Cholesterol in the cargo membrane amplifies tau inhibition of kinesin-1-based transport.

Intracellular cargos are often membrane-enclosed and transported by microtubule-based motors in the presence of microtubule-associated proteins (MAPs). Whereas increasing evidence reveals how MAPs impact the interactions between motors and microtubules, critical questions remain about the impact of the cargo membrane on transport. Here we combined in vitro optical trapping with theoretical approaches to determine the effect of a lipid cargo membrane on kinesin-based transport in the presence of MAP tau. Our results demonstrate that attaching kinesin to a fluid lipid membrane reduces the inhibitory effect of tau on kinesin. Moreover, adding cholesterol, which reduces kinesin diffusion in the cargo membrane, amplifies the inhibitory effect of tau on kinesin binding in a dosage-dependent manner. We propose that reduction of kinesin diffusion in the cargo membrane underlies the effect of cholesterol on kinesin binding in the presence of tau, and we provide a simple model for this proposed mechanism. Our study establishes a direct link between cargo membrane cholesterol and MAP-based regulation of kinesin-1. The cholesterol effects uncovered here may more broadly extend to other lipid alterations that impact motor diffusion in the cargo membrane, including those associated with aging and neurological diseases.

A simple rule governs the evolution and development of hominin tooth size.

The variation in molar tooth size in humans and our closest relatives (hominins) has strongly influenced our view of human evolution. The reduction in overall size and disproportionate decrease in third molar size have been noted for over a century, and have been attributed to reduced selection for large dentitions owing to changes in diet or the acquisition of cooking. The systematic pattern of size variation along the tooth row has been described as a 'morphogenetic gradient' in mammal, and more specifically hominin, teeth since Butler and Dahlberg. However, the underlying controls of tooth size have not been well understood, with hypotheses ranging from morphogenetic fields to the clone theory. In this study we address the following question: are there rules that govern how hominin tooth size evolves? Here we propose that the inhibitory cascade, an activator-inhibitor mechanism that affects relative tooth size in mammals, produces the default pattern of tooth sizes for all lower primary postcanine teeth (deciduous premolars and permanent molars) in hominins. This configuration is also equivalent to a morphogenetic gradient, finally pointing to a mechanism that can generate this gradient. The pattern of tooth size remains constant with absolute size in australopiths (including Ardipithecus, Australopithecus and Paranthropus). However, in species of Homo, including modern humans, there is a tight link between tooth proportions and absolute size such that a single developmental parameter can explain both the relative and absolute sizes of primary postcanine teeth. On the basis of the relationship of inhibitory cascade patterning with size, we can use the size at one tooth position to predict the sizes of the remaining four primary postcanine teeth in the row for hominins. Our study provides a development-based expectation to examine the evolution of the unique proportions of human teeth.

A fluid membrane enhances the velocity of cargo transport by small teams of kinesin-1.

Kinesin-1 (hereafter referred to as kinesin) is a major microtubule-based motor protein for plus-end-directed intracellular transport in live cells. While the single-molecule functions of kinesin are well characterized, the physiologically relevant transport of membranous cargos by small teams of kinesins remains poorly understood. A key experimental challenge remains in the quantitative control of the number of motors driving transport. Here we utilized "motile fraction" to overcome this challenge and experimentally accessed transport by a single kinesin through the physiologically relevant transport by a small team of kinesins. We used a fluid lipid bilayer to model the cellular membrane in vitro and employed optical trapping to quantify the transport of membrane-enclosed cargos versus traditional membrane-free cargos under identical conditions. We found that coupling motors via a fluid membrane significantly enhances the velocity of cargo transport by small teams of kinesins. Importantly, enclosing a cargo in a fluid lipid membrane did not impact single-kinesin transport, indicating that membrane-dependent velocity enhancement for team-based transport arises from altered interactions between kinesins. Our study demonstrates that membrane-based coupling between motors is a key determinant of kinesin-based transport. Enhanced velocity may be critical for fast delivery of cargos in live cells.

GSK-3ß Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility.

Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3ß in both neurons and non-neuronal cells. GSK-3ß coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3ß, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3ß inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.

Quantitative Determination of the Probability of Multiple-Motor Transport in Bead-Based Assays.

With their longest dimension typically being less than 100 nm, molecular motors are significantly below the optical-resolution limit. Despite substantial advances in fluorescence-based imaging methodologies, labeling with beads remains critical for optical-trapping-based investigations of molecular motors. A key experimental challenge in bead-based assays is that the number of motors on a bead is not well defined. Particularly for single-molecule investigations, the probability of single- versus multiple-motor events has not been experimentally investigated. Here, we used bead travel distance as an indicator of multiple-motor transport and determined the lower-bound probability of bead transport by two or more motors. We limited the ATP concentration to increase our detection sensitivity for multiple- versus single-kinesin transport. Surprisingly, for all but the lowest motor number examined, our measurements exceeded estimations of a previous model by ≥2-fold. To bridge this apparent gap between theory and experiment, we derived a closed-form expression for the probability of bead transport by multiple motors, and constrained the only free parameter in this model using our experimental measurements. Our data indicate that kinesin extends to ∼57 nm during bead transport, suggesting that kinesin exploits its conformational flexibility to interact with microtubules at highly curved interfaces such as those present for vesicle transport in cells. To our knowledge, our findings provide the first experimentally constrained guide for estimating the probability of multiple-motor transport in optical trapping studies. The experimental approach utilized here (limiting ATP concentration) may be generally applicable to studies in which molecular motors are labeled with cargos that are artificial or are purified from cellular extracts.

Microtubule Defects Influence Kinesin-Based Transport In Vitro.

Microtubules are protein polymers that form "molecular highways" for long-range transport within living cells. Molecular motors actively step along microtubules to shuttle cellular materials between the nucleus and the cell periphery; this transport is critical for the survival and health of all eukaryotic cells. Structural defects in microtubules exist, but whether these defects impact molecular motor-based transport remains unknown. Here, we report a new, to our knowledge, approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo.

What did Hadropithecus eat, and why should paleoanthropologists care?

Over 40 years ago, Clifford Jolly noted different ways in which Hadropithecus stenognathus converged in its craniodental anatomy with basal hominins and with geladas. The Malagasy subfossil lemur Hadropithecus departs from its sister taxon, Archaeolemur, in that it displays comparatively large molars, reduced incisors and canines, a shortened rostrum, and thickened mandibular corpus. Its molars, however, look nothing like those of basal hominins; rather, they much more closely resemble molars of grazers such as Theropithecus. A number of tools have been used to interpret these traits, including dental microwear and texture analysis, molar internal and external morphology, and finite element analysis of crania. These tools, however, have failed to provide support for a simple dietary interpretation; whereas there is some consistency in the inferences they support, dietary inferences (e.g., that it was graminivorous, or that it specialized on hard objects) have been downright contradictory. Cranial shape may correlate poorly with diet. But a fundamental question remains unresolved: why do the various cranial and dental convergences exemplified by Hadropithecus, basal hominins, and Theropithecus exist? In this paper we review prior hypotheses regarding the diet of Hadropithecus. We then use stable carbon and nitrogen isotope data to elucidate this species' diet, summarizing earlier stable isotope analyses and presenting new data for lemurs from the central highlands of Madagascar, where Hadropithecus exhibits an isotopic signature strikingly different from that seen in other parts of the island. We offer a dietary explanation for these differences. Hadropithecus likely specialized neither on grasses nor hard objects; its staples were probably the succulent leaves of CAM plants. Nevertheless, aspects of prior hypotheses regarding the ecological significance of its morphology can be supported. Am. J. Primatol. 78:1098-1112, 2016. © 2015 Wiley Periodicals, Inc.

Tuning multiple motor travel via single motor velocity.

Microtubule-based molecular motors often work in small groups to transport cargos in cells. A key question in understanding transport (and its regulation in vivo) is to identify the sensitivity of multiple-motor-based motion to various single molecule properties. Whereas both single-motor travel distance and microtubule binding rate have been demonstrated to contribute to cargo travel, the role of single-motor velocity is yet to be explored. Here, we recast a previous theoretical study, and make explicit a potential contribution of velocity to cargo travel. We test this possibility experimentally, and demonstrate a strong negative correlation between single-motor velocity and cargo travel for transport driven by two motors. Our study thus discovers a previously unappreciated role of single-motor velocity in regulating multiple-motor transport.

Interplay between velocity and travel distance of kinesin-based transport in the presence of tau.

Although the disease-relevant microtubule-associated protein tau is known to severely inhibit kinesin-based transport in vitro, the potential mechanisms for reversing this detrimental effect to maintain healthy transport in cells remain unknown. Here we report the unambiguous upregulation of multiple-kinesin travel distance despite the presence of tau, via decreased single-kinesin velocity. Interestingly, the presence of tau also modestly reduced cargo velocity in multiple-kinesin transport, and our stochastic simulations indicate that the tau-mediated reduction in single-kinesin travel underlies this observation. Taken together, our observations highlight a nontrivial interplay between velocity and travel distance for kinesin transport, and suggest that single-kinesin velocity is a promising experimental handle for tuning the effect of tau on multiple-kinesin travel distance.

A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules.

Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here, we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP-Gly family of proteins. Using single-particle microtubule-binding assays, we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors - a process we term 'skating'. In contrast, the previously described CAP-Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon, dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP-Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.

Dental topography indicates ecological contraction of lemur communities.

Understanding the paleoecology of extinct subfossil lemurs requires reconstruction of dietary preferences. Tooth morphology is strongly correlated with diet in living primates and is appropriate for inferring dietary ecology. Recently, dental topographic analysis has shown great promise in reconstructing diet from molar tooth form. Compared with traditionally used shearing metrics, dental topography is better suited for the extraordinary diversity of tooth form among subfossil lemurs and has been shown to be less sensitive to phylogenetic sources of shape variation. Specifically, we computed orientation patch counts rotated (OPCR) and Dirichlet normal energy (DNE) of molar teeth belonging to 14 species of subfossil lemurs and compared these values to those of an extant lemur sample. The two metrics succeeded in separating species in a manner that provides insights into both food processing and diet. We used them to examine the changes in lemur community ecology in Southern and Southwestern Madagascar that accompanied the extinction of giant lemurs. We show that the poverty of Madagascar's frugivore community is a long-standing phenomenon and that extinction of large-bodied lemurs in the South and Southwest resulted not merely in a loss of guild elements but also, most likely, in changes in the ecology of extant lemurs.

Lemur habitat and dental senescence in Ranomafana National Park, Madagascar.

Not only can teeth provide clues about diet, but they also can be indicators of habitat quality. Conspecific groups living in different habitats with different kinds of foods may exhibit different rates of dental attrition because their teeth are less well adapted to some foods than to others. Ecological disequilibrium describes the situation in which animals live in habitats to which they are relatively poorly adapted. We test whether dental senescence, the wear-related decrease in dental functionality that is associated with decreased survival of infants born to older Propithecus edwardsi females, can be explained by ecological disequilibrium. Specifically, we compare the rates of dental wear in sifaka groups living in nearby habitats that differ in the degree of anthropogenically induced disturbance. We hypothesize that sifakas living in disturbed areas have an unusual rate of tooth wear compared to those living in a more pristine area, and that dental senescence is a consequence of an atypically high wear rate in a degraded habitat. To test whether habitat quality affects tooth wear more generally, we compare rates of use-wear in two subsets of Microcebus rufus living in either relatively undisturbed or disturbed habitats. Contrary to our predictions, we did not detect different rates of tooth wear in disturbed versus undisturbed habitats for either species and consider that reproductively detrimental dental senescence in P. edwardsi females is unlikely to be a pathological consequence of ecological disequilibrium.

Changes in orientation of attritional wear facets with implications for jaw motion in a mixed longitudinal sample of Propithecus edwardsi from Ranomafana National Park, Madagascar.

In many mammalian species, the progressive wearing down of the teeth that occurs over an individual's lifetime has the potential to change dental function, jaw movements, or even feeding habits. The orientation of phase-I wear facets on molars reveals the direction of jaw movement during the power stroke of mastication. We investigated if and how molar wear facets change with increasing wear and/or age by examining a mixed longitudinal dataset of mandibular tooth molds from wild Propithecus edwardsi (N = 32 individuals, 86 samples). Measurements of the verticality of wear facets were obtained from three-dimensional digital models generated from µCT scans. Results show that verticality decreases over the lifetime of P. edwardsi, a change that implies an increasingly lateral translation of the jaw as the teeth move into occlusion. A more transverse phase-I power stroke supports the hypothesis that these animals chew to maximize longevity and functionality of their teeth, minimizing the "waste" of enamel, while maintaining sharp shearing crests. Results of this study indicate that wear facet verticality is more closely correlated with age than overall amount of tooth wear, measured as area of exposed dentin, suggesting that age-related changes in cranial morphology may be more responsible for adjustments in jaw motion over the lifetimes of Propithecus than wear-related changes inthe shape of occluding teeth. Finally, the rate of decrease in wear facet verticality with age is greater in males than in females suggesting differences in development and/or access to resources between the sexes in this species.

Frugivory in four sympatric lemurs: implications for the future of Madagascar's forests.

Although some conservationists accept that not all species can be saved, we illustrate the difficulty in deciding which species are dispensable. In this article, we examine the possibility that the integrity of a forest relies on its entire faunal assemblage. In Madagascar, one faunal group, the lemurs, accounts for the greatest biomass and species richness among frugivores. For example, 7 of the 13 sympatric lemur species in Madagascar's eastern rainforests consume primarily fruit. Because of this, we suggest that some tree species may rely heavily on particular lemur taxa for both seed dispersal and germination. In Ranomafana National Park, the diets for four of the day-active lemur frugivores have been documented during annual cycles over a 5-year period. We predicted that, although the fruit of some plant taxa would be exploited by multiple lemur species, the fruit of others would be eaten by one lemur species alone. Analyses reveal that while lemurs overlap in a number of fruit taxa exploited, 46% (16/35) of families and 56% (29/52) of genera are eaten exclusively by one lemur species. We, therefore, predict local changes in forest composition and structure if certain of these lemur species are eliminated from a forest owing to hunting, disease, or habitat disturbance. We also suggest that this result may be of global significance because carbon sequestration by the tropical forests in Madagascar may be reduced as a result of this predicted change in forest composition.

Morphometrics and pattern of growth in wild sifakas (Propithecus edwardsi) at Ranomafana National Park, Madagascar.

We summarize morphometric data collected over a period of 22 years from a natural population of rainforest sifakas (Propithecus edwardsi) at Ranomafana National Park, Madagascar, and we use those data to document patterns of growth and development. Individually identified, known-age sifakas were successfully captured, measured, and released. We found that body segment lengths increased faster during growth than did body mass, with individuals attaining adult lengths earlier than adult mass. Females can begin reproducing before they are fully grown, but this may not be common. With the exception of hand length, we found no significant sex difference in any adult metric including body mass, chest, and limb circumferences, body segment lengths, and canine tooth height; however, body masses of individual females fluctuated more, independently of pregnancy, than did those of males. We found considerable interannual fluctuation in body mass with single individuals differing more within the same season in different years than from season to season in the same year. Such body mass fluctuation may be a consequence of eastern Madagascar's variable and unpredictable environment in which rainfall during any selected month varies from year to year.

Tuning microtubule-based transport through filamentous MAPs: the problem of dynein.

We recently proposed that regulating the single-to-multiple motor transition was a likely strategy for regulating kinesin-based transport in vivo. In this study, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein-based transport. We show that tau's regulation of kinesin function can proceed without interfering with dynein-based transport. Surprisingly, at extremely high tau levels--where kinesin cannot bind microtubules (MTs)--dynein can still contact MTs. The difference between tau's effects on kinesin- and dynein-based motility suggests that tau can be used to tune relative amounts of plus-end and minus-end-directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to MTs. We show that this isoform-specific effect is not because of steric interference of tau's projection domains but rather because of tau's interactions with the motor at the MT surface. Nonetheless, we do observe a modest steric interference effect of tau away from the MT and discuss the potential implications of this for molecular motor structure.

Cytoplasmic dynein functions as a gear in response to load.

Cytoskeletal molecular motors belonging to the kinesin and dynein families transport cargos (for example, messenger RNA, endosomes, virus) on polymerized linear structures called microtubules in the cell. These 'nanomachines' use energy obtained from ATP hydrolysis to generate force, and move in a step-like manner on microtubules. Dynein has a complex and fundamentally different structure from other motor families. Thus, understanding dynein's force generation can yield new insight into the architecture and function of nanomachines. Here, we use an optical trap to quantify motion of polystyrene beads driven along microtubules by single cytoplasmic dynein motors. Under no load, dynein moves predominantly with a mixture of 24-nm and 32-nm steps. When moving against load applied by an optical trap, dynein can decrease step size to 8 nm and produce force up to 1.1 pN. This correlation between step size and force production is consistent with a molecular gear mechanism. The ability to take smaller but more powerful strokes under load--that is, to shift gears--depends on the availability of ATP. We propose a model whereby the gear is downshifted through load-induced binding of ATP at secondary sites in the dynein head.

Mice with an autosomal dominant Charcot-Marie-Tooth type 2O disease mutation in both dynein alleles display severe moto-sensory phenotypes.

Charcot-Marie-Tooth disease (CMT) is the most common peripheral neuromuscular disorder worldwide. The axonal degeneration in CMT causes distal muscle weakness and atrophy, resulting in gait problems and difficulties with basic motor coordination skills. A mutation in the cytoplasmic dynein heavy chain (DHC) gene was discovered to cause an autosomal dominant form of the disease designated Charcot-Marie-Tooth type 2O disease (CMT2O) in 2011. The mutation is a single amino acid change of histidine into arginine at amino acid 306 (H306R) in DHC. We previously generated a knock-in mouse carrying the corresponding CMT2O mutation (H304R) and examined the heterozygous H304R/+offspring in a variety of motor skills and histological assays. Here we report the initial characterization of the homozygous H304R/R mouse, which is the first homozygous mutant DHC mouse to survive past the neonatal stage. We show that H304R/R mice have significantly more severe disease symptoms than the heterozygous H304R/+mice. The H304R/R mice have significant defects in motor skills, including grip strength, motor coordination, and gait and also related defects in neuromuscular junction architecture. Furthermore, the mice have defects in sensation, another aspect of CMT disease. Our results show that the H304R/+ and H304R/R mice will be important models for studying the onset and progression of both heterozygous and homozygous CMT disease alleles.

Histone deacetylase 6 inhibition compensates for the transport deficit in Huntington's disease by increasing tubulin acetylation.

A defect in microtubule (MT)-based transport contributes to the neuronal toxicity observed in Huntington's disease (HD). Histone deacetylase (HDAC) inhibitors show neuroprotective effects in this devastating neurodegenerative disorder. We report here that HDAC inhibitors, including trichostatin A (TSA), increase vesicular transport of brain-derived neurotrophic factor (BDNF) by inhibiting HDAC6, thereby increasing acetylation at lysine 40 of alpha-tubulin. MT acetylation in vitro and in cells causes the recruitment of the molecular motors dynein and kinesin-1 to MTs. In neurons, acetylation at lysine 40 of alpha-tubulin increases the flux of vesicles and the subsequent release of BDNF. We show that tubulin acetylation is reduced in HD brains and that TSA compensates for the transport- and release-defect phenotypes that are observed in disease. Our findings reveal that HDAC6 inhibition and acetylation at lysine 40 of alpha-tubulin may be therapeutic targets of interest in disorders such as HD in which intracellular transport is altered.

A novel mouse model carrying a human cytoplasmic dynein mutation shows motor behavior deficits consistent with Charcot-Marie-Tooth type 2O disease.

Charcot-Marie-Tooth disease (CMT) is a peripheral neuromuscular disorder in which axonal degeneration causes progressive loss of motor and sensory nerve function. The loss of motor nerve function leads to distal muscle weakness and atrophy, resulting in gait problems and difficulties with walking, running, and balance. A mutation in the cytoplasmic dynein heavy chain (DHC) gene was discovered to cause an autosomal dominant form of the disease designated Charcot-Marie-Tooth type 2 O disease (CMT2O) in 2011. The mutation is a single amino acid change of histidine into arginine at amino acid 306 (H306R) in DHC. In order to understand the onset and progression of CMT2, we generated a knock-in mouse carrying the corresponding CMT2O mutation (H304R/+). We examined H304R/+ mouse cohorts in a 12-month longitudinal study of grip strength, tail suspension, and rotarod assays. H304R/+ mice displayed distal muscle weakness and loss of motor coordination phenotypes consistent with those of individuals with CMT2. Analysis of the gastrocnemius of H304R/+ male mice showed prominent defects in neuromuscular junction (NMJ) morphology including reduced size, branching, and complexity. Based on these results, the H304R/+ mouse will be an important model for uncovering functions of dynein in complex organisms, especially related to CMT onset and progression.