Bison phylogeography constrains dispersal and viability of the Ice Free Corridor in western Canada.

The Ice Free Corridor has been invoked as a route for Pleistocene human and animal dispersals between eastern Beringia and more southerly areas of North America. Despite the significance of the corridor, there are limited data for when and how this corridor was used. Hypothetical uses of the corridor include: the first expansion of humans from Beringia into the Americas, northward postglacial expansions of fluted point technologies into Beringia, and continued use of the corridor as a contact route between the north and south. Here, we use radiocarbon dates and ancient mitochondrial DNA from late Pleistocene bison fossils to determine the chronology for when the corridor was open and viable for biotic dispersals. The corridor was closed after ∼23,000 until 13,400 calendar years ago (cal y BP), after which we find the first evidence, to our knowledge, that bison used this route to disperse from the south, and by 13,000 y from the north. Our chronology supports a habitable and traversable corridor by at least 13,000 cal y BP, just before the first appearance of Clovis technology in interior North America, and indicates that the corridor would not have been available for significantly earlier southward human dispersal. Following the opening of the corridor, multiple dispersals of human groups between Beringia and interior North America may have continued throughout the latest Pleistocene and early Holocene. Our results highlight the utility of phylogeographic analyses to test hypotheses about paleoecological history and the viability of dispersal routes over time.

Cooperative responses of multiple kinesins to variable and constant loads.

Microtubule-dependent transport is most often driven by collections of kinesins and dyneins that function in either a concerted fashion or antagonistically. Several lines of evidence suggest that cargo transport may not be influenced appreciably by the combined action of multiple kinesins. Yet, as in previous optical trapping experiments, the forces imposed on cargos will vary spatially and temporally in cells depending on a number of local environmental factors, and the influence of these conditions has been largely overlooked. Here, we characterize the dynamics of structurally defined complexes containing multiple kinesins under the controlled loads of an optical force clamp. While demonstrating that there are generic kinetic barriers that restrict the ability of multiple kinesins to cooperate productively, the spatial and temporal properties of applied loads is found to play an important role in the collective dynamics of multiple motor systems. We propose this dependence has implications for intracellular transport processes, especially for bidirectional transport.

Collective dynamics of elastically coupled myosin V motors.

Characterization of the collective behaviors of different classes of processive motor proteins has become increasingly important to understand various intracellular trafficking and transport processes. This work examines the dynamics of structurally-defined motor complexes containing two myosin Va (myoVa) motors that are linked together via a molecular scaffold formed from a single duplex of DNA. Dynamic changes in the filament-bound configuration of these complexes due to motor binding, stepping, and detachment were monitored by tracking the positions of different color quantum dots that report the position of one head of each myoVa motor on actin. As in studies of multiple kinesins, the run lengths produced by two myosins are only slightly larger than those of single motor molecules. This suggests that internal strain within the complexes, due to asynchronous motor stepping and the resultant stretching of motor linkages, yields net negative cooperative behaviors. In contrast to multiple kinesins, multiple myosin complexes move with appreciably lower velocities than a single-myosin molecule. Although similar trends are predicted by a discrete state stochastic model of collective motor dynamics, these analyses also suggest that multiple myosin velocities and run lengths depend on both the compliance and the effective size of their cargo. Moreover, it is proposed that this unique collective behavior occurs because the large step size and relatively small stalling force of myoVa leads to a high sensitivity of motor stepping rates to strain.

Does the Locally-Adaptive Model of Archaeological Potential (LAMAP) work for hunter-gatherer sites? A test using data from the Tanana Valley, Alaska.

We report an assessment of the ability of the Locally-Adaptive Model of Archaeological Potential (LAMAP) to estimate archaeological potential in relation to hunter-gatherer sites. The sample comprised 182 known sites in the Tanana Valley, Alaska, which was occupied solely by hunter-gatherers for about 14,500 years. To estimate archaeological potential, we employed physiographic variables such as elevation and slope, rather than variables that are known to vary on short time scales, like vegetation cover. Two tests of LAMAP were carried out. In the first, we used the location of a random selection of 90 sites from all time periods to create a LAMAP model. We then evaluated the model with the remaining 92 sites. In the second test, we built a LAMAP model from 12 sites that pre-date 10,000 cal BP. This model was then tested with sites that post-date 10,000 cal BP. In both analyses, areas predicted to have higher archaeological potential contained higher frequencies of validation sites. The performance of LAMAP in the two tests was comparable to its performance in previous tests using archaeological sites occupied by agricultural societies. Thus, the study extends the use of LAMAP to the task of estimating archaeological potential of landscapes in relation to hunter-gatherer sites.

Productive cooperation among processive motors depends inversely on their mechanochemical efficiency.

Subcellular cargos are often transported by teams of processive molecular motors, which raises questions regarding the role of motor cooperation in intracellular transport. Although our ability to characterize the transport behaviors of multiple-motor systems has improved substantially, many aspects of multiple-motor dynamics are poorly understood. This work describes a transition rate model that predicts the load-dependent transport behaviors of multiple-motor complexes from detailed measurements of a single motor's elastic and mechanochemical properties. Transition rates are parameterized via analyses of single-motor stepping behaviors, load-rate-dependent motor-filament detachment kinetics, and strain-induced stiffening of motor-cargo linkages. The model reproduces key signatures found in optical trapping studies of structurally defined complexes composed of two kinesin motors, and predicts that multiple kinesins generally have difficulties in cooperating together. Although such behavior is influenced by the spatiotemporal dependence of the applied load, it appears to be directly linked to the efficiency of kinesin's stepping mechanism, and other types of less efficient and weaker processive motors are predicted to cooperate more productively. Thus, the mechanochemical efficiencies of different motor types may determine how effectively they cooperate together, and hence how motor copy number contributes to the regulation of cargo motion.

Two kinesins transport cargo primarily via the action of one motor: implications for intracellular transport.

The number of microtubule motors attached to vesicles, organelles, and other subcellular commodities is widely believed to influence their motile properties. There is also evidence that cells regulate intracellular transport by tuning the number and/or ratio of motor types on cargos. Yet, the number of motors responsible for cargo motion is not easily characterized, and the extent to which motor copy number affects intracellular transport remains controversial. Here, we examined the load-dependent properties of structurally defined motor assemblies composed of two kinesin-1 molecules. We found that a group of kinesins can produce forces and move with velocities beyond the abilities of single kinesin molecules. However, such capabilities are not typically harnessed by the system. Instead, two-kinesin assemblies adopt a range of microtubule-bound configurations while transporting cargos against an applied load. The binding arrangement of motors on their filament dictates how loads are distributed within the two-motor system, which in turn influences motor-microtubule affinities. Most configurations promote microtubule detachment and prevent both kinesins from contributing to force production. These results imply that cargos will tend to be carried by only a fraction of the total number of kinesins that are available for transport at any given time, and provide an alternative explanation for observations that intracellular transport depends weakly on kinesin number in vivo.

Coupling between motor proteins determines dynamic behaviors of motor protein assemblies.

Transport of intracellular cargos by multiple microtubule motor proteins is believed to be a common and significant phenomenon in vivo, yet signatures of the microscopic dynamics of multiple motor systems are only now beginning to be resolved. Understanding these mechanisms largely depends on determining how grouping motors affect their association with microtubules and stepping rates, and hence, cargo run lengths and velocities. We examined this problem using a discrete state transition rate model of collective transport. This model accounts for the structural and mechanical properties in binding/unbinding and stepping transitions between distinct microtubule-bound configurations of a multiple motor system. In agreement with previous experiments that examine the dynamics of two coupled kinesin-1 motors, the energetic costs associated with deformations of mechanical linkages within a multiple motor assembly are found to reduce the system's overall microtubule affinity, producing attenuated mean cargo run lengths compared to cases where motors are assumed to function independently. With our present treatment, this attenuation largely stems from reductions in the microtubule binding rate and occurs even when mechanical coupling between motors is weak. Thus, our model suggests that, at least for a variety of kinesin-dependent transport processes, the net 'gains' obtained by grouping motors together may be smaller than previously expected.

Archaeological assessment reveals Earth's early transformation through land use.

Environmentally transformative human use of land accelerated with the emergence of agriculture, but the extent, trajectory, and implications of these early changes are not well understood. An empirical global assessment of land use from 10,000 years before the present (yr B.P.) to 1850 CE reveals a planet largely transformed by hunter-gatherers, farmers, and pastoralists by 3000 years ago, considerably earlier than the dates in the land-use reconstructions commonly used by Earth scientists. Synthesis of knowledge contributed by more than 250 archaeologists highlighted gaps in archaeological expertise and data quality, which peaked for 2000 yr B.P. and in traditionally studied and wealthier regions. Archaeological reconstruction of global land-use history illuminates the deep roots of Earth's transformation and challenges the emerging Anthropocene paradigm that large-scale anthropogenic global environmental change is mostly a recent phenomenon.

Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips.

Disassembling microtubules can generate movement independently of motor enzymes, especially at kinetochores where they drive chromosome motility. A popular explanation is the 'conformational wave' model, in which protofilaments pull on the kinetochore as they curl outward from a disassembling tip. But whether protofilaments can work efficiently via this spring-like mechanism has been unclear. By modifying a previous assay to use recombinant tubulin and feedback-controlled laser trapping, we directly demonstrate the spring-like elasticity of curling protofilaments. Measuring their mechanical work output suggests they carry ~25% of the energy of GTP hydrolysis as bending strain, enabling them to drive movement with efficiency similar to conventional motors. Surprisingly, a ß-tubulin mutant that dramatically slows disassembly has no effect on work output, indicating an uncoupling of disassembly speed from protofilament strain. These results show the wave mechanism can make a major contribution to kinetochore motility and establish a direct approach for measuring tubulin mechano-chemistry.

Rapid measurement of mitotic spindle orientation in cultured mammalian cells.

Factors that influence the orientation of the mitotic spindle are important for the maintenance of stem cell populations and in cancer development. However, screening for these factors requires rapid quantification of alterations of the angle of the mitotic spindle in cultured cell lines. Here we describe a method to image mitotic cells and rapidly score the angle of the mitotic spindle using a simple MATLAB application to analyze a stack of Z-images.

Construction and analyses of elastically coupled multiple-motor systems.

Precision analyses of the collective motor behaviors have become important to dissecting mechanisms underlying the trafficking of subcellular commodities in eukaryotic cells. Here, we describe a synthetic approach to create structurally defined multiple protein complexes containing two elastically coupled motor molecules. Motors are connected using a simple DNA-scaffolding molecule and DNA-conjugated, artificial protein polymers that function as tunable elastic linkers. The procedure to self-assemble these components produces complexes in high synthetic yield and allows individual multiple-motor systems to be interrogated at the single-complex level. Methods to evaluate cooperative motor responses in a static optical trap are also discussed. While enabling the average transport properties of single/noninteracting and coupled motors to be compared, these procedures can provide insight into the extent to which motors cooperate productively via load sharing as well as the roles loading-rate-dependent phenomena play in collective motor functions.

Measuring kinetochore-microtubule interaction in vitro.

Many proteins and protein complexes perform sophisticated, regulated functions in vivo. Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause-effect relationships, for example, between a protein and its phosphorylating kinase. Here, we describe a protocol to purify kinetochores, the protein complexes that attach chromosomes to microtubules during mitosis, and quantitatively assay their microtubule-binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of microtubules to kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of kinetochore-microtubule attachments, which is believed to be critical for correcting erroneous kinetochore-spindle attachments and thereby avoiding chromosome missegregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology.

Analysis of Cooperative Behavior in Multiple Kinesins Motor Protein Transport by Varying Structural and Chemical Properties.

Intracellular transport is a fundamental biological process during which cellular materials are driven by enzymatic molecules called motor proteins. Recent optical trapping experiments and theoretical analysis have uncovered many features of cargo transport by multiple kinesin motor protein molecules under applied loads. These studies suggest that kinesins cooperate negatively under typical transport conditions, although some productive cooperation could be achieved under higher applied loads. However, the microscopic origins of this complex behavior are still not well understood. Using a discrete-state stochastic approach we analyze factors that affect the cooperativity among kinesin motors during cargo transport. Kinesin cooperation is shown to be largely unaffected by the structural and mechanical parameters of a multiple motor complex connected to a cargo, but much more sensitive to biochemical parameters affecting motor-filament affinities. While such behavior suggests the net negative cooperative responses of kinesins will persist across a relatively wide range of cargo types, it is also shown that the rates with which cargo velocities relax in time upon force perturbations are influenced by structural factors that affect the free energies of and load distributions within a multiple kinesin complex. The implications of these later results on transport phenomena where loads change temporally, as in the case of bidirectional transport, are discussed.

How the interplay between mechanical and nonmechanical interactions affects multiple kinesin dynamics.

Intracellular transport is supported by enzymes called motor proteins that are often coupled to the same cargo and function collectively. Recent experiments and theoretical advances have been able to explain certain behaviors of multiple motor systems by elucidating how unequal load sharing between coupled motors changes how they bind, step, and detach. However, nonmechanical interactions are typically overlooked despite several studies suggesting that microtubule-bound kinesins interact locally via short-range nonmechanical potentials. This work develops a new stochastic model to explore how these types of interactions influence multiple kinesin functions in addition to mechanical coupling. Nonmechanical interactions are assumed to affect kinesin mechanochemistry only when the motors are separated by less than three microtubule lattice sites, and it is shown that relatively weak interaction energies (~2 k(B)T) can have an appreciable influence over collective motor velocities and detachment rates. In agreement with optical trapping experiments on structurally defined kinesin complexes, the model predicts that these effects primarily occur when cargos are transported against loads exceeding single-kinesin stalling forces. Overall, these results highlight the interdependent nature of factors influencing collective motor functions, namely, that the way the bound configuration of a multiple motor system evolves under load determines how local nonmechanical interactions influence motor cooperation.

Negative interference dominates collective transport of kinesin motors in the absence of load.

The collective function of motor proteins is known to be important for the directed transport of many intracellular cargos. However, understanding how multiple motors function as a group remains challenging and requires new methods that enable determination of both the exact number of motors participating in motility and their organization on subcellular cargos. Here we present a biosynthetic method that enables exactly two kinesin-1 molecules to be organized on linear scaffolds that separate the motors by a distance of 50 nm. Tracking the motions of these complexes revealed that while two motors produce longer average run lengths than single kinesins, the system effectively behaves as though a single-motor attachment state dominates motility. It is proposed that negative motor interference derived from asynchronous motor stepping and the communication of forces between motors leads to this behavior by promoting the rapid exchange between different microtubule-bound configurations of the assemblies.

The use of fullerene substituted phenylalanine amino acid as a passport for peptides through cell membranes.

We report the formation of a fullerene-peptide conjugate via the incorporation of a fullerene substituted phenylalanine derivative, "Bucky amino acid" (Baa), to a cationic peptide, which acts as a passport for intracellular delivery, enabling transport of a range of sequences into HEK-293, HepG2, and neuroblastoma cells where the peptides in the absence of the fullerene amino acid cannot enter the cell. Delivery of the fullerene species to either the cytoplasm or nucleus of the cell is demonstrated. Fullerene peptides based on the nuclear localization sequence (NLS), H-Baa-Lys(FITC)-Lys-Lys-Arg-Lys-Val-OH, can actively cross over the cell membrane and accumulate significantly around the nucleus of HEK-293 and neuroblastoma cells, while H-Baa-Lys(FITC)-Lys8-OH accumulates in the cytoplasm. Cellular studies show that the uptake for the anionic peptide Baa-Lys(FITC)Glu4Gly3Ser-OH is greatly reduced in comparison with the cationic fullerene peptides of the same concentration. The hydrophobic nature of the fullerene assisting peptide transport is suggested by the effect of gamma-cyclodextrin (CD) in lowering the efficacy of transport. These data suggest that the incorporation of a fullerene-based amino acid provides a route for the intracellular delivery of peptides and as a consequence the creation of a new class of cell penetrating peptides.

Fullerene-derivatized amino acids: synthesis, characterization, antioxidant properties, and solid-phase peptide synthesis.

A series of [60]fullerene-substituted phenylalanine (Baa) and lysine derivatives have been prepared by the condensation of 1,2-(4'-oxocyclohexano)fullerene with the appropriately protected (4-amino)phenylalanine and lysine, respectively. Conversion of the imine to the corresponding amine is achieved by di-acid catalyzed hydroboration. The reduction of the imine is not accompanied by hydroboration of the fullerene cage. The [70]fullerene phenylalanine derivative has also been prepared as have the di-amino acid derivatives. The compounds were characterized by MALDI-TOF mass spectrometry, UV/Vis spectroscopy, and cyclic voltammetry. 1H and 13C NMR spectroscopy allowed the observation of diastereomers. Fullerene-substituted peptides may be synthesized on relatively large scale by solid-phase peptide synthesis. The presence of the C60-substituted amino acid in a peptide has a significant effect on the secondary structures and self-assembly properties of peptides as compared to the native peptide. The antioxidant assay of Baa and a Baa-derived anionic peptide was determined to be significantly more potent than Trolox.

Rise and fall of the Beringian steppe bison.

The widespread extinctions of large mammals at the end of the Pleistocene epoch have often been attributed to the depredations of humans; here we present genetic evidence that questions this assumption. We used ancient DNA and Bayesian techniques to reconstruct a detailed genetic history of bison throughout the late Pleistocene and Holocene epochs. Our analyses depict a large diverse population living throughout Beringia until around 37,000 years before the present, when the population's genetic diversity began to decline dramatically. The timing of this decline correlates with environmental changes associated with the onset of the last glacial cycle, whereas archaeological evidence does not support the presence of large populations of humans in Eastern Beringia until more than 15,000 years later.