Selective motor activation drives organelle transport in axons

Abstract

The active transport of organelles and other cargos along the axon is required to maintain neuronal health and function, but we are just beginning to understand the complex, context-specific regulatory mechanisms involved. The molecular motors cytoplasmic dynein and kinesin transport cargos along microtubules; this transport is tightly regulated by adaptors and effectors. Here, we review our current understanding of motor regulation in axonal transport. We discuss the mechanisms by which regulatory proteins induce or repress the activity of dynein or kinesin motors, and explore how this regulation plays out during organelle trafficking in the axon, where motor activity is both cargo-specific and dependent upon subaxonal location. We survey several well-characterized examples of axonal transport—including autophagosomes, endolysosomes, signaling endosomes, mitochondria, and synaptic vesicle precursors—and highlight the specific mechanisms that regulate motor activity to provide localized trafficking within the neuron. Defects in axonal transport have been implicated in conditions ranging from cortical malformation to neurodegenerative disease, making it essential that we better understand the underlying mechanisms in order to develop more effective options for treatment.

Introduction

Neurons display highly specialized morphologies befitting their role in information processing and long-distance communication. Signals are received by the dendrites, integrated in the cell body, and then conducted down the axon to signal target cells including neurons and muscle fibers¹ (Fig. 1). The nucleus is in the cell body, also known as the soma; therefore, gene transcription as well as most protein synthesis and organelle biogenesis are primarily localized to this subcellular compartment^2–4. The active delivery of new proteins and organelles from the soma (anterograde transport) is required to establish and maintain the signaling regions, known as synapses, in both the dendrites and axon⁵. Protein and organelle turnover at synapses also requires active transport back toward the soma (retrograde transport) for recycling⁶. Additionally, signalling endosomes containing activated receptors must be transported along the axon to regulate transcription and cellular response⁷. Trafficking in neuronal processes is therefore essential for function.

Fig. 1: Microtubule and motor organization in neurons.

Neurons can be divided into three functional regions: the soma, where the nucleus and most translation and biogenesis occur; the long extended axon, which sends signals to other cells at pre-synapses; and the shorter highly branched dendrites, which receive signals at post-synapses. In either the axon or dendrites, transport away from the soma is anterograde, while transport toward the soma is retrograde. Microtubules in the axon are predominantly plus-end-out, while microtubules in mammalian dendrites are mixed polarity (plus- and minus-ends-out). Dynein (red), kinesin-1 (blue), and kinesin-3 (green) are the focus of this review.

The fastest intracellular transport is achieved by molecular motors moving along the microtubule cytoskeleton⁸. Microtubules form polarized tracks in axons: the faster growing plus-ends of the microtubules extend preferentially away from the soma, while the slower growing minus-ends point toward the soma⁹ (Fig. 1). By contrast, microtubules in vertebrate dendrites exhibit mixed polarity that can vary with distance from the soma^10,11 (Fig. 1). On the plus-end-out microtubules that predominate in axons, plus-end-directed kinesin motors move cargo from the soma to presynapses while the minus-end-directed microtubule motor cytoplasmic dynein 1 exclusively moves cargo from presynapses back to the soma^12–15. Here we focus on the mechanisms regulating the fast axonal transport of organelles and vesicles. Microtubule motors also mediate slow axonal transport, whereby mostly cytoplasmic components are trafficked 3–4 orders of magnitude more slowly along the axon; this transport mechanism has been recently reviewed¹⁶ and will not be discussed here.

Despite the opposing action of dynein and kinesin, most organelles copurify from brain with both motors bound^17,18. How then can unidirectional transport be achieved? Further, how are the microtubule motors regulated on specific cargos to achieve efficient delivery to, and retrieval from, specific destinations? In this review we discuss the coordinate regulation of microtubule motors bound to cargos moving along the axon, focusing specifically on new findings. We first explore recent advances in our understanding of motor coordination, activation, and inactivation. We then review the latest developments in the field of axonal transport, using what we know about motor regulation to better understand the motile behaviors of multiple organelles. Axonal transport is compromised in many neurodegenerative and neurodevelopmental disorders^19,20,5; thus a thorough understanding of its regulation is vital.

Mechanisms of motor regulation

Kinesins are encoded by a superfamily of 44 distinct genes in humans (referred to as KIFs) that can be grouped by similarity into 14 classes²¹. Some but not all kinesins are processive motors that move cargo toward microtubule plus ends²². Kinesin-1 (KIF5B), a ubiquitously expressed and canonical member of the kinesin superfamily, is typically a heterotetramer of two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs)²³. KHC is composed of a microtubule-binding motor head that can hydrolyse ATP, linked by a coiled-coil stalk domain that mediates dimerization and inter-head coordination to a tail domain²⁴. Kinesin tail domains are of varying length and may bind to regulatory proteins including KLCs and/or cargo-associated proteins²⁵ (Fig. 2, a–b). The main kinesins driving long-distance transport in neurons are kinesin-1—both the ubiquitous KIF5B isoform and the neuron-specific KIF5A and KIF5C isoforms— kinesin-3 (KIF1A, KIF1B, KIF13, KIF16B), kinesin-2 (KIF3, KIF17), and kinesin-4 (KIF4, KIF21A, KIF21B)^26–34. Each of these kinesins transports distinct cargos along axons and/or dendrites. Some kinesins localize preferentially to the axon, e.g. KIF5C or KIF13B, or to dendrites, e.g. KIF17 or KIF13A^5,35,36 (Fig. 1). In the axon this subcellular specificity may be driven by the motor itself, while in the dendrite the attached cargo seems to direct the motor localization³⁷.

Fig. 2: Motor autoinhibition and activation.

(a) Kinesin-1 autoinhibition is mediated by a head-tail interaction in the heavy chain (KIF5), which is stabilized/exacerbated by the light chains (KLC). Relief from autoinhibition is mediated by effector proteins, such as SKIP, binding to both KLC and the tail of KIF5 to fully free the motor heads. (b) Kinesin-3 (KIF1A) is autoinhibited by the stalk coiled-coil (CC) domain 1 binding to the motor heads. Relief from autoinhibition is mediated by effector proteins, such as MADD, binding to the stalk CC domain 3, which destabilizes the stalk-head interaction and frees the motor heads. KIF1A is further inhibited by kinesin binding protein (KBP) sterically blocking motor domain binding to the microtubule, which may be released by dephosphorylation of KBP. (c) Dynein alone oscillates between a closed “phi” conformation and an open anti-parallel conformation that can bind microtubules but not move. The effector LIS1 stabilizes the open conformation to facilitate binding with both dynein’s obligate partner complex dynactin and an activating adaptor protein, e.g. Hook1. Dynactin induces a conformational change such that the motor heads become parallel, and provides an additional microtubule-binding domain. The activating adaptor stabilizes the dynein-dynactin interaction and mediates contact with cargo. Finally, LIS1 and the activating adaptor can promote the addition of extra dynein dimer, increasing velocity and processivity. The effector proteins are often themselves regulated. (a) SKIP is head-tail autoinhibited but binding to GTP-Arl8 on membranes stabilizes its active open conformation; the BORC complex serves as an Arl8 GEF. (b) MADD is recruited to membranes cooperatively with Rab3 by serving as a Rab3 GEF. (c) Hook membrane contact is mediated by an interaction between GTP-Rab5, FHIP1B, and FTS. Note that the stoichiometry of the interactions displayed here are not all known.

In contrast to the specialization seen within the kinesin superfamily, cytoplasmic dynein 1 is ubiquitous and required for the vast majority of minus-end-directed transport in animal cells^38,39. Dynein heavy chain (DHC) consists of an ATP-hydrolysing AAA+ ring and a microtubule-binding domain separated by a coiled-coil stalk, plus an N-terminal tail necessary for dimerization and binding to additional proteins⁴⁰. Dynein light chains (DLC), light intermediate chains (DLIC), and intermediate chains (DIC) co-assemble with DHC to form the full 1.4 MDa complex⁴¹. Dynein assembles with its obligate partner complex dynactin, itself a multi-subunit 1.1 MDa complex⁴². Additional activating adaptors are required to enhance the interaction between dynein and dynactin and to link the megacomplex to cargo⁴³ (Fig. 2 c). Recent reviews detail the biophysical dynamics of microtubule motors^22,24,41,44; here we discuss the most recent findings regarding motor activation and the coordinated regulation of multiple motors bound to a single cargo.

Common themes in motor regulation.

While kinesin and dynein motors differ both structurally and mechanically, there are similarities in their regulatory mechanisms: both are activated by relief from autoinhibition and complex formation. Autoinhibition is mediated by diverse intra- and inter-molecular interactions, but uniformly induces a conformation that is incapable of binding and/or moving along the microtubule^23,45–48. Complex formation includes interaction with both ubiquitous obligate partners and scaffolding proteins that mediate cargo interactions and/or interact with opposing motors to facilitate unidirectional transport^41,49–52. In many cases, there are additional upstream regulators such as cargo-specific GTPase effectors or targeted kinases and phosphatases^53–60. Together, these mechanisms ensure that motors are appropriately activated when bound to cargo, but also ensure that opposing motors can be coordinately regulated to prevent an inefficient tug-of-war, as discussed below. Additional mechanisms are also critical to the spatiotemporal control of cellular motors in the neuron, including regulation by microtubule-associated proteins (MAPs) and the tubulin code (see Box 1). These cellular aspects of motor regulation have been recently reviewed^61–63; here we focus on cargo-specific mechanisms.

Box 1: Multilevel regulation of motor function.

Here we focus on the regulation of motor proteins by autoinhibition and complex formation on organelles; however, motors can also be regulated by microtubule associated proteins (MAPs) and the microtubules themselves. Motor recruitment to microtubules is regulated dynamically in cells by the ‘tubulin code,’ the ‘language’ used by cells to differentially and dynamically label microtubule tracks^167–169. This includes the use of distinct tubulin isoforms, the nucleotide state of the tubulin dimers in the lattice, and any post-translational modifications (PTMs) of the microtubule (recently reviewed^62,63). For example, the differential affinity of kinesin-1 for acetylated detyrosinated microtubules and kinesin-3 for tyrosinated microtubules is thought to bias the trafficking of specific cargos toward either the axon or dendrite^11,170. Similarly, tyrosinated microtubules are specifically enriched in the distal tip of the axon; the dynactin subunit p150^Glued and the dynein regulator CLIP-170 preferentially bind to tyrosinated tubulin, thus dynein-dynactin binds more readily to microtubules in the distal tip of the axon to initiate minus-end- (soma) directed runs^60,167. As another example, the kinesin-3 motor KIF1A preferentially binds to GDP-tubulin over GTP-tubulin; newly nucleated microtubule plus-ends, which contain GTP-tubulin, are enriched in presynaptic zones, facilitating KIF1A detachment from microtubules in this region^134,139.

The related ‘MAP code’ refers to the differential binding of MAPs to the microtubule track, which can likewise affect motor binding (recently reviewed^61,135). In neurons, different subcellular compartments develop distinct sets of MAPs and PTMs to differentially direct motor activity. For instance, MAPs associated with the dendrite (DCX, DCKL1, MAP9, SEPT9) specifically inhibit kinesin-1 but have no effect on kinesin-3 transport, causing kinesin-1 to preferentially enter the axon as compared to the dendrites, while kinesin-3 localizes to both compartments^37,61,171. Thus, microtubules and MAPs regulate run initiation and termination to direct specific activity and localization of motors.

Disease-causing mutations can disrupt microtubule organization, PTMs, or dynamics, thus affecting axonal transport. Mutations in tubulin itself cause severe brain malformations, although studying their mechanistic roles are difficult; at least some are known to directly affect motor binding¹⁷². Mutations in the microtubule-severing protein spastin cause HSP; many spastin mutations are loss-of-function, and spastin-depleted mice demonstrate longer microtubules with more polyglutamylation, which disrupts KIF5C transport¹⁷³. The mice also have fewer synapses, which may be due to reduced GTP-tubulin and/or increased polyglutamylation along the lattice preventing KIF1A delivery of synaptic cargo to en passant synapses^134,173,174. Disease-associated arginine-rich dipeptide repeats (DPRs) generated from ALS-causing mutations in the C9orf72 gene decrease microtubule-based motility by forming “roadblocks” along the microtubule lattice¹⁷⁵. In vitro, the positively-charged DPRs bind robustly to the negatively-charged C-terminal tails of tubulin along the microtubule lattice preventing motor binding, which results in reduced transport of a range of cargos inside neurons¹⁷⁵. Motor proteins are thus self-regulated, regulated by their cargo, and by the tracks along which they travel. Further, disruptions at any of these levels can cause loss of or aberrant axonal transport and severe neurological disorders.

Activation of kinesin by unfolding.

Kinesin-1 autoinhibition is mediated by two primary interactions: the KHC tail folds onto the KHC motor domain to block interaction with the microtubule, and addition of KLC leads to further separation of the KHC motor domains in the inactive conformation⁴⁶ (Fig. 2 a). Many kinesin-1 activating proteins bind to the tetratricopeptide repeats (TPR) or the adjacent leucine-phenylalanine-proline (LFP) motif of KLC; this binding destabilizes the interaction between KLC and the KHC motor heads partially relieving autoinhibition^48,51,52,64. Many activators also bind the exposed KHC tail destabilizing its interaction with the KHC motor domains, thus freeing the motor domain to bind the microtubule^52,64,65 (Fig. 2 a).

Kinesin-3 is autoinhibited when its neck or stalk region bind to its motor domains^47,66 (Fig. 2 b). Kinesin-3 activating proteins bind to the heavy chain stalk region, which destabilizes the interaction with the motor domains, freeing them to bind and transit along the microtubule^66,67 (Fig. 2 b). Kinesin-3 and several other kinesins are additionally regulated by kinesin binding protein (KBP; also known as KIF1BP), which binds the motor domain through TPR motifs to sterically block microtubule attachment, akin to KLC^68,69 (Fig. 2 b). KBP phosphorylation enhances its interaction with kinesins, so kinesin-3 activation may require local phosphatase activity⁶⁸.

Most kinesins bind their cargo through light chains or an activating protein, although some can directly bind cargo membranes through interactions mediated by their PH/PX domains^52,70,71. There is an emerging role for small GTPases in motor recruitment to cargo. Many GTPases, including Rabs, are recruited to cargo by a membrane-associated guanine nucleotide exchange factor (GEF) where they in turn recruit motor proteins^72–74. Most small GTPases recruit motor proteins indirectly through interaction with a motor regulatory protein; however, some GTPases, such as Arl8 binding to kinesin-3, can directly activate motor proteins by relieving autoinhibition^75–77 (Fig. 2, 4).

Fig. 4: Many motor effectors induce both anterograde and retrograde transport.

Some effectors, e.g. TRAK2 (a) and Hook3 (b), can simultaneously complex with dynein-dynactin and kinesin to coordinate the bidirectional transport of organelles. For at least TRAK2, processive unidirectional transport actually requires the formation of these multi-motor complexes. Other effectors, e.g. JIP1, drive differential transport depending upon phosphorylation status. (c) Dephosphorylated JIP1 induces inhibition of KIF5C facilitating retrograde transport; although JIP1 directly binds dynactin p150^Glued it is unknown whether it activates dynein-dynactin transport. (d) JIP1 phosphorylation at site S421 by JNK induces activation of KIF5C and anterograde transport of cargo. Some effectors, e.g. HAP1, activate dynein or kinesin motility depending upon interactions with other proteins. (e) HAP1 activates dynein-dynactin motility in coordination with its partner Htt, which binds both HAP1 and DIC. (f) HAP1 complexes with GRIP1 to relieve KIF5C autoinhibition. Note that the stoichiometry of the interactions displayed here are not all known.

Activation of dynein by conformational change and complex assembly.

Dynein autoinhibition and activation (reviewed recently⁴¹) are even more complicated. Dynein alone stochastically switches between two inactive conformations: an autoinhibited conformation known as phi wherein the stalk domains, AAA+ rings, and N-terminal linkers interact, and an open conformation wherein the free motor domains are available but unaligned for processive motility^45,78 (Fig. 2 c). Lissencephaly 1 (LIS1) binds to and stabilizes the open conformation, allowing the dynactin complex to bind and orient the dynein motor domains into a parallel conformation that permits more efficient stepping along the microtubule^78–81 (Fig. 2 c). The addition of a coiled-coil (CC)-containing dynein activating adaptor stabilizes the dynein-dynactin interaction by binding both dynein (via interactions with DLIC) and dynactin (via interactions with the Arp1 pointed end and/or p150Glued) and promoting a rigid parallel conformation^{49,50,82–88}. Dynein activating adaptors are relatively cargo-specific, linking the motor complex either directly or indirectly to the organelle³⁹. Some activating adaptors induce the recruitment of a second dynein dimer to the complex to induce greater velocity, processivity, and force production by the macro-motor complex (Fig. 2 c); thus dynein motile behavior can be tuned for the specific organelle’s transport needs^83,84,89,90.

Coordination of opposing motors.

The opposing activities of kinesin and dynein motors must be modulated to achieve rapid, processive transport along the axon (Fig. 3). The net directionality of transport is determined by the relationship between plus- and minus-end directed motors on a cargo. This relationship may be competitive. For example, dynein, either alone or in complex with dynactin, is incapable of outcompeting kinesin-1 in a tug-of-war; however, the addition of activating adaptors that induces the binding of a second dynein dimer allows dynein to ‘win’^79,84,91. These larger complexes exhibit higher forces and slower load-dependent detachment kinetics, indicating these factors are particularly important in competition^84,91. In microtubule gliding assays, the number of active kinesin or dynein motors engaging a single microtubule had the largest impact on the net directionality, while ATP concentration, MAPs on the microtubule, and stepping kinetics had little impact⁹². Dynein also appears to cluster in teams on membranes; for example, multiple dynein complexes cluster in cholesterol-rich microdomains on the surface of phagosomes leading to large force production and retrograde transport^93,94.

Fig. 3: Models of inter-motor coordination.

To transport cargo unidirectionally along microtubules, opposing motors must be coordinated. Three main models for this coordination have been proposed. Selective recruitment entails minus- or plus-end-directed motors being recruited to a given cargo separately, such that only motors the move in the desired direction are recruited to cargo. Testing of this model in vitro yields cargo that move at a similar velocity to that seen in cells and in vivo. However, organelles pulled out from the brain copurify with both direction motors, suggesting association of one motor type at a time is nonphysiological. The tug-of-war model posits that opposite direction motors bound to a single cargo compete to pull the cargo in their direction of movement. Generally a single kinesin can out-compete a single dynein dimer, but the formation of a dynein tetramer and/or dynein “teams” (multiple dynein complexes) allows dynein to “win.” While this fits the finding that cargo bind both direction motors simultaneously, the velocities seen in tug-of-war experiments are much slower than what is seen in cells, presumably due to the force produced in the non-motile direction. The selective activation model, wherein both direction motors are present on cargo but they are differentially activated to induce unidirectional movement, is thus the most accurate.

Although the tug-of-war model is appealing, and straightforward to model both in vitro and in cells, this model has important limitations in advancing our understanding of cargo transport in vivo. For example, because of the opposing force generated by the ‘loser’ motor, the velocities observed in tug-of-war assays are usually slower than those seen for endogenous cargos moving in vivo, suggesting that this model may not satisfactorily explain the rapid organelle transport observed in cells (Fig. 3)⁹⁵. Instead, as more studies are performed either in live cell assays, or in vitro using motility assays with more physiological motor complexes, accumulating evidence points towards models of selective activation and motor interdependency^95–97 (Fig. 3). Efficient unidirectional transport may actually require both motors⁹⁸. At a mechanistic level, many motor adaptor and/or activator proteins bind both kinesin and dynein (Fig. 4). Some bind opposing motors simultaneously to coordinate transport (Fig. 4, a–c), while others bind opposing motors in a mutually exclusive fashion whereby their motor binding state can be regulated by upstream effectors^57,67,88,99 (Fig. 4, e–f). Our understanding of these diverse motor regulatory mechanisms has substantially expanded in recent years, emphasizing the importance of distinct regulatory mechanisms specific to individual cellular cargos.

Cargo-specific regulation of axonal transport

The efficient delivery of cargo into and out of the axon is a particularly complex task, with the axon being several orders of magnitude longer than the soma or other cells. Initiation, continuance, and cessation of axonal organelle motility must be tightly regulated to develop and sustain presynaptic regions and maintain local energy levels and degradative capacity. Here we highlight mechanisms of transport—from relief of motor autoinhibition to upstream regulation by kinases—that have been illuminated by recent progress in axonal transport research.

Mitochondrial trafficking.

Axons and presynaptic sites have high metabolic demands and require precise regulation of calcium ions, necessitating the highly regulated trafficking of mitochondria¹⁰⁰. Microtubule motors are scaffolded on mitochondria by interactions between Miro proteins and the motor-binding proteins TRAKs and metaxins (MTX)^97,101 (Fig. 5 a). Miro proteins, Ca²⁺-sensitive GTPases embedded in the mitochondrial outer membrane, mediate both long-distance microtubule-based transport of mitochondria and anchoring of mitochondria to the cytoskeleton, where they can generate high local ATP levels¹⁰². TRAK1/2 (Milton in Drosophila) are canonical CC-enriched dynein activating adaptors, which bind DLIC helix-1 via a CC1 box motif, dynactin pointed end complex via a spindly motif, and dynactin p150^Glued via a Glued motif^50,88,97. They also bind to kinesin-1 heavy chain using their N-terminal CC domain; however the binding to opposing motors does not appear to be mutually exclusive^97,103 Both TRAKs have been shown to activate kinesin and dynein motility in vitro^{88,97,104,105}. TRAK1/2 can form co-complexes with both dynein-dynactin and kinesin; in a minimal recombinant model, TRAK1/2 complexes containing both motors move only in the anterograde direction, but in lysate-based in vitro experiments TRAK2 activation of either kinesin or dynein is dependent upon the presence of the opposing motor, indicating cooperativity between the motors (Fig. 4 a)^97,105. TRAK1/2 knockdown in neurons led to a loss of mitochondrial motility in both the anterograde and retrograde direction¹⁰¹. By contrast, MTX-1/2 knockdown disrupted mitochondrial motility almost exclusively in the anterograde direction, consistent with MTX1/2 interaction with KLC-1¹⁰¹. This interaction presumably relieves kinesin inhibition, but the mechanism by which MTX1/2 affects anterograde transport is not yet known.

Fig. 5: Axonal transport of mitochondria, endolysosomes, and synaptic components.

(a) The anterograde and retrograde transport of mitochondria is controlled by interactions between th small GTPase Miro and the motor binding proteins TRAK1/2 and Metaxin1/2. (b) Early endosomal transport is mediated by the motor activators Hook1/3 binding to dynein-dynactin and kinesin-3 (KIF1C). Late endosome (c) and lysosome (d) anterograde transport are both driven by the KIF5-SKIP-Arl8b-BORC complex. Late endosome (c) retrograde transport involves RILP, but the actual mechanism of dynein activation is not known. Synaptic vesicle precursor (SVP) and dense core vesicle (DCV) anterograde transport are both driven by kinesin-3. The primary regulators of kinesin-3 on SVPs are MADD and Rab3 (e), while kinesin-3 on DCVs (f) is regulated by Calmodulin, Arl8a, PTPN21, and/or Hook3. DCV retrograde transport is likely scaffolded by Hook3. (g) APP-containing vesicles are transported anterogradely by KIF5, scaffolded either by phosphorylated JIP1 and/or phosphorylated Htt. Note that multiple motor complexes may be simultaneously bound to a single cargo, and that the interplay between motor complexes is not well understood.

Cessation of mitochondrial transport is equally important for local function in presynapses and other locations. Mitochondria are locally anchored through associations with either the microtubule or actin cytoskeleton^106,107. For example, high local glucose concentrations induce O-GlcNAcylation of TRAK1/2, which induces TRAK1 binding to the actin-binding protein FHL2 and subsequent halting¹⁰⁸. At presynapses, microtubule-bound syntaphilin can halt mitochondrial transport either through association with the mitochondrial outer membrane and/or by sterically blocking kinesin binding to the microtubule^106,109. Miro binding to Ca²⁺ also reduces mitochondrial transport, but there is controversy over how this effect is mediated¹¹⁰. While the mechanisms underlying both mitochondrial transport and anchoring are still being determined, the dynamics of mitochondrial motility are key to meeting local energy demands.

Endosomal motility.

Axonal endolysosomes are important for a variety of functions including degradation, protein sorting, and even local translation^57,111,112. The bidirectional transport of endolysosomal vesicles in axons differs based upon the organelle’s maturation state. Nondegradative EEA1- and Rab5-labelled early endosomes mature into Rab7-labelled late endosomes which further mature into highly acidified and degradative LAMP-labelled lysosomes. In both nonneuronal and neuronal cells, a gradient can be observed whereby endolysosomes in the perinuclear or somatic region are more degradatively competent than those in the cell periphery or distal processes^113,114.

It is somewhat unclear whether EEA1-positive early endosomes localize to axons. Their transport is controlled by a direct interaction between phosphatidylinositol 3-phosphate, generated by the Rab5 effector Vps34, and the kinesin-3 KIF16B¹¹⁵. KIF16B and EEA1 are enriched in the somatodendritic region and excluded from the axon, likely due to autoinhibition of KIF16B²⁷. However, Rab5-containing early endosomes do localize to axons, possibly generated in the axon by endocytosis¹¹⁶. The retrograde transport of Rab5-containing endosomes is regulated by Hook1-dynein-dynactin (Fig. 5 b), which is recruited to microtubule plus-ends to initiate transport by an indirect interaction between dynactin and EB1 through the protein STIM1^116,117. STIM1 is expressed in neurons but this mechanism has not been confirmed therein¹¹⁸. Hooks are dynein activating adaptors that bind DLIC through their N-terminal Hook domain and dynactin through their spindly and Glued motifs^49,86,88,117 (Fig. 2 c). The Hooks are recruited to the endosomal membrane by their C-terminal cargo binding domain, likely via interaction with Fused Toes (FTS) and FTS-Hook-interacting protein (FHIP) 1B, which in turn bind Rab5-GTP (Fig. 5 b)^112,119. Rab5-containing vesicles also move towards the distal tip in axons, although the mechanism is not known¹¹⁶. In vitro Hook3 can form a co-complex with both dynein-dynactin and the kinesin-3 motor KIF1C and can activate transport in both directions (Fig. 4 b), thus Hook3 could activate both the anterograde and retrograde motility of endosomes^67,120.

Rab7-positive late endosomes are present in axons and traffic primarily in the retrograde direction^121,122 (Fig. 5 c). Dynein is recruited to Rab7-containing late endosomes via interaction with RILP, which specifically interacts with GTP-Rab7 on membranes^73,117,121. Like early endosomes, the RILP-dynein-dynactin complex is recruited to microtubule plus-ends by STIM1 in non-neuronal cells¹¹⁷. Despite structural similarities with other dynein activators, RILP has not been shown to activate dynein motility in vitro or in cells. In zebrafish axons, a subset of Rab7 retrograde endosomes are regulated by the transmembrane scaffolding protein vezatin, possibly functioning through a Hook protein¹²³.

Late endosomal anterograde motility is mediated by kinesin-1, scaffolded by the SKIP-Arl8b-BORC effector complex^73,124. In C. elegans, the multi-subunit BORC complex recruits the small GTPase Arl8 to lysosomal membranes because its subunit SAM-4 functions as an Arl8 guanine nucleotide exchange factor (GEF)⁷⁴. The SAM-4 mammalian ortholog is BORCS5/Myrlysin; however, recombinant mammalian BORC complex was unable to induce Arl8b nucleotide exchange in vitro¹²⁵. GTP-Arl8 binds autoinhibited SKIP to relieve its head-tail autoinhibition⁷⁰ (Fig. 2 a). The conserved acidic tryptophan motif in SKIP then binds the LFP motif of KLC and the tail domain of KHC to relieve kinesin autoinhibition and activate transport^48,52 (Fig. 2 a). The number of anterograde-moving late endosomes seems limited by quantifying Rab7; however, non-neuronal work has shown that Arl8b-SKIP localization on late endosomal membranes recruits the Rab7 GTPase-activating protein (GAP) TBC1D15 (Fig. 5 c), which induces Rab7 GTP hydrolysis and subsequent release of Rab7-GDP into the cytosol⁷³. Thus, there may be an undetected population of Rab7-negative late endosomes in axons moving predominantly anterograde. Thus, the diverse modes of transit for endosomes continues to expand as we discover even greater diversity in the endosomal population.

Lysosomal trafficking.

There is some disagreement as to whether mature degradative lysosomes are present in the axon. In C. elegans DA9 neurons, lysosomes are excluded from the axon, although the mechanism is unknown⁷⁴. In mammalian neurons, some LAMP1-labeled axonal vesicles are unacidified transport carrier vesicles derived from the trans Golgi network; these vesicles move anterograde in the axon, driven by an unknown kinesin, and fuse with endosomes and autophagosomes to supply maturation factors to these compartments¹²². Additionally, a subpopulation of fully mature lysosomes with low pH and active degradative enzymes have also been observed in mammalian axons; these lysosomes are delivered into the axon by the same kinesin-Arl8b-SKIP complex (Fig. 2 a, 5 d) that mediates late endosomal transport^4,124,126. In nonneuronal cells, lysosomal transport can also be driven by kinesin-3-Arl8b-BORC, although that transport is SKIP-independent; instead Arl8b binds directly to the stalk region of kinesin-3 to relieve its autoinhibition^66,75. There is some evidence for kinesin-3 activation of lysosomal motility in neurons, although it appears to be dependent upon Arl8a rather than Arl8b³³.

The BORC complex and/or its related complex BLOC appears to be involved in not only the anterograde transport, but also the retrograde transport of lysosomes (and endosomes)^127,128. The BORC/BLOC subunit SNAPIN binds the middle domain of DIC using a series of nonpolar residues near its C-terminus; however, given its size and specific interaction with DIC, it seems likely SNAPIN serves only as an adaptor rather than an activator of dynein^127,128. In nonneuronal cells, the minus-end-directed transport of dynein is driven by the motor regulator JIP4 binding to dynein-dynactin and the lysosomal membrane protein TMEM55B^71,77,129. Although this has not been directly tested in neurons, the loss of both JIP4 and its related protein JIP3 leads to the accumulation of lysosomes in axons, suggesting a block in transport^130,131. In zebrafish axons, the retrograde transport of LAMP1-containing vesicles requires JIP3¹³². JIP3/4 function on retrograde lysosomes requires the small GTPase Arf6: specifically, JIP3/4 binding to GTP-ARF6 shifts JIP3/4 from interacting with kinesin to interacting with dynein^77,132. It is not known whether or how JIP3/4 directly activates dynein motility. Maintaining bidirectional trafficking of lysosomal structures is key to maintaining the degradative capacity of the neuron.

Synaptic vesicle precursor trafficking.

Most synaptic proteins and vesicles are produced in the soma and transported into the axon using either slow or fast microtubule-based axonal transport^133,134. There are three partially overlapping classes of organelles transported from the soma to pre-synapses: (1) synaptic vesicle precursors (SVPs) which mature into the neurotransmitter-containing synaptic vesicles involved in canonical fast synaptic signaling; (2) dense core vesicles (DCVs) which release neuropeptides and hormones from synapses to exert temporally and spatially wider signals; and (3) organelles containing the active zone proteins that form the infrastructure of the presynapse¹³⁵. Proteins may also be translated locally for use at synapses, which requires the anterograde transport of mRNA and ribosomes from the soma to synapses; we will not discuss this transport here as it was recently reviewed elsewhere¹³⁶.

SVPs move predominantly anterograde, as evidenced by their predominant accumulation on the somal (proximal) side of a dissected nerve¹³⁷. The anterograde transport of SVPs is primarily facilitated by the kinesin-3 KIF1A¹³⁸ (Fig. 5 e). KIF1A facilitates the transport of SVPs into axons and specifically delivers them to presynaptic sites by sensing the nucleotide state of the microtubule array¹³⁴. Newly nucleated microtubules and microtubule plus-ends, which contain GTP-tubulin, are enriched in both the axon tip and en passant synapses (Fig. 1)^134,139. In vitro KIF1A binds more weakly to GTP-tubulin-containing lattices than to GDP-tubulin lattices facilitating microtubule detachment and SVP delivery to GTP-tubulin-enriched synapses¹³⁴.

In C. elegans, kinesin-3 (UNC-104) can directly bind to the SVP membrane through its PH domain¹⁴⁰. Kinesin-3 can also be scaffolded on SVPs by the Arl8-BORC complex, the same complex that moves lysosomes into axons in mammalian cells⁷⁴. This led to the hypothesis that SVPs may in fact be transported with and/or as part of lysosomes in axons. In C. elegans and Drosophila, the majority of SVPs and active zone proteins cotransport with lysosomal markers in an Arl8-dependent fashion^141,142. However, in mammalian cells this cotransport is contested: as much as 85% and as little as 20% comigration has been observed between lysosomal and synaptic structures in the same mammalian cell type^76,141. Furthermore, in mammalian axons SVP transport does not seem to rely on Arl8 scaffolding⁷⁶. The GEF MADD (also known as DENN) activates the small GTPase Rab3 on SVP membranes and also directly binds the KIF1A CC3 domain^33,143 (Fig. 2 b). Additionally, the bidirectional motor scaffold JIP3 could play a role, as C. elegans null mutants for its ortholog (UNC-16) show disruption in the anterograde transport of SVPs¹⁴⁴. While JIP3 activation of kinesin-3 (KIF1A) is not known, JIP3 is known to activate kinesin-1 by binding both the TPR domain of KLC and the tail domain of KIF5C to relieve autoinhibition^{51,64,65,71,145}. More studies, especially in mammalian neurons, are necessary to settle the many debates over SVP transport.

Dense core vesicle transport.

DCVs exhibit an unusual method of transport known as circulation and sporadic capture. Rather than being delivered by kinesins directly from the soma to the numerous presynapses, DCVs are delivered initially all the way to the axon tip, with some stopping to localize at en passant synapses as they pass; at the tip, excess DCVs switch to retrograde transport, returning to the axon initial segment where they again switch to anterograde transport^146,147. This phenomenon has been best studied in Drosophila but appears to be conserved in mammalian neurons^146,147. DCV anterograde transport is driven by KIF1A, which is activated either by Arl8a binding to its CC3 domain and/or Ca²⁺-dependent binding of calmodulin to the region between its CC2 and CC3^33,148 (Fig. 5 f). Ca²⁺-dependent binding of calmodulin to KIF1A activates the motor both by changing the conformation of the tail domains and possibly by inducing Ca²⁺ and calmodulin-dependent kinase II phosphorylation of the PH domain, which increases KIF1A affinity for DCV and SVP membranes^33,148. It is not known whether Arl8 and calmodulin can bind KIF1A simultaneously.

Fellow kinesin-3 KIF1C has also been shown to drive anterograde transport of DCVs²⁸, although the regulation is wholly different. KIF1C is autoinhibited by interactions between its stalk region and its microtubule-binding domain, and autoinhibition is relieved upon binding to the phosphatase PTPN21 and/or the motor adaptor Hook3^67,120 (Fig. 5 f). Because Hook3 is also known to activate dynein-dynactin motility, it is possibly responsible for retrograde transport of DCVs in axons, although the actual mechanism of DCV retrograde transport is unknown^67,82. Rab2 loss in Drosophila causes a loss of bidirectional DCV transport, with a particularly strong phenotype in the retrograde direction, thus perhaps Rab2 is involved in recruiting dynein to the DCV membrane⁷² (Fig. 5 f).

Importantly, there appear to be several methods by which transport is halted at synapses to facilitate the retention of DCVs at these sites. In addition to KIF1A’s reduced affinity for GTP-enriched microtubule plus-ends at presynapses, KIF1A-dependent capture of DCVs can also be regulated by neuronal activity through a phosphorylation dependent event¹⁴⁷. Neuronal activity increases JNK activity in synapses, leading JNK to phosphorylate the active zone protein synaptotagmin-4 (Syt4), which cotraffics with DCVs¹⁴⁷. Activity-dependent phosphorylation of Syt4 at site S135 disrupts the direct interaction between Syt4 and KIF1A, halting the transport of DCVs at sites of neuronal activity¹⁴⁷. It is worth noting that the direct interaction between Syt4 and KIF1A also appears to be modulated by and/or downstream of the Ca²⁺-dependent binding of calmodulin to KIF1A¹⁴⁸. DCV capture at synapses may additionally rely on the direct binding between KIF1A and the active zone protein liprin-α; however whether this mechanism depends on liprin-α activation of KIF1A or on liprin-α anchoring KIF1A to actin to stop motility is unclear^148,149. In conclusion, motor inactivation may be as important as activation to facilitate delivery to presynaptic sites.

Delivery of other synaptic components.

Many active zone proteins appear to be transported in concert with SVPs or DCVs, as mentioned above. However, they can also be transported independently, for example as Piccolo-Bassoon transport vesicles³⁴. At least one active zone protein liprin-α interacts directly with and may activate KIF1A, binding to the region between its CC2 and CC3 domains^148,149. The active zone protein syntaxin-1 indirectly interacts with KIF5B through the kinesin adaptors FEZ1 and/or syntabulin, although neither adaptor has been shown to independently activate kinesin motility^34,150,151. Additionally, the microtubule associated protein MARK/Par-1 and the small GTPase Rab2 appear to play a role in the axonal transport of active zone proteins in Drosophila, although the specific mechanisms are unknown^72,152.

Amyloid precursor protein (APP) is also necessary for presynaptic homeostasis and is transported with an anterograde bias in axons^53,56. APP transport relies on the phosphorylation state of two motor scaffolding proteins, JIP1 and Huntingtin (Htt). JIP1 phosphorylation at S421 by JNK induces preferential binding to kinesin-1 over dynein, and Htt phosphorylation at S421 by Akt promotes binding to kinesin-1, both events favoring the anterograde transport of APP-containing vesicles^53,56 (Fig. 4 c). Phospho-JIP1 activates kinesin-1 motility by relieving both KLC-TPR mediated autoinhibition and head-tail autoinhibition^56,64, but the mechanism by which phospho-Htt affects kinesin motility is unclear⁵⁴.

Transport of autophagic vacuoles.

Because neurons are postmitotic and long-lived, protein and organelle turnover and recycling are essential to maintain homeostasis. One degradative pathway occurring continuously in axons is autophagy; autophagosomes engulf proteins and organelles in the distal axon and fuse with late endosomes and lysosomes during transport to the soma to degrade their cargo¹⁸. Despite trafficking continuously in the retrograde direction, autophagic vacuoles require distinct dynein regulatory proteins depending upon their subaxonal location, presumably because the external membrane and associated proteins change as the autophagic vacuole matures⁸⁸ (Fig. 6 a). An early maturation step involves the cleavage of LC3 and other Atg8 orthologs from the external surface; hence only the first autophagosomal motor regulator JIP1 binds the autophagosome via LC3¹⁵³ (Fig. 6 a). JIP1 specifically inactivates kinesin-1 when bound to LC3 (Fig. 4 c), thus allowing the initiation of retrograde transport from the distal axon^153,88. Subsequently, dynein scaffolding on autophagosomes in the mid-axon is controlled by the scaffolding protein Htt and its interacting protein HAP1⁸⁸ (Fig. 6 a). HAP1 is a dynein activating adaptor that binds DLIC using its CC1 box motif and dynactin via the spindly and Glued motifs to induce formation of a motile complex^85,88. Htt links the dynein-dynactin-HAP1 complex to the autophagosomal membrane, presumably through putative interactions with lysosomal membrane proteins (e.g. Vps35, multiple vATPase subunits)^154,155 (Fig. 4 e). Interestingly, HAP1 in complex with a different scaffold protein, GRIP1, can activate kinesin-1 motility; this is required for the transport of neurotransmitter receptors into dendrites^99,156 (Fig. 4 f).

Fig. 6: Axonal transport of autophagic vacuoles and signaling endosomes.

Autophagic vacuoles and signaling endosomes both move retrograde through the axon, maturing during transit either through fusion or Rab conversion. During this maturation, it is known (a) or proposed (b) that multiple dynein effectors sequentially drive transport. (a) Autophagic vacuole transport is initiated by JIP1 inactivating kinesin and driven along the mid-axon by HAP1 activating dynein. Dynein is scaffolded on mature autolysosomes by JIP3, although whether and how JIP3 activates dynein is unknown. (b) Nascent signaling endosomes are formed at presynaptic sites following neurotrophin (e.g. BDNF) binding to a receptor (e.g. TrkB, p75). This binding induces local calcium signaling and BDNF phosphorylation, which may induce calcineurin to dephosphorylate the motor scaffold Htt. Dephosphorylated Htt binds less kinesin, potentially helping initiate retrograde transport. Dynein activator HAP1 has also been detected colocalizing with signaling endosomes, thus it may activate their retrograde transport. Hook1 activates dynein on signaling endosomes in the distal and mid-axon, which may involve the motor scaffold vezatin. BICD1 in coordination with ESCRT-interacting protein PTPN23 appears to regulate the transit of signaling endosomes, possibly during sorting for recycling or degradation. Note that multiple motor complexes may be simultaneously bound to a single cargo, and that the interplay between motor complexes is not well understood.

Finally, mature degradative autolysosomes in the mid- and proximal axon require the bidirectional motor scaffold JIP3 to continue motility near the soma⁸⁸ (Fig. 6 a). JIP3 is required for autophagic vacuole transport in both C. elegans and mammalian neurons, and can bind both kinesin and dynein-dynactin; however, the mechanism by which it drives retrograde transport is not yet known^51,71,88,157. It may interact with the autolysosome membrane via its binding partner and small GTPase Arf6, which is important for autophagosomal trafficking in zebrafish^77,158. The function of JIP3 on autolysosomes and the association of JIP1 and HAP1 with the autophagic vacuole membrane is dependent upon the organelle’s maturation state⁸⁸. Further, the function of all three effectors is directly or indirectly regulated by phosphorylation: JIP1 dephosphorylation by MKP1 phosphatase induces it to inactivate kinesin^56,153 (Fig 4 c); the HAP1 effector Htt’s dephosphorylation by calcineurin leads to less interaction with kinesin^54,55; and JIP3 may be downstream of LRRK2 phosphorylation of Rab GTPases on the autolysosomal membrane⁵⁷. Additional proteins including SNAPIN and RILP have been suggested to regulate dynein on axonal autophagic vacuoles, but their specific roles in transport are poorly understood^121,159. The tightly regulated handoff between motor effector proteins on a single cargo during unidirectional transport has so far only been observed for autophagic vacuoles, but may be relevant for other cargo that mature or traverse long distances.

Signaling endosome transport.

Nascent Rab5-positive signaling endosomes form in the distal axon by endocytosing activated neurotrophin receptors and their bound ligands, which are then transported to the soma to modulate transcription. Like autophagosomes, signaling endosomes mature in the axon by acquiring Rab7, and this maturation is important for their retrograde transport¹⁶⁰. Multiple dynein regulatory proteins have been implicated in the transport of signaling endosomes, thus we predict they may use multiple sequential motor effectors to scaffold their long unidirectional transport. However, the different motor effectors may instead be specific for different types of signaling endosomes, for example TrkB receptor-containing vs. p75 receptor-containing, although recent work suggests these neurotrophin receptors are internalized and possibly trafficked together¹⁶¹.

The earliest step in retrograde signaling endosome transport may be initiated by local calcium signaling triggered by neurotrophins binding to receptors at the presynapse⁵⁵. This local increase in calcium triggers the activity of the phosphatase calcineurin, which seems to dephosphorylate S421 of Htt, inducing less kinesin recruitment to the newly formed endosome⁵⁵ (Fig. 6 b). Htt interactor HAP1 also localizes to neurotrophin receptors upon neurotrophin binding and appears to be required for their signaling to the nucleus¹⁶². Thus, HAP1 may activate dynein on signaling endosomes⁸⁸. Hook1 has been shown to drive the retrograde motility of both Rab5- and Rab7-positive signaling endosomes in the distal and mid-axon, where it likely binds via FTS-FHIP1B-Rab5^112,116,119 (Fig. 6 b). In Zebrafish or Drosophila neurons, loss of the protein vezatin or vezatin-like (Vezl) inhibits the transport of signaling endosomes along the mid-axon; the mechanism by which vezatin/Vezl controls motility is unknown, but it may be Hook-dependent^123,163.

Additionally, BICD1, a dynein activating adaptor that binds dynein-dynactin through a CC1 box and a spindly motif, was shown to be involved in the axonal transport of p75-containing signaling endosomes, although the subaxonal specificity was not examined^85,86,164. BICD1 appears to be recruited to signaling endosomal compartments via interaction with PTPN23, an ESCRT-interacting protein¹⁶⁴. Interestingly, loss of PTPN23 led to buildup of neurotrophin receptors in vacuoles where they became highly ubiquitinated, suggesting perhaps BICD1 plays a role at later stages of the signaling endosome pathway when the neurotrophins and their receptors are sorted for recycling or degradation^164,165. The scaffold SNAPIN may also help recruit dynein to signaling endosomes¹⁶⁶. Despite the many motor regulators involved, it remains to be seen whether individual signaling endosomes actually cycle between different motor effector proteins depending upon their localization, contents, and maturity.

Conclusion

Properly orchestrated organellar transport in axons is integral for neuronal development and homeostasis, and dysfunction at any level can result in neurodevelopmental or neurodegenerative disease (see Box 2). The tight regulation of microtubule motors not only via autoinhibition but also via cargo interaction is key to organizing the cytoplasm, especially in cells as complex as neurons. Between high resolution structural studies of purified complexes, high temporal resolution imaging, and in vitro reconstituted motility, we have made massive strides in recent years understanding the regulation of fast microtubule-based motility. The next stage of understanding axonal transport is just beginning. For every cargo examined here, multiple motor-containing complexes have been identified: further investigations of the interplay between these complexes should be undertaken. For example, sequential motor activation schemes as observed for autophagic vacuoles⁸⁸ likely exist for other cargos that mature or traverse long distances.

Box 2: Dysfunction of axonal trafficking.

Disruption of axonal transport at either the level of the motor proteins themselves or their regulators can result in disease; here we highlight here just a few examples. Mutations in the gene encoding DHC (DYNC1H1) lead to cortical malformations, muscle weakness, and/or neurodegeneration^176,177. Very few disease-causing mutations are seen in the AAA+ motor domain, likely because they would be lethal early in development, due to essential roles for cytoplasmic dynein in cell division and cell polarity¹⁷⁷. Neurodegeneration-causing mutations, such as those seen in Spinal Muscular Atrophy with lower extremity predominance (SMA-LED), are primarily located in the tail, which affects dimerization and complex formation with other dynein subunits, dynactin, and cargo adaptors^176,177. Lissencephaly, a specific form of cortical malformation, can be caused by mutations in the dynein regulatory protein LIS1, which is important for relieving dynein autoinhibition⁷⁸.

Disease-causing mutations in the kinesin-1 KIF5A are mostly in the motor domain, where they primarily affect ATPase activity and microtubule binding¹⁷⁸. A newly identified group of mutations in the cargo-binding domain cause amyotrophic lateral sclerosis, which appear to result in a truncated protein unable to bind cargo^179,180. Mutations in the kinesin-3 KIF1A result in progressive juvenile neurodegeneration¹⁸¹. The specific symptoms vary based upon the location of the mutation, most of which are clustered in the motor domain, although some mutations have been identified in the protein C-terminus¹⁸¹. Many KIF1A mutations are loss-of-function, showing less ATPase activity, microtubule interaction, or cargo binding^182,183. Others can be classified as gain-of-function, leading to increased microtubule binding or motor hyperactivity^134,181,184. Rare missense mutations in the kinesin-4 KIF21B are also gain-of-function and seem to disrupt normal autoinhibition, leading to hyperactive KIF21B and subsequent neuronal migration and neurodevelopmental phenotypes, including microcephaly¹⁸⁵.

Mutations in cargo adaptor proteins and their upstream regulatory proteins may lead to more specific disruptions in the axonal transport of particular organelles. Mutations in the scaffolding protein Htt, which interacts with the dynein activating adaptor HAP1, disrupt the axonal transport of autophagosomes, which inhibits protein and organelle turnover and possibly contributes to the development of Huntington’s disease^88,155. Autophagosome motility can also be disrupted by Parkinson’s disease-causing mutations in LRRK2 kinase; LRRK2 mutants are hyperactive leading to increased phosphorylation of Rabs, including Rab35 and Rab10^57,186. Rab hyperphosphorylation appears to increase autophagosomal membrane localization, which increases the recruitment of the putative kinesin activator JIP4 to the autophagic vacuole membrane⁵⁷. The increased kinesin activation on the autophagosomal membrane then presumably reduces autophagosomal axonal transport through aberrant competition with dynein⁵⁷. Mutations in the lysosomal membrane proteins NPC1/2 lead to a childhood neurodegenerative disorder called Niemann-Pick disease type C, a lysosomal storage disorder¹⁸⁷. Loss of NPC1/2 leads to excess lipids, especially cholesterol, accumulating on the lysosomal membrane, which impairs axonal lysosome transport either due to increased dynein clustering driving lysosomes to remain at the microtubule minus-ends in the soma and/or reduced interaction of kinesin-1 with its effector proteins on the lysosomal membrane^93,94,187. Although there are many other disease-causing mutations that affect axonal transport, these examples demonstrate the wide effects on transport induced by mutations in motor proteins and the relatively specific, though still devastating, effects of mutations in cargo adaptors and regulators.

Additionally, most cargo appear to bind both plus-end-directed and minus-end-directed motors, but still relatively little is known about how the motors are coordinated to drive unidirectional transport. Further motility and binding experiments are necessary to understand how motor activators simultaneously and/or exclusively interact with and regulate motors, especially in the larger cellular context wherein motors and activators are regulated by upstream kinases/phosphatases and small GTPases. As protein purification and in vitro reconstitution methods improve, we must also continually return to cell-based assays to identify the additional regulatory proteins and lipids used by diverse cellular cargo. Altogether, the recent results both in vitro and in vivo reveal distinct but comparable motor activation schemes on different cargo, whereby each cargo can be transported with specificity to its subcellular destination for function.

Glossary

Active zone

The region of the presynapse where synaptic vesicle fusion and neurotransmitter release occurs.

Akt kinase

Also known as protein kinase B, a family of serine/threonine kinases involved in cell survival, proliferation, and metabolism.

Amyloid precursor protein (APP)

A transmembrane protein enriched at synapses believed to be important for synaptic formation and plasticity. APP can be differentially cleaved and the cleavage product β-amyloid accumulates in neurodegenerative diseases including Alzheimer’s disease.

Amyotrophic lateral sclerosis (ALS)

A progressive nervous system disease resulting in muscle weakness and other motor symptoms. Causes vary and onset is typically between 40 and 65 years of age. Also known as Lou Gehrig’s disease.

Atg8

A family of ubiquitin-like proteins localized primarily to the autophagosomal membrane and necessary for both selective and bulk autophagy, autophagosome biogenesis, and autophagosome-lysosome fusion. LC3B is a well-characterized member of this family commonly used as a marker for autophagosomes in mammalian cells.

Axon initial segment

The short (20–60μm) region of axon immediately adjacent to the soma which acts as a selective filter to limit axonal transport and initiates action potentials (electrical signaling).

Calmodulin

A secondary messenger protein activated by the binding of Ca²⁺ involved in numerous cell signaling pathways.

Coiled-coil (CC)

Structural motif in which multiple alpha helices are coiled together to form a supercoil. CC domains are often stiff rods that function as molecular spacers.

Dynein activating adaptor

Dynein effectors that activate dynein motility and link dynein—directly or indirectly—to cargo. Most dynein activating adaptors contain an extended coiled-coil motif flanked by conserved motifs for dynein and dynactin binding.

en passant synapse

A presynapse located along the axon shaft.

Huntington’s disease

A neurodegenerative disease resulting from a polyglutamine expansion in the gene encoding huntingtin (Htt). Symptoms typically appear in early adulthood, include both motor and cognitive problems, and worsen over time.

JNK (c-Jun N-terminal Kinase)

A family of mitogen-activate protein (MAP) kinases that respond to stress stimuli and trigger signaling cascades implicated in inflammation, gene expression, DNA repair, neuronal plasticity, and cell death or senescence.

KIF1A-Associated Neurological Disorder

A severe progressive childhood neurodegenerative disorder resulting from primarily de novo mutations in the kinesin-3 KIF1A. The severity and symptoms—including epilepsy, cognitive problems, and muscle spasticity—vary depending on the specific mutation.

Lissencephaly

A neurodevelopmental disorder characterized by a “smooth brain” absent of normal cortical folds.

Lysosomal storage disorder

A class of inherited metabolic disorders wherein lysosomal degradation is defective. They can affect a range of tissues including the brain, eyes, muscles, and kidneys. Most patients develop symptoms during childhood and worsen over time.

Microcephaly

A birth defect wherein a baby’s head appears smaller than normal due to abnormal brain development.

Phox homology (PX) or pleckstrin homology (PH) domains

Lipid-binding domains that interact with phosphoinositides, facilitating membrane localization.

Piccolo-Bassoon transport vesicles

Trans-Golgi-derived vesicles that transport Piccolo and Bassoon, two large scaffolding proteins that help form the active zone, from the soma to the presynapses.

Post-synaptic terminal

The receiving side of a synapse where neurotransmitter receptors are located. Most post-synaptic sites are in the dendrites or soma of a neuron.

Pre-synaptic site

The signalling side of a synapse that releases neurotransmitters into the synaptic cleft. Most pre-synaptic sites are in the axon of a neuron.

Spinal muscular atrophy with lower extremity predominance (SMA-LED)

An inherited neuromuscular disorder characterized by muscle weakness and wasting in the lower limbs, which primarily appears in childhood.