Cytoskeleton

The cytoskeleton"s ability to contract and propagate forces is the fundamental mechanism behind cell morphology, division and migration. This can only occur if the network is sufficiently connected, yet a rigorous description of the connectivity requirements has never been provided. In this work we focused on the polarity-sorting contraction mechanism and showed that connectivity is not determined by the spatial distribution of filaments alone, but by the interconnectivity between the dual network of filaments and motors. We developed a method to quantify filament-motor connectivity as a function of motor length, filament length distributions, and the densities of each component. Using this framework, we derived a general theory that predicts when a network is sufficiently connected to allow global or local contraction. We validated our predictions with computer simulations and introduced a novel metric to distinguish between these outcomes. Our findings show that the conditions for local and global contraction in the presence of fiber dynamics correspond, respectively, to the pulsatile and steady-state contraction behaviors observed in vivo. All results are independent of filament rigidity, making our conclusions applicable to both actin and microtubule networks. Lastly, we discuss how those outcomes are affected by the introduction of crosslinking proteins, which - despite not actively generating forces of their own - can promote global contractility at small concentrations even in networks made of short and/or rigid filaments.

Neurofilaments are space-filling cytoskeletal polymers that are transported into axons where they accumulate during development to expand axon caliber. We previously described novel severing and end-to-end annealing mechanisms in neurons that alter neurofilament length. To explore the functional significance of neurofilament length, we developed a long-term multi-field time-lapse method to track the movement of fluorescently tagged neurofilaments in axons of cultured neurons for up to 30 minutes. All filaments moved rapidly, but long filaments paused and reversed more often, resulting in little net movement, whereas short filaments moved persistently for long distances, pausing and reversing less often. Long filaments severed more frequently, generating shorter filaments, and short filaments annealed more frequently, generating longer filaments. Thus, neurofilament length is regulated by a dynamic cycle of severing and annealing and this influences neurofilament transport. Site-directed mutagenesis to mimic phosphorylation at four known phosphorylation sites in the head domain of neurofilament protein L generated shorter neurofilaments that moved more frequently. A non-phosphorylatable mutant had the opposite effect. Treatment of cultured neurons with activators of protein kinase A, which phosphorylates three of these sites, increased neurofilament severing. This effect was blocked by the non-phosphorylatable mutant. We propose that focal destabilization of intermediate filaments by N-terminal phosphorylation of their constituent polypeptides at specific locations along their length may be a general enzymatic mechanism for severing this class of cytoskeletal polymers. Our data suggest a novel mechanism for the control of neurofilament transport and accumulation in axons based on regulation of neurofilament polymer length. SUMMARYNeurofilaments are space-filling cytoskeletal polymers that are transported into axons where they accumulate to expand axon caliber, which is an important determinant of axonal conduction velocity. We reported previously that neurofilaments can lengthen and shorten by novel end-to-end annealing and severing mechanisms. Here, we show that neurofilament annealing and severing are robust phenomena in cultured neurons that act antagonistically to dynamically regulate neurofilament length, which in turn regulates their transport. In addition, we present evidence for a novel enzymatic mechanism of intermediate filament severing based on site-directed phosphorylation of the neurofilament subunit proteins. We propose that modulation of neurofilament length by annealing and severing may be a mechanism for the regulation of neurofilament transport and accumulation in axons.

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in {beta}-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus- pseudocamptodactyly syndrome. It is not known if their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human {beta}, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins, with the most dramatic in perinatal, but minimal effects in {beta} myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing ATPase cycle rate. In contrast, the only measured effect of R671C in {beta} myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not {beta}, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents the first direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are yet another testament to myosins highly allosteric nature.

Cells need intracellular forces for their physiological functions, such as migration, cytokinesis, and morphogenesis. The actin cytoskeleton generates a large fraction of the forces via interactions between cytoskeletal components, such as actin filament (F-actin), myosin, and actin cross-linking proteins (ACPs). Myosin II plays the most important role in cellular force generation. Myosin II molecules self-assemble into filaments with different structures depending on myosin II isoforms and other conditions such as pH and ionic concentration. It has remained elusive how force generation in actomyosin structures is affected by the architecture of myosin II filaments. In this study, we employed an agent-based model to investigate the effects of the structural properties of myosin II filaments on force generation in disorganized actomyosin structures. We demonstrated that the magnitude of forces and the efficiency of force generation can vary over a wide range depending on the number and spatial distribution of myosin II filaments. Further, we showed that the number of myosin heads and the length of a bare zone at the center of myosin II filaments without heads highly affect the force generation process in bundles and networks. Our study provides insights into understanding the roles of the structural properties of myosin II filaments in actomyosin contractility.

Molecular motors like kinesin are critical for cellular organization and biological function including in neurons. There is detailed understanding of how they move and how factors such as applied force and the presence of microtubule-associated proteins can alter this single-motor travel. In order to walk, the cargo-motor complex must first attach to a microtubule. This attachment process is less studied. Here, we use a combination of single-molecule bead experiments, modeling, and simulation to examine how cargos with kinesin-1 bind to microtubules. In experiment, we find that increasing cargo size and environment viscosity both signficantly slow cargo binding time. We use modeling and simulation to examine how the single motor on rate translates to the on rate of the cargo. Combining experiment and modeling allows us to estimate the single motor on rate as 100 s-1. This is a much higher value than previous estimates. We attribute the difference between our measurements and previous estimates to two factors: first, we are directly measuring initial motor attachment (as opposed to re-binding of a second motor) and second, the theoretical framework allows us to account for missed events (i.e. binding events not detected by the experiments due to their short duration). This indicates that the mobility of the cargo itself, determined by its size and interaction with the cytoplasmic environment, play a previously underestimated role in determining intracellular transport kinetics.

Skeletal myosins II are non-processive molecular motors, that work in ensembles to produce muscle contraction while binding to the actin filament. Although the molecular properties of myosin II are well known, there is still debate about the collective work of the motors: is there cooperativity between myosin motors while binding to the actin filaments? In this study, we used high-speed AFM to evaluate this issue. We observed that the initial binding of small arrays of myosin heads to the non-regulated actin filaments did not affect the cooperative probability of subsequent bindings to neighboring sites and did not lead to an increase in the fractional occupancy of the actin binding sites. These results suggest that myosin motors are independent force generators when connected in small arrays, and that the binding of one myosin does not alter the kinetics of other myosins. In contrast, the probability of binding of myosin heads to regulated thin filaments under activating conditions (at high Ca2+ concentration and with 2 M ATP) was increased with the initial binding of one myosin, leading to a larger occupancy of neighboring available binding sites. The result suggests that myosin cooperativity is defined by the activation status of the thin filaments. eLife digestMuscle contraction is the result of large ensembles of the molecular motor myosin II working in coordination while attached to actin. Myosin II produces the power stroke, responsible for force generation. In this paper, we used High-Speed Atomic Force Microscopy (HS-AFM) to determine the potential cooperativity between myosin motors bound to non-regulated and regulated thin filaments. Based on the direct visualization of myosin-actin interaction, probability of myosin binding, and the myosin fractional occupancy of binding sites along non-regulated and regulated actin filaments, our results show no cooperative effects over [~]100 nm of the actin filament length. In contrast, there is myosin cooperativity within the activated thin filament, that induces a high affinity of myosin heads to the filaments. Our results support the independent behaviour of myosin heads while attached to actin filaments, but a cooperative behavior when attached to regulated thin filaments.

At the core of cilia are microtubules which are important for establishing length and assisting ciliary assembly and disassembly; however, another role for microtubule regulation on ciliogenesis lies outside of the cilium. The microtubule cytoskeleton is a highly dynamic structure which polymerizes and depolymerizes rapidly to assist in cellular processes. These processes have been studied across various organisms with chemical as well as genetic perturbations. However, these have generated conflicting data in terms of the role of cytoplasmic microtubules (CytoMTs) and free tubulin dynamics during ciliogenesis. Here we look at the relationship between ciliogenesis and cytoplasmic microtubule dynamics in Chlamydomonas reinhardtii using chemical and mechanical perturbations. We find that not only can stabilized CytoMTs allow for normal ciliary assembly, but high calcium concentrations and low pH-induced deciliation cause CytoMTs to depolymerize separately from ciliary shedding. In addition, we find that ciliary shedding through mechanical shearing, cilia regenerate earlier despite intact CytoMTs. Our data suggests that cytoplasmic microtubules are not a sink for a limiting pool of cytoplasmic tubulin in Chlamydomonas, depolymerization that occurs following deciliation is a consequence rather than a requirement for ciliogenesis, and intact CytoMTs in the cytoplasm and the proximal cilium support more efficient ciliary assembly.

During Drosophila myognesis, myonuclei are actively moved during embryogenesis, and their spacing is maintained through an anchoring mechanism in the fully differentiated myofiber. While we have identified microtubule associated proteins, motors, and nuclear envelope proteins that regulate myonuclear spacing, the developmental time during which each gene functions has not been tested. Here we have identified a Dystrophin as required only for the maintenance of myonuclear spacing. Furthermore, we demonstrate that Dystrophin genetically interacts with the KASH-domain protein Msp300 to maintain myonuclear spacing. Mechanistically, both Dystrophin and Msp300 regulate microtubule organization. Specifically, in animals with disrupted expression of both Dystrophin and Msp300, microtubule colocalization with sarcomeres is reduced. Taken altogether, these data indicate that the peripheral membrane protein Dystrophin, and the outer nuclear membrane protein Msp300, together regulate the organization of the microtubule network which then acts as an anchor to restrict myonuclear movement in contractile myofibers. These data are consistent with growing evidence that myonuclear movement and myonuclear spacing are critical to muscle development, muscle function, and muscle repair and provide a mechanism to connect disparate muscle diseases. Summary StatementHere we show that Dystrophin is required to maintain the spacing of nuclei in differentiated myofibers. Furthermore, Dystrophin achieves this function via a genetic interaction with Msp300 which regulates microtubule organization.

Microtubule polymerization dynamics result from the biochemical interactions of {beta}-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of {beta}-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently poisoned growth, yielding a wider range of growth rate compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP tubulin at the growing microtubule end may be an important early event determining catastrophe.

Subcellular localisation of mitochondria provides a spatial and temporal organisation for cellular energy demands. Long-range mitochondrial transport is mediated by microtubule tracks and associated dynein and kinesin motor proteins. The actin cytoskeleton has a more versatile role and provides transport, tethering, and anchoring functions. SPIRE actin nucleators organise actin filament networks at vesicle membranes, which serve as tracks for myosin 5 motor protein-driven transport processes. Following alternative splicing, SPIRE1 is targeted to mitochondria. In analogy to vesicular SPIRE functions, we have analysed whether SPIRE1 regulates mitochondrial motility. By tracking mitochondria of living fibroblast cells from SPIRE1 mutant mice and splice-variant specific mitochondrial SPIRE1 knockout mice, we determined that the loss of SPIRE1 function increased mitochondrial motility. The SPIRE1 mutant phenotype was reversed by transient overexpression of mitochondrial SPIRE1, which almost completely inhibited motility. Conserved myosin 5 and formin interaction motifs contributed to this inhibition. Consistently, mitochondrial SPIRE1 targeted myosin 5 motors and formin actin filament generators to mitochondria. Our results indicate that SPIRE1 organises an actin/myosin network at mitochondria, which opposes mitochondrial motility. Summary statementThe mitochondrial SPIRE1 protein targets myosin 5 motor proteins and formin actin-filament nucleators/elongators towards mitochondria and negatively regulates mitochondrial motility.

The ability to generate active stresses within filamentous actin matrices is a fundamental and evolutionarily conserved process driving locomotion and morphogenetic changes in cells. The generation of pushing forces by actin polymerization is reasonably well understood, and is known to drive lamellipodia based motility and filopodial extension. Actin filaments decorated with myosin motors can also generate contractile stresses as in the cell cortex or in cytokinetic rings. In this article we use membrane nanotubes pulled out of axonal shaft to investigate actin dynamics and force generation. We report cyclic growth and retraction dynamics of actin within the tube and correlated contraction events giving rise to sustained load and fail cycles. The contraction mechanism operate independent of myosin II motor proteins. Furthermore, we analyzed the dynamics of actin within the tube, including under various biochemical or genetic perturbations. By combining these results with physical modeling, we argue that stresses generated in the actin filaments by the binding of actin depolymerizing factor (ADF/cofilin) proteins can explain the cyclic load-fail behavior.

Animal cell shape is largely determined by the organization of the actin cytoskeleton. Spread shapes result from a balance between protrusive actin networks and contractile stress fibers, while rounded shapes are supported by a contractile actomyosin cortex. The assembly and regulation of distinct types of actin networks have been extensively studied, yet, what determines which networks dominate in a given cell remains unclear. In this Brief Report, we explore the molecular regulation of overall actin organization and resulting cell shape. We use our recently published comparison of the F-actin interactome in spread interphase and rounded mitotic cells to establish a list of candidate regulators of actin networks in spread cells. Utilizing micropatterning and automated image analysis we quantitatively analyze how these candidates affect actin organization. Out of our initial 16 candidates, we identify subsets of proteins promoting stress fibers or regulating their arrangement. Interestingly, no single regulator depletion caused significant cell shape change. However, perturbing two hits simultaneously, supervillin and myosin II, led to stress fiber disassembly and cell rounding. Overall, our systematic investigation shows that actin networks are robust to perturbations, and identifies regulatory modules controlling overall actin organization and resulting cell shape.

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Although variations in elongation rates produce modest changes in the equilibrium lengths of formin-bound filaments, nucleation constitutes the primary mode of monomer consumption over the course of assembly. Sustained elongation of small numbers of formin-bound filaments therefore requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formins nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.

A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitment of the LIM domain proteins TRIP6 and LIMD1 to adherens junctions. Mechanical tension also triggers TRIP6 and LIMD1 to bind and inhibit the Hippo pathway kinase LATS1. How TRIP6 and LIMD1 are recruited to adherens junctions in response to tension is not clear, but previous studies suggested that they could be regulated by the known mechanosensory proteins -catenin and vinculin at adherens junctions. We found that the three LIM domains of TRIP6 and LIMD1 are necessary and sufficient for tension-dependent localization to adherens junctions. The LIM domains of TRIP6, LIMD1, and certain other LIM domain proteins have been shown to bind to actin networks under strain/tension. Consistent with this, we show that TRIP6 and LIMD1 colocalize with the ends of actin fibers at adherens junctions. Point mutations in a key conserved residue in each LIM domain that are predicted to impair binding to f-actin under strain inhibits TRIP6 and LIMD1 localization to adherens junctions and their ability to bind to and recruit LATS1 to adherens junctions. Together these results show that the ability of TRIP6 and LIMD1 to bind to strained actin underlies their ability to localize to adherens junctions and regulate LATS1 in response to mechanical tension.

The actin cytoskeleton is a potent regulator of tenocyte homeostasis. However, the mechanisms by which actin regulates tendon homeostasis are not entirely known. This study examined the regulation of tenocyte molecule expression by actin polymerization via the globular (G-) actin-binding transcription factor, myocardin-related transcription factor-a (MRTF). We determined that decreasing the proportion of G-actin in tenocytes by treatment with TGF{beta}1 increases nuclear MRTF. These alterations in actin polymerization and MRTF localization coincided with favorable alterations to tenocyte gene expression. In contrast, latrunculin A increases the proportion of G-actin in tenocytes and reduces nuclear MRTF, causing cells to acquire a tendinosis-like phenotype. To parse out the effects of F-actin depolymerization from regulation by MRTF, we treated tenocytes with cytochalasin D. Similar to latrunculin A treatment, exposure of cells to cytochalasin D increases the proportion of G-actin in tenocytes. However, unlike latrunculin A treatment, cytochalasin D increases nuclear MRTF. Compared to latrunculin A treatment, cytochalasin D led to opposing effects on the expression of a subset of genes. The differential regulation of genes by latrunculin A and cytochalasin D suggests that actin signals through MRTF to regulate a specific subset of genes. By targeting the deactivation of MRTF through the inhibitor CCG1423, we verify that MRTF regulates Type I Collagen, Tenascin C, Scleraxis, and -smooth muscle actin in tenocytes. Actin polymerization status is a potent regulator of tenocyte homeostasis through the modulation of several downstream pathways, including MRTF. Understanding the regulation of tenocyte homeostasis by actin may lead to new therapeutic interventions against tendinopathies, such as tendinosis.

Cellular cargos, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. Many experimental and computational studies of cargos with rigidly attached motors, in contrast to many biological cargos that have lipid surfaces that may allow surface mobility of motors. We extend a mechanochemical 3D computational model by adding coupled-viscosity effects to compare different motor arrangements and mobilities. We show that organizational changes can optimize for different objectives: Cargos with clustered motors are transported efficiently, but are slow to bind to microtubules, whereas those with motors dispersed rigidly on their surface bind microtubules quickly, but are transported inefficiently. Finally, cargos with freely-diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results suggest that experimentally observed changes in motor organization may be a control point for transport.

Integrin adhesion complexes (IACs) are a network of many proteins that serve as anchors of the cell to the extracellular matrix (ECM). In muscle, IACs located at costameres, also serve to transmit the force of muscle contraction to the outside of the cell. We have reported that IACs, which are found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the RacGEF PIX-1 for their proper assembly or maintenance. We have reported that a RacGAP for the PIX pathway is RRC-1, is in a complex with PIX-1, and that RRC-1 is required for assembly or maintenance of IACs at MCBs. Our previous studies suggested that RRC-1 might be associated with the muscle cell membrane, and here we present evidence that this occurs via its PX domain, a domain that is known to bind to membrane phosphoinositides (PIPs). We predict the existence of a PX domain based on bioinformatic analysis and AlphaFold3, which includes conserved residues characteristic of most PX domains and a PIP binding site. This region of RRC-1 binds to phosphoinositides in vitro. Analysis of a nematode strain that has an in-frame deletion of the PX domain, indicates that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffold protein GIT-1. Lastly, we show that the overexpression of the full length RRC-1, but not RRC-1 with an in-frame deletion of its PX domain, results in reduced accumulation of IAC components and reduced whole animal movement. Our study highlights the importance of RRC-1s lipid interactions at the cell membrane for proper assembly and function of IACs in C. elegans muscle. Article SummaryIntegrin adhesion complexes (IACs) facilitate the transmission of force of muscle contraction to the outside of the cell. IACs, found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the PIX-1 (a RacGEF) signaling pathway for their assembly or maintenance. A RacGAP for the PIX pathway is RRC-1. Here, we show that RRC-1 has a PX domain that binds to membrane phosphoinositides. We also show that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffolding protein GIT-1.

Cytoskeletal structures aid in cell polarization, motility, and intracellular transport. Their functions are predicated on the rapid turnover of cytoskeleton proteins, which is achieved by the coordinated effort of multiple regulatory proteins whose dynamics are not well understood. In-vitro experiments have shown that free tubulin can repair nanoscale damages of microtubules created by severing proteins. Based on this observation, we propose a model for microtubule severing as a competition between the processes of damage spreading and tubulin-induced repair. Using theory and simulations, we demonstrate that this model is in quantitative agreement with in vitro experiments. We predict the existence of a critical tubulin concentration above which severing becomes rare but fast, and hypersensitive to the concentration of free tubulin. Further we show that this hypersensitivity leads to a dramatic increase in the dynamic range of steady-state microtubule lengths, when lengths are controlled by severing. Our work demonstrates how synergy between tubulin and severing proteins can lead to novel dynamical properties of microtubules.

During normal anaphase in animal cells, elastic tethers connect partner telomeres of segregating chromosomes and exert backward (anti-poleward) forces on those chromosomes. The experiments reported herein test whether microtubules need to be present in order for tethers to produce backwards forces. We disassembled spindle microtubules by treating anaphase crane-fly primary spermatocytes separately with nocodazole, colcemid, or podophyllotoxin. The drug treatments caused anaphase chromosomes to stop moving poleward; almost immediately thereafter they moved backward. The characteristics of the backward movements of the half-bivalents match those of the backwards movements of arm fragments formed by cutting chromosome arms during anaphase - for example the occurrence and lengths of backward movements were a function of tether length. The only difference from movement of arm fragments is that the chromosomes in the treated cells moved backwards slower than arm fragments did. Immunofluorescence of spindle tubulin after the drug treatments indicated that acetylated kinetochore microtubules were not depolymerized by the drugs, though the non-kinetochore spindle microtubules were depolymerized. Our data indicate that tethers move anaphase chromosomes backwards in the absence of functioning spindle microtubules. We suggest that the backward movements that take place when poleward forces are absent are due to tethers, and that the backward movements are slowed by the presence of acetylated kinetochore microtubules.

Kinesin-5 motors are bipolar tetramers that crosslink and slide antiparallel microtubules during mitotic spindle assembly. Fungal kinesin-5 motors, such as Cin8, exhibit bidirectional motility, switching between minus- and plus-end-directed stepping in response to environmental conditions; however, the molecular basis of this directional switching remains unclear. To better understand the origin of this bidirectional behavior, we investigated the motility and ATPase kinetics of two Cin8 dimers, created by fusing the motor domains to a stable coiled-coil domain from kinesin-1. To investigate the role of the proximal neck coiled-coil region in coordinating motor activity, we compared Cin8 dimers that included or lacked the first four heptads of the Cin8 neck-coil domain. By analyzing the stepping kinetics, microtubule residence times, and directional switching dynamics, we found that these Cin8 dimers move processively with a net plus-end directionality along with undirected movements, behaviors that mimic the plus-ended motility state of wild-type Cin8. However, fast minus-ended motility seen in wild-type Cin8 tetramers was not observed in the dimers. The instantaneous velocity distributions and ATPase rates were inconsistent with the undirected movement being solely due to passive diffusion, suggesting that they reflect random bidirectional stepping. Fewer undirected movements were seen on yeast microtubules, their native physiological substrate, compared to on bovine microtubules. Replacing the Cin8 neck-coil domain with a stable coiled-coil led to faster plus-end stepping, fewer undirected movements, a reduction in the microtubule binding duration, and enhanced coupling between ATP hydrolysis and plus-end stepping. Our results suggest that the native Cin8 neck coil confers flexibility between the two motor domains that contributes to bidirectional stepping, and that sustained minus-end movement requires regions outside the motor domain. Statement of SignificanceThe kinesin-5 family of motors, which contain two pairs of heads located on either end of a long stalk domain, power mitotic spindle formation by sliding antiparallel microtubules. The yeast kinesin-5, Cin8 moves to microtubule minus-ends under specific conditions, breaking the dogma that N-terminal kinesins move to microtubule plus-ends. To gain insight into how Cin8 changes its walking direction, we analyzed engineered Cin8 dimers, models of one half of full-length Cin8. The dimers step erratically, consistent with stepping in both directions, but have a plus-end bias. We find evidence that the two heads poorly coordinate, which contrasts with other kinesins, and suggest that the stepping direction may be regulated by altering the degree of inter-head coordination.