving particle may also have one or more segments of retrograde movement along its track and vice-versa; a net stationary particle may have one or more segments of anterograde or retrograde movement along its track).
Centrosome-centrosome axis tilt in the one-cell embryo. After maximum intensity projection of z-stacks, the angle between the centrosome-centrosome axis and anterior-posterior axis at NEBD was calculated from the coordinates of the two centrosomes and the two outermost points along the embryo long axis, using the cytoplasmic GFP signal to visualize the embryo outline.

Statistical analysis
Statistical analysis was performed with GraphPad Prism 7.0 software. Values in figures and text are reported as mean ± SEM. The type of statistical analysis performed is described in each figure legend. Differences were considered significant at P < 0.05. For NIN, only the first 1,000 of 2,090 residues are represented. Predictions of α-helical and coiled-coil segment were performed using the JPred Secondary Structure Prediction server. Protein domains (Hook, RILP homology 1 and 2, proline rich, EF-hand) are shown as annotated in the UniProt database. The CC1 box, which is required for the interaction with LIC in BICD2, SPDL1, and presumably TRAK1, is also highlighted. Dashed rectangles indicate the recombinant purified protein fragments used for in vitro assays. For HOOK3, fragment 2-552 was used for GST pull-downs experiments in Fig 3 and Fig 4. For NMR (Fig 2D), SPR (Fig 2G), and MST (S5C Fig) experiments, which required more protein, we used HOOK3(2-239), because we obtained a higher yield from bacteria with this fragment than with HOOK3(2-552). HOOK3(2-239) had previously been shown to be sufficient for LIC binding [17]. BICD2, bicaudal D homolog 2; GST, glutathione S-transferase; HOOK3, Hook homolog 3; LIC, light intermediate chain; MST, microscale thermophoresis; NIN, ninein; NMR, nuclear magnetic resonance; RAB11FIP3, RAB11 familyinteracting protein 3; RILP, RAB-interacting lysosomal protein; SPDL1, Spindly; SPR, surface plasmon resonance; TRAK1, trafficking kinesin-binding protein 1. Note that GFP-tagged DHC-1 is expressed at higher levels than DHC-1 in either wild-type or dli-1(Δ369-443) animals. Since DHC-1::GFP animals do not exhibit any obvious defects [37], the slight increase in DHC-1 levels in dli-1 mutants relative to wild-type animals is unlikely to be the cause for the phenotype. DHC-1, dynein heavy chain 1; GFP, green fluorescent protein; RNAi, RNA interference.