Microtubule motors of opposite polarity cooperate rather than compete in cargo transport

Abstract

Microtubule-based cargo transport relies on the actions of dynein and kinesins, motors that walk in opposite directions yet act together to ensure appropriate distribution of cargos in cells. Research now provides mechanistic insights into how these seemingly antagonistic motors collaborate, rather than compete, to promote each other’s activities.

Microtubule-based transport of cargoes to their requisite cellular destinations requires the activity of two distinct classes of motors: kinesins and dynein. Whereas the former class comprises a large family of ~45 different members that mostly walk along microtubules toward their plus ends, the latter class constitutes just one (cytoplasmic dynein-1) that transports cargoes toward the minus ends. The mechanisms by which these motors associate with and transport cargoes has been an active area of study for many years. As such, our understanding of how they function has improved enormously. However, many questions remain unanswered, including how these two classes of presumably antagonistic motors work together to ensure appropriate cargo transport in the complex cellular environment.

It is well established that many cellular cargoes do not associate with only dynein or only kinesin but are instead simultaneously bound to both¹. Consistent with this, live cell imaging of native cargoes has revealed that most undergo bidirectional movement, in which unidirectional excursions are interrupted by occasional directional switches². This confounding phenomenon has raised many questions, including: how do these cargoes achieve a net ‘forward’ motion? Do the motors antagonize each other (that is, engage in a tug-of-war), or do they cooperate? Is only one of them engaged with the microtubule at a given time, and if so, what governs their activity? Finally, what advantages are there to cargoes possessing both classes of motors? A preponderance of evidence from in-cell and ex vivo studies indicates that dynein and kinesin rely on each other for cargo transport, as depletion of either one results in a reduction in all transport^3,4. In a study in Nature Structural and Molecular Biology, Abid Ali et al.⁵ now show that, despite their opposing directional preferences, dynein and kinesin work together to ensure productive cargo transport.

Several efforts to study the interplay between dynein and kinesin have relied on the artificial tethering of these motors together (for example, using DNA-based scaffolds or tethers^6,7). Although useful for studying the biophysical properties of motors, these systems do not accurately recapitulate the natural arrangement of cargo-bound dynein and kinesins. A growing family of cargo adaptor proteins have been found to recruit and activate both dynein and kinesin family members. In the case of dynein, these adaptors help link dynein to dynactin, thereby promoting its motility. Although the role of cargo adaptors in kinesin activity is less clear, some studies indeed show that adaptors can relieve kinesin autoinhibition and thereby promote microtubule binding and motility⁸. Notably, several adaptor proteins can simultaneously bind dynein and kinesin, indicating that both classes of motors are not just present on the same cargo, but are linked to cargoes by the same adaptor molecules. Thus, an understanding of the interdependent relationship between dynein and kinesin requires a ‘bottom-up’ approach in which their motility is reconstituted with such shared adaptors.

Using a complement of biochemical reconstitutions, single-molecule motility assays and structural biological approaches, Abid Ali et al.⁵ identify the mechanism by which dynein and the kinesin-3 KIF1C collaborate to promote cargo transport. As in other recent studies^4,9,10, the authors found that a single cargo adaptor (HOOK3 in this case) simultaneously recruits active dynein–dynactin and KIF1C. These complexes moved either toward the minus ends (~84% of all events) or the plus ends (~13%), with very little apparent directional switching (~1.5%), indicating that only one of the bound motors at a time is active and engaged in motility. Thus, even though both classes of motors can be present on a single cargo adaptor scaffold, they observed no apparent tug-of-war, which would result in little to no net movement. In fact, addition of KIF1C (or the HOOK3-binding ‘stalk’ domain of KIF5C) increased the number of minus-end-directed motility events undergone by dynein–dynactin–HOOK3 (DDH) complexes, and, vice versa, addition of DDH increased the number of KIF1C motility events. This is consistent with a recent study that showed a similar reliance of dynein and KIF5C (a kinesin-1) on the opposing motor when each is bound to the mitochondrial adaptor protein TRAK2⁴. These findings argue against a tug-of-war model, instead suggesting that dynein and kinesin exhibit a co-dependence for motility that can be recapitulated with single cargo-adaptor-linked complexes.

The linkage between HOOK3 and its native early endosomal cargo requires a group of proteins that include Fused-Toes (FTS) and FTS and HOOK-interacting protein (FHIP). To better understand the linkage between HOOK3 and cargo, Abid Ali et al.⁵ solved a high-resolution cryo-EM structure of the FTS–HOOK3–FHIP (FHF) complex. In addition to revealing the structural arrangement and contact surfaces for this complex, the structure shows that HOOK3 is unlikely to bind directly to cargo, but rather does so via a highly conserved region of FHIP to which the small G protein RAB5 likely binds (Fig. 1).

Fig. 1 |. Activation of cargo transport by kinesin.

Cartoon model depicting the mechanism by which KIF1C activates its own motility or that of dynein–dynactin. KIF1C binding to autoinhibited endosome-associated HOOK3 (via RAB5–FTS–FHIP) results in HOOK3 adopting an open, uninhibited conformation. It is unclear whether HOOK3 binding leads to KIF1C adopting an uninhibited conformation, or if KIF1C adopts this state before binding HOOK3. The resulting endosome-associated RAB5–FHF–KIF1C complex drives transport of endosomes toward microtubule plus ends. Data from Abid Ali et al⁵ indicate that KIF1C–HOOK3 binding (via the KIF1C stalk globular domain) relieves the autoinhibited state of HOOK3, thereby promoting dynein–dynactin binding. Note their data also support a reciprocal model whereby dynein–dynactin binding to HOOK3 also relieves its autoinhibited state, thereby promoting subsequent recruitment of KIF1C (not pictured here). The resulting FHF–KIF1C–DD complex exhibits a strong bias toward the microtubule minus ends, at least in the conditions used by Abid Ali et al.⁵. It is likely that there are many factors that can to shift the balance of dynein- versus kinesin-driven motility, including MAPs, post-translational modifications to tubulin and other factors, as indicated.

Several cargo adaptor proteins are autoinhibited, including JIP3 and Spindly, which link dynein to lysosomes and autophagosomes and to kinetochores, respectively. Although it is unclear what relieves the autoinhibited state of cargo adaptors, it has been proposed that cargo binding might trigger this change. Abid Ali et al.⁵ found that HOOK3 also adopts an autoinhibited state that precludes its assembly into motile dynein–dynactin complexes. Although the addition of FTS and FHIP is not sufficient to relieve this autoinhibition, addition of the KIF1C stalk domain is. Using a combination of biochemical reconstitutions, cryo-EM and AlphaFold 2-based structural predictions, the authors showed that the KIF1C stalk (specifically the stalk globular domain) binds to an intact FHF complex, and in so doing, it disrupts the contacts between the N and C termini of HOOK3 that hold it in its autoinhibited state. These observations explain, at least in part, the mechanism by which dynein relies on kinesin in cells. Although Abid Ali et al.⁵ did not unequivocally demonstrate a reciprocal mechanism for dynein activating kinesin activity, it is reasonable to hypothesize that dynein–dynactin binding to HOOK3 similarly disrupts HOOK3 autoinhibition, thereby promoting its binding to KIF1C and its consequent activation (as was observed by the authors).

Finally, Abid Ali et al.⁵ inquired about the status of the non-motile motor in processively moving DDH–KIF1C complexes. By measuring the motility parameters (run length, velocity and dwell time) of either minus- or plus-end-directed complexes, they determined that the opposing motor in such configurations indeed affects the motile behavior of these complexes. Specifically, they showed that the presence of the opposing motor reduces the velocity of DDH–KIF1C complexes. Whereas this reduces run length values for KIF1C-driven motility, it increases those for dynein–dynactin-driven events. These findings show that both families of motors are actively engaged with the microtubule during such runs (as passive tethers), which can improve motility behavior in the case of dynein-driven events. The authors posit that, in addition to modulating the motility characteristics, the presence of both classes of motors on a single cargo adaptor scaffold offers several advantages, including the ability to quickly switch direction if the need should arise in cells (for example, in response to obstacles or cellular cues).

The findings from this study⁵ strongly support a model whereby kinesin and dynein collaborate to promote each other’s activity and suggest that the target for this mechanism is the cargo adaptor itself (Fig. 1). If this is true, a constitutively active HOOK3 mutant (with mutations that destabilize the autoinhibited state) would be expected to reduce the reliance of dynein on kinesin (and vice versa) for in-cell cargo transport. In addition to suggesting other testable hypotheses, this study also raises many questions. For example, the authors of this and another recent study found that reconstituted DDH–KIF1C complexes are predominantly moved by dynein¹⁰. Although some studies find a similar imbalance of dynein compared to kinesin transport in neurons¹¹, others have found a more equal balance of kinesin- and dynein-mediated transport¹. Several potential culprits may have the potential to shift the relative activities of these two motors. These include the large family of noncatalytic microtubule-associated proteins (MAPs), such as MAP4, MAP7 and MAP9, all of which either preclude dynein–dynactin and/or promote kinesin motility^12,13 and would thus be expected to shift the balance toward kinesin’s favor. This is supported by a recent study indicating that addition of MAP7 indeed shifts the balance of DD–TRAK1–KIF5B complexes toward kinesin-driven motility⁹. Microtubules are also targets for numerous types of post-translational modifications, some of which are known to directly influence the binding of either motors or MAPs. For example, dynein–dynactin–adaptor complexes are known to preferentially bind and walk along tyrosinated microtubules (that is, those composed of α-tubulin with its C-terminal tyrosine intact¹⁴), whereas kinesins exhibit better motility along microtubules that are polyglutamylated¹⁵. Thus, enzymatic addition or removal of these or other modifications on α- or β-tubulin could have a large role in regulating the balance of dynein versus kinesin motility in DDH–kinesin complexes. Other mechanisms may also shift this balance, including the dynein activators NDE1, NDEL1 or LIS1 (the latter of which was included in most assays by Abid Ali et al.⁵) and kinases, some of which (such as CDK5) have been found to affect the activity of these motors in cells.

One intriguing inference from this study is that all dynein activity in cells may similarly rely on a corresponding kinesin for maximal activity. However, it is currently unclear whether this mechanism of co-dependence is universal, as not all adaptors are thought to adopt an autoinhibited state. It is also unclear whether all adaptors have the capacity to simultaneously bind dynein and kinesin, although, as noted by the authors, evidence suggests that many may indeed do so. Future studies that similarly employ a ‘bottom-up’ reconstitution with dynein, kinesin, scaffolding cargo adaptors and other factors will be needed to reveal how these motors — once believed to exhibit an antagonistic relationship — collaborate to ensure the appropriate distribution and delivery of cargoes to their requisite cellular destination.