Huntington's disease is caused by polyglutamine (polyQ) expansions in huntingtin (HTT). PolyQ lengths >35Q lead to neurodegeneration, and longer repeats correspond to earlier onset of symptoms. HTT scaffolds kinesin-1 and dynein to organelles directly and through adaptors. We tracked BDNF vesicles, mitochondria, and lysosomes in stem-cell-derived neurons engineered to express HTT with polyQ lengths of 30, 45, 65, and 81. BDNF endosomes were more motile in HTT-45Q and HTT-65Q neurons and misdirected toward the distal tip in HTT-81Q neurons. Under neuroinflammatory stress, polyQ expansions resulted in fewer BDNF cargoes and more lysosomes. We next isolated BDNF endosomes from neurons and counted the associated motors and adaptors. We found BDNF endosomes associated with greater numbers of kinesin-1 and HAP1 molecules in HTT-81Q neurons. Together, these results show that polyQ expansions in HTT alter the motors and adaptors recruited to cargoes, resulting in dysregulated transport and responses to neuroinflammatory stress.

Doublecortin is a neuronal microtubule-associated protein that regulates microtubule structure in neurons. Mutations in Doublecortin cause lissencephaly and subcortical band heterotopia by impairing neuronal migration. We use CRISPR/Cas9 to knock-out the Doublecortin gene in induced pluripotent stem cells and differentiate the cells into cortical neurons. DCX-KO neurons show reduced velocities of nuclear movements and an increased number of neurites early in neuronal development, consistent with previous findings. Neurite branching is regulated by a host of microtubule-associated proteins, as well as by microtubule polymerization dynamics. However, EB comet dynamics are unchanged in DCX-KO neurons. Rather, we observe a significant reduction in α-tubulin polyglutamylation in DCX-KO neurons. Polyglutamylation levels and neuronal branching are rescued by expression of Doublecortin or of TTLL11, an α-tubulin glutamylase. Using U2OS cells as an orthogonal model system, we show that DCX and TTLL11 act synergistically to promote polyglutamylation. We propose that Doublecortin acts as a positive regulator of α-tubulin polyglutamylation and restricts neurite branching. Our results indicate an unexpected role for Doublecortin in the homeostasis of the tubulin code. Lissencephaly is a severe neurodevelopmental disease often caused by mutations in the Dcx gene. Using a human cellular model of lissencephaly, the authors report that DCX restricts neuronal branching by activating tubulin polyglutamylation.

In neurons, patterns of different microtubule types are essential for neurite extension and nucleokinesis. Cellular model systems such as rodent primary cultures and induced pluripotent stem cells (iPSC)-derived neurons have provided key insights into how these patterns are created and maintained through the action of microtubule-associated proteins, motor proteins, and regulatory enzymes. iPSC-derived models show tremendous promise but lack benchmarking and validation relative to rodent primary cultures. Here we have characterized a recent iPSC-derived model, in which doxycycline-induced expression of Neurogenin-2 drives consistent transdifferentiation into the neuronal state (EBiSC-NEUR1 neurons, referred to as NGN2 neurons below). We developed a suite of open-access, semiautomated methods to measure neurite extension and nucleokinesis of NGN2 neurons, which compare favorably to published data from other models. Then, we challenged NGN2 neurons with a panel of drugs that perturb microtubule physiology. NGN2 neurons extension and nucleokinesis were significantly perturbed by two microtubule-targeting drugs, namely a taxane (paclitaxel) and a vinca alkaloid (DZ-2384). In contrast, inhibition of microtubule severing (spastazoline) or of deacetylation (trichostatin A) had a limited effect on nucleokinesis only. Our results support the primary importance of microtubule dynamics in neuronal development and demonstrate the power of NGN2 neurons as a model system.

Microtubules exist in expanded and compacted states, as defined by the lattice spacing of αβ-tubulin dimers. Changes in lattice spacing have been linked to factors such as GTP-hydrolysis, the binding of microtubule-associated proteins (MAPs), the tubulin code, and microtubule bending. These diverse factors exert opposing molecular driving forces on the microtubule lattice that push lattice spacing toward expanded or compacted states. To better understand how these opposing forces are reconciled, we developed in vitro and cell-based model systems for the competition between a microtubule expander (paclitaxel) and a microtubule compactor (doublecortin or DCX). Using an in vitro reconstitution approach, we show that paclitaxel expands microtubules cooperatively. In cells, high concentrations of paclitaxel cause DCX to relocalize to compacted lattices found at concave bends. When the concentration of DCX is increased, however, we find that DCX re-compacts the previously expanded microtubules in vitro. Consistently, high expression levels of DCX prevent its relocalization in paclitaxel-treated cells. When the competition between paclitaxel and DCX is "balanced," we observe a complex phenotype: DCX simultaneously localizes to both long, straight clusters and concave bends, whereas other regions on the microtubule network remain DCX free. We conclude that multiple lattice spacings can coexist in cells. Our results indicate that competition for microtubule lattice spacing is a critical aspect of microtubule physiology.

Tau, a neuronal microtubule-associated protein (MAP), organizes the axonal cytoskeleton and regulates intracellular transport. Tau hyperphosphorylation is linked to neurodegeneration in tauopathies including Alzheimer’s disease. Tau binds microtubules cooperatively to form cohesive envelopes, which are thought to control access to the microtubule lattice and regulate the activity of motor proteins and other microtubule-associated proteins. However, how disease-related perturbations affect tau dynamics and its function as a selective barrier to intracellular transport remains unclear. Using tau phospho-variants in vitro and in live neurons, we show that tau hyperphosphorylation disrupts cooperative microtubule binding and dysregulates lysosome transport. Hyperphosphorylated tau does not form envelopes, distributes more uniformly along the axon, and dissociates faster from microtubules. Tau weakly inhibits KIF5C motility, but strongly inhibits KIF1A. Hyperphosphorylation reduces KIF5C inhibition but increases KIF1A inhibition by decreasing processivity and accelerating detachment. Consistent with these effects, hyperphosphorylated tau alters lysosome transport in neurons. While phospho-resistant tau inhibits processive lysosome motility, hyperphosphorylated tau weakens tau-mediated regulation of lysosome transport, mimicking tau knockout neurons that exhibit enhanced processivity. Altogether, these findings show that hyperphosphorylation disrupts tau envelopes and impairs lysosome trafficking, likely contributing to early defects in degradative pathways that drive neurodegeneration.

A dynamic network of scaffolding molecules, adaptor proteins, and motor proteins work together to orchestrate the movement of proteins, mRNA, and vesicular cargoes. Defects in intracellular transport can often lead to neurodegeneration. Huntingtin (HTT) is a ubiquitously expressed scaffolding protein with a multitude of cellular roles, including regulating the transport of various organelles. HTT is remarkable in its ability to regulate the transport of a wide range of cargoes, including BDNF vesicles, APP vesicles, early endosomes, autophagosomes, lysosomes, and mitochondria. This interaction network allows huntingtin to control microtubule-based transport by kinesin and dynein, as well as actin-based transport by myosin VI. By forming complexes with multiple motor adaptors, huntingtin regulates a variety of cargoes and guides cargoes through the different stages of biosynthesis, signaling, and degradation. Accordingly, pathogenic polyglutamine expansions seen in Huntington's Disease (HD) dysregulate huntingtin transport complexes, resulting in defects in transport and neurodegeneration.

The transport of organelles is important to maintain cellular organization and function. Efficient retrograde transport of large organelles with a size of several micrometers requires high collective forces from multiple dynein motors. However, the exact transport forces and their dependence on the cargo size are unknown for large organelles. Furthermore, it is not known how many dynein motors are active during this transport and how they generate high collective forces sufficient to overcome the cytoplasmic drag. We measured forces generated during retrograde transport of phagosomes with diameters between 1 and 5 μm. Forces increased with phagosome volume and ranged from under 10 pN for the smallest up to 160 pN for the largest phagosomes. These forces matched the cytoplasmic drag to achieve equally fast transport with a velocity of 25 ± 4 nm s-1 for phagosomes of all sizes. To confirm the need for many dynein motors to generate such high forces, we labeled and quantified dynein on isolated phagosomes. We found up to 250 dyneins on the largest phagosomes and a dynein surface density that was independent of the phagosome size. We connected the dynein numbers and transport forces with a theoretical model of the microtubule distribution around the organelles. The model implies that, because larger organelles displace and bend the microtubules, disproportionately large numbers of dyneins can be active and contribute to the high transport forces of large phagosomes. Our results indicate that, during the transport of large organelles, many dyneins interact with multiple microtubules in a cargo-size-dependent manner to achieve sufficiently large transport forces.

Fluid protein condensates are used as precursor phases for fabricating extracellular protein-based materials including elastin, spider silk, and mussel byssus. The byssus, utilized by mussels for anchoring in marine environments, consists of tough, self-healing adhesive fibers. Byssus formation involves the secretion of protein condensate droplets under acidic conditions that subsequently solidify under basic seawater conditions. We currently have a poor understanding of the physicochemical triggers and molecular-level interactions at play, in particular the role of pH and sulfate anions previously identified during native fabrication. Here, we investigated the pH and sulfate-dependent structural and mechanical response of condensates made from a recombinant byssus protein (mfp-1) using optical tweezers microrheology, FRAP, confocal Raman spectroscopy, NMR, and cryo-EM. We found that the protein concentration in condensates increased, and the viscoelastic response became more rigid under basic conditions in the presence of sulfate ions compared with chloride ions, consistent with spectroscopic analysis indicating different molecular interactions under these different chemical conditions. These studies highlight the crucial interplay between sulfate anions and pH in tuning condensate viscoelasticity via control of intermolecular interactions, providing insights into the natural byssus formation process with relevance for bio-inspired materials processing of sustainable plastics and materials for tissue engineering.

Axons experience strong mechanical forces due to animal movement. While these forces serve as sensory cues in mechanosensory neurons, their impact on other neuron types remains poorly defined. Here, we uncover signaling that controls an axonal cytoskeletal response to external physiological forces and plays a key role in axonal integrity. Live imaging of microtubules at single-polymer resolution in a C. elegans motor neuron reveals local oscillatory movements that fine-tune polymer positioning. Combining cell-specific chemogenetic silencing with targeted degradation alleles to distinguish neuron-intrinsic from extrinsic regulators of these movements, we find that they are driven by muscle contractions and require the mechanosensitive protein Talin, the small GTPase RhoA, and actomyosin activity in the axon. Genetic perturbation of the axon's ability to buffer tension by disrupting the spectrin-based membrane-associated skeleton leads to RhoA hyperactivation, actomyosin relocalization to foci at microtubule ends, and converts local oscillations into processive bidirectional movements. This results in large gaps between microtubules, disrupting coverage of the axon and leading to its breakage and degeneration. Notably, hyperpolarizing muscle or degrading components of the mechanotransduction signaling pathway in the axon rescues cytoskeletal defects in spectrin-deficient axons. These results identify mechanisms of an axonal cytoskeletal response to physiological forces and highlight the importance of force-buffering and mechanotransduction signaling for axonal integrity.

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins -1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein-complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.

In iPSC-derived neurons and in vitro motility assays of isolated organelles, phosphomutations and point mutations of tau, which are associated with neurodegenerative diseases, influence the teams of motors that transport organelles and vesicular cargoes along microtubules. Tau, a neuronal microtubule-associated protein, directs intracellular transport by acting as a selective barrier along microtubules to modulate the trafficking of cargoes bound by different sets of motors. Tauopathies, a group of neurodegenerative disorders linked to tau, are often characterized by mutations and hyperphosphorylation that cause tau to misfold and aggregate.

Doublecortin (DCX) is a neuronal microtubule-associated protein (MAP) that binds directly to microtubules via two Doublecortin (DC) domains. The DC domains sense the nucleotide state, longitudinal curvature, and protofilament number of the microtubule lattice, indicating a role in the regulation of microtubule structure in neurons. Mutations in DCX cause lissencephaly and subcortical band heterotopia (also known as double-cortex syndrome) due to impaired neuronal migration. To better understand the role of DCX in neuronal migration, we developed a model system based on induced pluripotent stem cells (iPSCs). We used CRISPR/Cas9 to knock out the Dcx gene in iPSCs and differentiated the cells into cortical neurons. Compared to control neurons, the DCX-KO neurons showed reduced velocities of nuclear movements. The reduced velocities correlated with an increase in the number of neurites early in the neuronal development process, consistent with a neuronal migration phenotype and previous findings in a DCX-KO mouse model. Neurite branching is regulated by a host of MAPs and other factors, as well as by microtubule polymerization dynamics. However, microtubule dynamics were unchanged in DCX-KO neurons, with similar growth rates, lifetimes, and numbers. Rather, we observe a significant reduction in tubulin polyglutamylation in DCX-KO neurons. Polyglutamylation is usually abundant in neurons and regulates microtubule severing enzymes and intracellular trafficking by molecular motors. Consistently, we observe that lysosomes in DCX-KO neurons show a reduction of their processivity. We propose that the reduction of polyglutamylation leads to increased neurite branching and thus reduced neuronal migration. Our results indicate an unexpected role for DCX in the homeostasis of the tubulin code.### Competing Interest StatementThe authors have declared no competing interest.

Huntingtin scaffolds motor proteins and adaptors to organelles. Polyglutamine expansions in huntingtin cause neurodegenerative disease, where the severity and onset of the disease correspond to the polyglutamine repeat length. We examined the transport of BDNF, lysosomes, and mitochondria in neurons derived from isogenic embryonic stem cell lines engineered to express huntingtin with varying polyglutamine repeat lengths of 30Q (control), 45Q, 65Q, and 81Q. In neurons expressing polyglutamine-expanded huntingtin (polyQ-htt), the BDNF-quantum dots exhibit more anterograde motility, and increased total flux.

Each cargo in a cell employs a unique set of motor proteins for its transport. To dissect the roles of each type of motor, we developed optogenetic inhibitors of endogenous kinesin-1, -2, -3 and dynein motors and examined their effect on the transport of early endosomes, late endosomes, and lysosomes. While kinesin-1, -3, and dynein transport vesicles at all stages of endocytosis, kinesin-2 primarily drives late endosomes and lysosomes. Transient optogenetic inhibition of kinesin-1 or dynein causes both early and late endosomes to move more processively by relieving competition with opposing motors. Kinesin-2 and -3 support long-range transport, and optogenetic inhibition reduces the distances that their cargoes move. These results suggest that the directionality of transport is controlled through regulating kinesin-1 and dynein activity. On vesicles transported by several kinesin and dynein motors, modulating the activity of a single type of motor on the cargo is sufficient to direct motility.

Huntingtin (HTT) is a scaffolding protein that recruits motor proteins to vesicular cargoes, enabling it to regulate kinesin-1, dynein, and myosin-VI-dependent transport. To maintain the native stoichiometry of HTT with its interacting partners, we used CRISPR/Cas9 to induce a phosphomimetic mutation of the endogenous HTT at S421 (HTT-S421D). Using single-particle tracking, optical tweezers, and immunofluorescence, we examined the effects of this mutation on the motility of early endosomes and lysosomes. In HTT-S421D cells, lysosomes exhibit longer displacements and higher processive fractions compared with wild-type (HTT-WT) cells. Kinesins and dyneins exert greater forces on early endosomes and lysosomes in cells expressing HTT-S421D. In addition, endosomes bind to microtubules faster and are more resistant to detachment under load. The recruitment of kinesins and dyneins to microtubules is enhanced in HTT-S421D cells. In contrast, overexpression of HTT had variable effects on the processivity, displacement, and directional bias of both early endosomes and lysosomes. These data indicate that phosphorylation of the endogenous HTT causes early endosomes and lysosomes to move longer distances and more processively by recruiting and activating both kinesin-1 and dynein.

Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, we constructed a minimal kinesin-5 tetramer (mini-tetramer). We determined the x-ray structure of the extended, 34-nm BASS domain. Guided by these structural studies, we generated active bipolar kinesin-5 mini-tetramer motors from Drosophila melanogastor and human orthologues which are half the length of native kinesin-5. We then used these kinesin-5 mini-tetramers to examine the role of two unique structural adaptations of kinesin-5: 1) the length and flexibility of the tetramer, and 2) the Cterminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, we find that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.

Huntingtin scaffolds motor proteins and adaptors to vesicular cargoes. Through interactions with kinesin, dynein, and myosin-VI, huntingtin regulates the direction of transport along microtubules and switching between microtubules and actin filaments. Huntington's disease (HD) is caused by polyglutamine (polyQhtt) expansion of length >35Q at Huntington's N-terminus, with longer repeats leading to more severe neurodegeneration. Defects in transport of brain-derived neurotrophic factor (BDNF) and lysosomes have been reported in cells expressing polyQhtt. We track signalling BDNF vesicles and degradative lysosomes in induced neurons from isogenic human stem cell lines with repeat lengths of 18, 30, 45, 65, and 81Q to understand how transport of signalling and degradative cargoes are affected in HD. Preliminary data indicate that lysosomes have lower processivity and similar run lengths with pathological polyQhtt. Interestingly, the 45Q cells have lower processivity and run length compared to all other conditions, indicating divergent mechanisms might contribute to mild and severe disease phenotypes. Lysosomes are typically transported towards the soma. However, with polyQhtt we observe increased outward motility, suggesting huntingtin's ability to regulate transport direction is impaired. To examine how polyQ huntingtin alters the activity of kinesin and dynein, we use optical tweezers to measure the forces exerted on endocytosed BDNF-coated nanodiamonds in control compared to 81Q neurons. BDNF-coated nanodiamonds (∼100 nm) are readily endocytosed by neurons and enable reliable force measurements due to their high refractive index. In parallel, we developed methods to isolate BDNF-phagosomes from neurons and reconstitute their motility along microtubules. Together, live-cell imaging, optical trapping, and in vitro reconstitution reveal how HD mutations impact the transport of signalling and degradative cargoes by misregulating the recruitment and activity of kinesin and dynein.

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. Tau is a neuronal MAP that stabilizes microtubules within the axon. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. To dissect tau's role in regulating transport, we isolate endocytic organelles from cells and reconstitute their motility along tau-decorated microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility characteristics and response to tau. Here, we found that early phagosomes (EPs) undergo unidirectional retrograde transport while late phagosomes (LPs) move bidirectionally. Correspondingly, different numbers and combinations of kinesins-1, -2, -3, and dynein are bound to EPs and LPs. Previous studies showed that tau preferentially inhibits kinesin motors, which biases the transport of LPs towards the microtubule minus-end. Recently, we showed that tau strongly inhibits long-range, dynein-mediated EP motility. Dynein is the dominant motor in EP transport, and tau strongly impedes forces generated by teams of multiple dynein motors and accelerates dynein unbinding under load. These results are surprising, as previous single-molecule studies showed that dynein is less sensitive to tau compared to kinesin. Our results show that specific cargoes differentially respond to tau, where dynein-complexes on EPs are more sensitive to tau inhibition than those on LPs, suggesting that pathological changes in tau associated with Alzheimer's disease and other tauopathies will also have disparate impact on cargoes. We will next compare the effects of pathogenic tau mutations on the transport of different cargoes in iPSC-derived neurons with the aim of identifying the pathways most disrupted in neurodegenerative disease.

In this chapter, we describe methods for reconstituting and analyzing the transport of isolated endogenous cargoes in vitro. Intracellular cargoes are transported along microtubules by teams of kinesin and dynein motors and their cargo-specific adaptor proteins. Observations from living cells show that organelles and vesicular cargoes exhibit diverse motility characteristics. Yet, our knowledge of the molecular mechanisms by which intracellular transport is regulated is not well understood. Here, we describe step-by-step protocols for the extraction of phagosomes from cells at different stages of maturation, and reconstitution of their motility along microtubules in vitro. Quantitative immunofluorescence and photobleaching techniques are also described to measure the number of motors and adaptor proteins on these isolated cargoes. In addition, we describe techniques for tracking the motility of isolated cargoes along microtubules using TIRF microscopy and quantitative force measurements using an optical trap. These methods enable us to study how the sets of motors and adaptors that drive the transport of endogenous cargoes regulate their trafficking in cells.