Dimerization of hub protein DYNLL1 and bZIP transcription factor CREB3L1 enhances transcriptional activation of CREB3L1 target genes like arginine vasopressin.

bZIP transcription factors can function as homodimers or heterodimers through interactions with their disordered coiled-coil domain. Such dimer assemblies are known to influence DNA-binding specificity and/or the recruitment of binding partners, which can cause a functional switch of a transcription factor from being an activator to a repressor. We recently identified the genomic targets of a bZIP transcription factor called CREB3L1 in rat hypothalamic supraoptic nucleus by ChIP-seq. The objective of this study was to investigate the CREB3L1 protein-to-protein interactome of which little is known. For this approach, we created and screened a rat supraoptic nucleus yeast two-hybrid prey library with the bZIP region of rat CREB3L1 as the bait. Our yeast two-hybrid approach captured five putative CREB3L1 interacting prey proteins in the supraoptic nucleus. One interactor was selected by bioinformatic analyses for more detailed investigation by co-immunoprecipitation, immunofluorescent cellular localisation, and reporter assays in vitro. Here we identify dimerisation hub protein Dynein Light Chain LC8-Type 1 as a CREB3L1 interacting protein that in vitro enhances CREB3L1 activation of target genes.

Patient-specific mutation of Dync1h1 in mice causes brain and behavioral deficits.

AIMS: Cytoplasmic dynein heavy chain (DYNC1H1) is a multi-subunit protein complex that provides motor force for movement of cargo on microtubules and traffics them back to the soma. In humans, mutations along the DYNC1H1 gene result in intellectual disabilities, cognitive delays, and neurologic and motor deficits. The aim of the study was to generate a mouse model to a newly identified de novo heterozygous DYNC1H1 mutation, within a functional ATPase domain (c9052C > T(P3018S)), identified in a child with motor deficits, and intellectual disabilities. RESULTS: P3018S heterozygous (HET) knockin mice are viable; homozygotes are lethal. Metabolic and EchoMRI™ testing show that HET mice have a higher metabolic rate, are more active, and have less body fat compared to wildtype mice. Neurobehavioral studies show that HET mice perform worse when traversing elevated balance beams, and on the negative geotaxis test. Immunofluorescent staining shows neuronal migration abnormalities in the dorsal and lateral neocortex with heterotopia in layer I. Neuron-subtype specific transcription factors CUX1 and CTGF identified neurons from layers II/III and VI respectively in cortical layer I, and abnormal pyramidal neurons with MAP2+ dendrites projecting downward from the pial surface. CONCLUSION: The HET mice are a good model for the motor deficits seen in the child, and highlights the importance of cytoplasmic dynein in the maintenance of cortical function and dendritic orientation relative to the pial surface. Our results are discussed in the context of other dynein mutant mice and in relation to clinical presentation in humans with DYNC1H1 mutations.

DYNLL1 accelerates cell cycle via ILF2/CDK4 axis to promote hepatocellular carcinoma development and palbociclib sensitivity.

BACKGROUND: Disorder of cell cycle represents as a major driver of hepatocarcinogenesis and constitutes an attractive therapeutic target. However, identifying key genes that respond to cell cycle-dependent treatments still facing critical challenges in hepatocellular carcinoma (HCC). Increasing evidence indicates that dynein light chain 1 (DYNLL1) is closely related to cell cycle progression and plays a critical role in tumorigenesis. In this study, we explored the role of DYNLL1 in the regulation of cell cycle progression in HCC. METHODS: We analysed clinical specimens to assess the expression and predictive value of DYNLL1 in HCC. The oncogenic role of DYNLL1 was determined by gain or loss-of-function experiments in vitro, and xenograft tumour, liver orthotopic, and DEN/CCl4-induced mouse models in vivo. Mass spectrometry analysis, RNA sequencing, co-immunoprecipitation assays, and forward and reverse experiments were performed to clarify the mechanism by which DYNLL1 activates the interleukin-2 enhancer-binding factor 2 (ILF2)/CDK4 signalling axis. Finally, the sensitivity of HCC cells to palbociclib and sorafenib was assessed by apoptosis, cell counting kit-8, and colony formation assays in vitro, and xenograft tumour models and liver orthotopic models in vivo. RESULTS: DYNLL1 was significantly higher in HCC tissues than that in normal liver tissues and closely related to the clinicopathological features and prognosis of patients with HCC. Importantly, DYNLL1 was identified as a novel hepatocarcinogenesis gene from both in vitro and in vivo evidence. Mechanistically, DYNLL1 could interact with ILF2 and facilitate the expression of ILF2, then ILF2 could interact with CDK4 mRNA and delay its degradation, which in turn activates downstream G1/S cell cycle target genes CDK4. Furthermore, palbociclib, a selective CDK4/6 inhibitor, represents as a promising therapeutic strategy for DYNLL1-overexpressed HCC, alone or particularly in combination with sorafenib. CONCLUSIONS: Our work uncovers a novel function of DYNLL1 in orchestrating cell cycle to promote HCC development and suggests a potential synergy of CDK4/6 inhibitor and sorafenib for the treatment of HCC patients, especially those with increased DYNLL1.

The BICD2 dynein cargo adaptor binds to the HPV16 L2 capsid protein and promotes HPV infection.

During entry, human papillomavirus (HPV) traffics from the endosome to the trans Golgi network (TGN) and Golgi and then the nucleus to cause infection. Although dynein is thought to play a role in HPV infection, how this host motor recruits the virus to support infection and which entry step(s) requires dynein are unclear. Here we show that the dynein cargo adaptor BICD2 binds to the HPV L2 capsid protein during entry, recruiting HPV to dynein for transport of the virus along the endosome-TGN/Golgi axis to promote infection. In the absence of BICD2 function, HPV accumulates in the endosome and TGN and infection is inhibited. Cell-based and in vitro binding studies identified a short segment near the C-terminus of L2 that can directly interact with BICD2. Our results reveal the molecular basis by which the dynein motor captures HPV to promote infection and identify this virus as a novel cargo of the BICD2 dynein adaptor.

Cargo specificity, regulation, and therapeutic potential of cytoplasmic dynein.

Intracellular retrograde transport in eukaryotic cells relies exclusively on the molecular motor cytoplasmic dynein 1. Unlike its counterpart, kinesin, dynein has a single isoform, which raises questions about its cargo specificity and regulatory mechanisms. The precision of dynein-mediated cargo transport is governed by a multitude of factors, including temperature, phosphorylation, the microtubule track, and interactions with a family of activating adaptor proteins. Activating adaptors are of particular importance because they not only activate the unidirectional motility of the motor but also connect a diverse array of cargoes with the dynein motor. Therefore, it is unsurprising that dysregulation of the dynein-activating adaptor transport machinery can lead to diseases such as spinal muscular atrophy, lower extremity, and dominant. Here, we discuss dynein motor motility within cells and in in vitro, and we present several methodologies employed to track the motion of the motor. We highlight several newly identified activating adaptors and their roles in regulating dynein. Finally, we explore the potential therapeutic applications of manipulating dynein transport to address diseases linked to dynein malfunction.

Dynein Light Intermediate Chains Exhibit Different Arginine Methylation Patterns.

BACKGROUND: The motor protein dynein is integral to retrograde transport along microtubules and interacts with numerous cargoes through the recruitment of cargo-specific adaptor proteins. This interaction is mediated by dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which govern the adaptor binding and are present in distinct dynein complexes with overlapping and unique functions. METHODS: Using bioinformatics, we analyzed the C-terminal domains (CTDs) of LIC1 and LIC2, revealing similar structural features but diverse post-translational modifications (PTMs). The methylation status of LIC2 and the proteins involved in this modification were examined through immunoprecipitation and immunoblotting analyses. The specific methylation sites on LIC2 were identified through a site-directed mutagenesis analysis, contributing to a deeper understanding of the regulatory mechanisms of the dynein complex. RESULTS: We found that LIC2 is specifically methylated at the arginine 397 residue, a reaction that is catalyzed by protein arginine methyltransferase 1 (PRMT1). CONCLUSIONS: The distinct PTMs of the LIC subunits offer a versatile mechanism for dynein to transport diverse cargoes efficiently. Understanding how these PTMs influence the functions of LIC2, and how they differ from LIC1, is crucial for elucidating the role of dynein-related transport pathways in a range of diseases. The discovery of the arginine 397 methylation site on LIC2 enhances our insight into the regulatory PTMs of dynein functions.

Structure and Function of Dynein's Non-Catalytic Subunits.

Dynein, an ancient microtubule-based motor protein, performs diverse cellular functions in nearly all eukaryotic cells, with the exception of land plants. It has evolved into three subfamilies-cytoplasmic dynein-1, cytoplasmic dynein-2, and axonemal dyneins-each differentiated by their cellular functions. These megadalton complexes consist of multiple subunits, with the heavy chain being the largest subunit that generates motion and force along microtubules by converting the chemical energy of ATP hydrolysis into mechanical work. Beyond this catalytic core, the functionality of dynein is significantly enhanced by numerous non-catalytic subunits. These subunits are integral to the complex, contributing to its stability, regulating its enzymatic activities, targeting it to specific cellular locations, and mediating its interactions with other cofactors. The diversity of non-catalytic subunits expands dynein's cellular roles, enabling it to perform critical tasks despite the conservation of its heavy chains. In this review, we discuss recent findings and insights regarding these non-catalytic subunits.

Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells.

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein activation occurs is unclear since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualize and track fluorescently tagged dynein. First, we observed that only ∼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of ∼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein's frequent MT binding-unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo and that complex cargo behavior emerges from stochastic motor-cargo interactions.

Lis1 slows force-induced detachment of cytoplasmic dynein from microtubules.

Lis1 is a key cofactor for the assembly of active cytoplasmic dynein complexes that transport cargo along microtubules. Lis1 binds to the AAA+ ring and stalk of dynein and slows dynein motility, but the underlying mechanism has remained unclear. Using single-molecule imaging and optical trapping assays, we investigated how Lis1 binding affects the motility and force generation of yeast dynein in vitro. We showed that Lis1 slows motility by binding to the AAA+ ring of dynein, not by serving as a roadblock or tethering dynein to microtubules. Lis1 binding also does not affect force generation, but it induces prolonged stalls and reduces the asymmetry in the force-induced detachment of dynein from microtubules. The mutagenesis of the Lis1-binding sites on the dynein stalk partially recovers this asymmetry but does not restore dynein velocity. These results suggest that Lis1-stalk interaction slows the detachment of dynein from microtubules by interfering with the stalk sliding mechanism.

Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis.

Actin-related proteins (Arps) are classified according to their similarity to actin and are involved in diverse cellular processes. ACTL7B is a testis-specific Arp, and is highly conserved in rodents and primates. ACTL7B is specifically expressed in round and elongating spermatids during spermiogenesis. Here, we have generated an Actl7b-null allele in mice to unravel the role of ACTL7B in sperm formation. Male mice homozygous for the Actl7b-null allele (Actl7b-/-) were infertile, whereas heterozygous males (Actl7b+/-) were fertile. Severe spermatid defects, such as detached acrosomes, disrupted membranes and flagella malformations start to appear after spermiogenesis step 9 in Actl7b-/- mice, finally resulting in spermatogenic arrest. Abnormal spermatids were degraded and levels of autophagy markers were increased. Co-immunoprecipitation with mass spectrometry experiments identified an interaction between ACTL7B and the LC8 dynein light chains DYNLL1 and DYNLL2, which are first detected in step 9 spermatids and mislocalized when ACTL7B is absent. Our data unequivocally establish that mutations in ACTL7B are directly related to male infertility, pressing for additional research in humans.

N-Acetylglucosamine Kinase-Small Nuclear Ribonucleoprotein Polypeptide N Interaction Promotes Axodendritic Branching in Neurons via Dynein-Mediated Microtubule Transport.

N-acetylglucosamine kinase (NAGK) has been identified as an anchor protein that facilitates neurodevelopment with its non-canonical structural role. Similarly, small nuclear ribonucleoprotein polypeptide N (SNRPN) regulates neurodevelopment and cognitive ability. In our previous study, we revealed the interaction between NAGK and SNRPN in the neuron. However, the precise role in neurodevelopment is elusive. In this study, we investigate the role of NAGK and SNRPN in the axodendritic development of neurons. NAGK and SNRPN interaction is significantly increased in neurons at the crucial stages of neurodevelopment. Furthermore, overexpression of the NAGK and SNRPN proteins increases axodendritic branching and neuronal complexity, whereas the knockdown inhibits neurodevelopment. We also observe the interaction of NAGK and SNRPN with the dynein light-chain roadblock type 1 (DYNLRB1) protein variably during neurodevelopment, revealing the microtubule-associated delivery of the complex. Interestingly, NAGK and SNRPN proteins rescued impaired axodendritic development in an SNRPN depletion model of Prader-Willi syndrome (PWS) patient-derived induced pluripotent stem cell neurons. Taken together, these findings are crucial in developing therapeutic approaches for neurodegenerative diseases.

Pain sensitivity related to gamma oscillation of parvalbumin interneuron in primary somatosensory cortex in Dync1i1-/- mice.

Cytoplasmic dynein is an important intracellular motor protein that plays an important role in neuronal growth, axonal polarity formation, dendritic differentiation, and dendritic spine development among others. The intermediate chain of dynein, encoded by Dync1i1, plays a vital role in the dynein complex. Therefore, we assessed the behavioral and related neuronal activities in mice with dync1i1 gene knockout. Neuronal activities in primary somatosensory cortex were recorded by in vivo electrophysiology and manipulated by optogenetic and chemogenetics. Nociception of mechanical, thermal, and cold pain in Dync1i1-/- mice were impaired. The activities of parvalbumin (PV) interneurons and gamma oscillation in primary somatosensory were also impaired when exposed to mechanical nociceptive stimulation. This neuronal dysfunction was rescued by optogenetic activation of PV neurons in Dync1i1-/- mice, and mimicked by suppressing PV neurons using chemogenetics in WT mice. Impaired pain sensations in Dync1i1-/- mice were correlated with impaired gamma oscillations due to a loss of interneurons, especially the PV type. This genotype-driven approach revealed an association between impaired pain sensation and cytoplasmic dynein complex.

A human dynein heavy chain mutation impacts cortical progenitor cells causing developmental defects, reduced brain size and altered brain architecture.

Dynein heavy chain (DYNC1H1) mutations can either lead to severe cerebral cortical malformations, or alternatively may be associated with the development of spinal muscular atrophy with lower extremity predominance (SMA-LED). To assess the origin of such differences, we studied a new Dync1h1 knock-in mouse carrying the cortical malformation p.Lys3334Asn mutation. Comparing with an existing neurodegenerative Dync1h1 mutant (Legs at odd angles, Loa, p.Phe580Tyr/+), we assessed Dync1h1's roles in cortical progenitor and especially radial glia functions during embryogenesis, and assessed neuronal differentiation. p.Lys3334Asn /+ mice exhibit reduced brain and body size. Embryonic brains show increased and disorganized radial glia: interkinetic nuclear migration occurs in mutants, however there are increased basally positioned cells and abventricular mitoses. The ventricular boundary is disorganized potentially contributing to progenitor mislocalization and death. Morphologies of mitochondria and Golgi apparatus are perturbed in vitro, with different effects also in Loa mice. Perturbations of neuronal migration and layering are also observed in p.Lys3334Asn /+ mutants. Overall, we identify specific developmental effects due to a severe cortical malformation mutation in Dync1h1, highlighting the differences with a mutation known instead to primarily affect motor function.

The meiotic LINC complex component KASH5 is an activating adaptor for cytoplasmic dynein.

Cytoplasmic dynein-driven movement of chromosomes during prophase I of mammalian meiosis is essential for synapsis and genetic exchange. Dynein connects to chromosome telomeres via KASH5 and SUN1 or SUN2, which together span the nuclear envelope. Here, we show that KASH5 promotes dynein motility in vitro, and cytosolic KASH5 inhibits dynein's interphase functions. KASH5 interacts with a dynein light intermediate chain (DYNC1LI1 or DYNC1LI2) via a conserved helix in the LIC C-terminal, and this region is also needed for dynein's recruitment to other cellular membranes. KASH5's N-terminal EF-hands are essential as the interaction with dynein is disrupted by mutation of key calcium-binding residues, although it is not regulated by cellular calcium levels. Dynein can be recruited to KASH5 at the nuclear envelope independently of dynactin, while LIS1 is essential for dynactin incorporation into the KASH5-dynein complex. Altogether, we show that the transmembrane protein KASH5 is an activating adaptor for dynein and shed light on the hierarchy of assembly of KASH5-dynein-dynactin complexes.

DNALI1 deficiency causes male infertility with severe asthenozoospermia in humans and mice by disrupting the assembly of the flagellar inner dynein arms and fibrous sheath.

The axonemal dynein arms (outer (ODA) and inner dynein arms (IDAs)) are multiprotein structures organized by light, intermediate, light intermediate (LIC), and heavy chain proteins. They hydrolyze ATP to promote ciliary and flagellar movement. Till now, a variety of dynein protein deficiencies have been linked with asthenospermia (ASZ), highlighting the significance of these structures in human sperm motility. Herein, we detected bi-allelic DNALI1 mutations [c.663_666del (p.Glu221fs)], in an ASZ patient, which resulted in the complete loss of the DNALI1 in the patient's sperm. We identified loss of sperm DNAH1 and DNAH7 rather than DNAH10 in both DNALI1663_666del patient and Dnali1-/- mice, demonstrating that mammalian DNALI1 is a LIC protein of a partial IDA subspecies. More importantly, we revealed that DNALI1 loss contributed to asymmetries in the most fibrous sheath (FS) of the sperm flagellum in both species. Immunoprecipitation revealed that DNALI1 might interact with the cytoplasmic dynein complex proteins in the testes. Furthermore, DNALI1 loss severely disrupted the transport and assembly of the FS proteins, especially AKAP3 and AKAP4, during flagellogenesis. Hence, DNALI1 may possess a non-classical molecular function, whereby it regulates the cytoplasmic dynein complex that assembles the flagella. We conclude that a DNALI deficiency-induced IDAs injury and an asymmetric FS-driven tail rigid structure alteration may simultaneously cause flagellum immotility. Finally, intracytoplasmic sperm injection (ICSI) can effectively resolve patient infertility. Collectively, we demonstrate that DNALI1 is a newly causative gene for AZS in both humans and mice, which possesses multiple crucial roles in modulating flagellar assembly and motility.

Knockout of DLIC1 leads to retinal cone degeneration via disturbing Rab8 transport in zebrafish.

Retinal photoreceptors execute phototransduction functions and require an efficient system for the transport of materials (e.g. proteins and lipids) from inner segments to outer segments. Cytoplasmic dynein 1 is a minus-end-directed microtubule motor and participates in cargo transport in the cytoplasm. However, the roles of dynein 1 motor in photoreceptor cargo transport and retinal development are still ambiguous. In our present study, the light intermediate chain protein DLIC1 (encoded by dync1li1), links activating adaptors to bind diverse cargos in the dynein 1 motor, was depleted using CRISPR-Cas9 technology in zebrafish. The dync1li1-/- zebrafish displayed progressive degeneration of retinal cone photoreceptors, especially blue cones. The retinal rods were not affected in dync1li1-/- zebrafish. Knockout of DLIC1 resulted in abnormal expression and localization of cone opsins in dync1li1-/- retinas. TUNEL staining suggested that apoptosis was induced after aberrant accumulation of cone opsins in photoreceptors of dync1li1-/- zebrafish. Instead of Rab11 transport, Rab8 transport was disturbed in dync1li1-/- retinas. Our data demonstrate that DLIC1 is required for function maintenance and survival of cone photoreceptors, and hint at an essential role of the cytoplasmic dynein 1 motor in photoreceptor cargo transport.

A conditional null allele of Dync1h1 enables targeted analyses of dynein roles in neuronal length sensing.

Size homeostasis is a fundamental process in biology and is particularly important for large cells such as neurons. We previously proposed a motor-dependent length-sensing mechanism wherein reductions in microtubule motor levels would be expected to accelerate neuronal growth, and validated this prediction in dynein heavy chain 1 Loa mutant (Dync1h1Loa) sensory neurons. Here, we describe a new mouse model with a conditional deletion allele of exons 24 and 25 in Dync1h1. Homozygous Islet1-Cre-mediated deletion of Dync1h1 (Isl1-Dync1h1-/-), which deletes protein from the motor and sensory neurons, is embryonic lethal, but heterozygous animals (Isl1-Dync1h1+/-) survive to adulthood with ∼50% dynein expression in targeted cells. Isl1-Dync1h1+/- sensory neurons reveal accelerated growth, as previously reported in Dync1h1Loa neurons. Moreover, Isl1-Dync1h1+/- mice show mild impairments in gait, proprioception and tactile sensation, similar to what is seen in Dync1h1Loa mice, confirming that specific aspects of the Loa phenotype are due to reduced dynein levels. Isl1-Dync1h1+/- mice also show delayed recovery from peripheral nerve injury, likely due to reduced injury signal delivery from axonal lesion sites. Thus, conditional deletion of Dync1h1 exons 24 and 25 enables targeted studies of the role of dynein in neuronal growth.

Structure of dynein-dynactin on microtubules shows tandem adaptor binding.

Cytoplasmic dynein is a microtubule motor that is activated by its cofactor dynactin and a coiled-coil cargo adaptor1-3. Up to two dynein dimers can be recruited per dynactin, and interactions between them affect their combined motile behaviour4-6. Different coiled-coil adaptors are linked to different cargos7,8, and some share motifs known to contact sites on dynein and dynactin4,9-13. There is limited structural information on how the resulting complex interacts with microtubules and how adaptors are recruited. Here we develop a cryo-electron microscopy processing pipeline to solve the high-resolution structure of dynein-dynactin and the adaptor BICDR1 bound to microtubules. This reveals the asymmetric interactions between neighbouring dynein motor domains and how they relate to motile behaviour. We found that two adaptors occupy the complex. Both adaptors make similar interactions with the dyneins but diverge in their contacts with each other and dynactin. Our structure has implications for the stability and stoichiometry of motor recruitment by cargos.

Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin.

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end-directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.

Kinesin-1, -2, and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport.

Bidirectional cargo transport in neurons requires competing activity of motors from the kinesin-1, -2, and -3 superfamilies against cytoplasmic dynein-1. Previous studies demonstrated that when kinesin-1 attached to dynein-dynactin-BicD2 (DDB) complex, the tethered motors move slowly with a slight plus-end bias, suggesting kinesin-1 overpowers DDB but DDB generates a substantial hindering load. Compared to kinesin-1, motors from the kinesin-2 and -3 families display a higher sensitivity to load in single-molecule assays and are thus predicted to be overpowered by dynein complexes in cargo transport. To test this prediction, we used a DNA scaffold to pair DDB with members of the kinesin-1, -2, and -3 families to recreate bidirectional transport in vitro, and tracked the motor pairs using two-channel TIRF microscopy. Unexpectedly, we find that when both kinesin and dynein are engaged and stepping on the microtubule, kinesin-1, -2, and -3 motors are able to effectively withstand hindering loads generated by DDB. Stochastic stepping simulations reveal that kinesin-2 and -3 motors compensate for their faster detachment rates under load with faster reattachment kinetics. The similar performance between the three kinesin transport families highlights how motor kinetics play critical roles in balancing forces between kinesin and dynein, and emphasizes the importance of motor regulation by cargo adaptors, regulatory proteins, and the microtubule track for tuning the speed and directionality of cargo transport in cells.

Nerve cells in the human body can reach up to one meter in length. Different regions of a nerve cell require different materials to perform their roles. The motor proteins kinesins and dynein help to transport the required 'cargo', by moving in opposite directions along tracks called microtubules. However, many cargos have both motors attached, resulting in a tug-of-war to determine which direction and how fast the cargo will travel. In many neurodegenerative diseases, including Alzheimer's, this cargo transport goes awry, so a better understanding of exactly how this process works may help to develop new therapies. There are three families of kinesin motors, for a total of about a dozen different kinesins that engage in this process. Motors in each of the three families have different mechanical properties. Specific cargos also tend to have specific kinesins attached to them. Here Gicking et al. hypothesized that when pulling against dynein in a tug-of-war, kinesins from the three families would behave differently. To test this hypothesis, Gicking et al. linked one kinesin to one dynein motor, one at a time in a test tube, and then observed how these two-motor complexes moved using fluorescence microscopy techniques. Unexpectedly, kinesins from the three different families competed similarly against dynein: there were no clear winners and losers. By incorporating previously published data describing the different motor behaviors, Gicking et al. developed a computational model that provided deeper insight into how this mechanical tug-of-war works. The modeling indicated that kinesins from the three families use different approaches for competing against dynein. Kinesin-1 motors tended to pull steadily against dynein, only detaching relatively rarely, but then take some time to attach back to the microtubule track. In contrast, kinesin-3 motors detached easily when they pull against dynein, but they attach back to the microtubule track quickly, taking only about a millisecond to start moving again. Kinesin-2 motors exhibited an intermediate behavior. Overall, these experiments suggest that the mechanical properties of the motor proteins are not the main factors determining the direction and speed of the cargo. In other words, the outcome of this molecular tug-of-war does not necessarily depend on which motor is stronger or faster. Rather, further mechanisms, including regulation of the adapter molecules that connect the motors to their cargo, may help to regulate which cargo go where in branched nerve cells. A better knowledge of how all these different factors work together will be important for understanding how cargo transport in nerve cells is disrupted in neurodegenerative diseases.