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Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP-MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.

Acidification of phagosomes is essential for the bactericidal activity of macrophages. Targeting machinery that regulates pH within the phagosomes is a prominent strategy employed by various pathogens that have emerged as major threats to public health. Nascent phagosomes acquire the machinery for pH regulation through a graded maturation process involving fusion with endolysosomes. Meticulous coordination between proton pumping and leakage mechanisms is crucial for maintaining optimal pH within the phagosome. However, relative to mechanisms involved in acidifying the phagosome lumen, little is known about proton leakage pathways in this organelle. Sodium proton transporter NHE9 is a known proton leakage pathway located on the endosomes. As phagosomes acquire proteins through fusions with endosomes during maturation, NHE9 seemed a promising candidate for regulating proton fluxes on the phagosome. Here, using genetic and biophysical approaches, we show NHE9 is an important proton leakage pathway associated with the maturing phagosome. NHE9 is highly expressed in immune cells, specifically macrophages; however, NHE9 expression is strongly downregulated upon bacterial infection. We show that compensatory ectopic NHE9 expression hinders the directed motion of phagosomes along microtubules and promotes early detachment from the microtubule tracks. As a result, these phagosomes have shorter run lengths and are not successful in reaching the lysosome. In accordance with this observation, we demonstrate that NHE9 expression levels negatively correlate with bacterial survival. Together, our findings show that NHE9 regulates lumenal pH to affect phagosome maturation, and consequently, microbicidal activity in macrophages.

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. We thank F. I. Ataullakhanov for help with the laser trap and data analysis; A. Kiyatkin, V. Mustyatsa, M. Molodtsov, A. Gautreau, G. Lakisic, and M. Barisic for technical assistance; and members of our laboratories for stimulating discussions. This work was supported by National Institutes of Health grant R01-GM098389 and RSG-14-018-01-CCG from the American Cancer Society to E.L.G.; by the Institut Curie, the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the L'Agence Nationale de la Recherche (ANR) award ANR-12-BSV2-0007, INCA_6517, ANR-10-LBX-0038, part of the IDEX Idex PSL, ANR-10-IDEX-0001-02 PSL to C.J.; and Fundacao Luso-Americana para o Desenvolvimento (FLAD) Life Science 2020 and PRECISE grant from the European Research Council to H.M. A.V.Z. is supported by the RAS Presidium Grants "Mechanisms of the Molecular Systems Integration," " Molecular and Cell Biology programs," and Russian Fund for Basic Research Grant 12-04-00111-a and 13-00-40188. R.S.S. is supported by a fellowship from the Programa Graduado em Areas da Biologia Basica e Aplicada (GABBA) PhD program from the University of Porto. A.L.P. is supported by fellowship SFRH/BPD/66707/2009 from Fundacao para a Ciencia e a Tecnologia of Portugal. M.B., R.S.S., S.K.T., M.M.M., C.J., E.L.G., and H.M. designed the experiments; M.B. performed all experiments in cells; M. M. M. established and performed the tubulin purification protocol from HeLa cells; R.S.S. performed single-molecule experiments; S.K.T. performed force measurements; A.L.P. provided reagents; all authors analyzed data; H.M., E.L.G., and M.B. wrote the paper, with contributions from all authors; H.M. conceived and coordinated the project. Data described can be found in the main figures and supplementary materials. The authors declare no conflict of interests.

Dynactin is the longest known cytoplasmic dynein regulator, with roles in dynein recruitment to subcellular cargo and in stimulating processive dynein movement. The latter function was thought to involve the N-terminal microtubule-binding region of the major dynactin polypeptide p150(Glued), although recent results disputed this. To understand how dynactin regulates dynein we generated recombinant fragments of the N-terminal half of p150(Glued). We find that the dynein-binding coiled-coil α-helical domain CC1B is sufficient to stimulate dynein processivity, which it accomplishes by increasing average dynein step size and forward-step frequency, while decreasing lateral stepping and microtubule detachment. In contrast, the immediate upstream coiled-coil domain, CC1A, activates a surprising diffusive dynein state. CC1A interacts physically with CC1B and interferes with its effect on dynein processivity. We also identify a role for the N-terminal portion of p150(Glued) in coordinating these activities. Our results reveal an unexpected form of long-range allosteric control of dynein motor function by internal p150(Glued) sequences, and evidence for p150(Glued) autoregulation.

There is significant interest in quantifying force production inside cells, but since conditions in vivo are less well controlled than those in vitro, in vivo measurements are challenging. In particular, the in vivo environment may vary locally as far as its optical properties, and the organelles manipulated by the optical trap frequently vary in size and shape. Several methods have been proposed to overcome these difficulties. We evaluate the relative merits of these methods and directly compare two of them, a refractive index matching method, and a light-momentum-change method. Since in vivo forces are frequently relatively high (e.g., can exceed 15 pN for lipid droplets), a high-power laser is employed. We discover that this high-powered trap induces local temperature changes, and we develop an approach to compensate for uncertainties in the magnitude of applied force due to such temperature variations.

We review our recent results obtained on an AlN/GaN-based high-electron-mobility transistor. The temperature of the electrons drifting under a relatively-high electric field is significantly higher than the lattice temperature (i.e., the hot electrons are generated). These hot electrons are produced through the Frohlich interaction between the drifting electrons and long-lived longitudinal-optical phonons. By fitting electric field vs. electron temperature deduced from the measurements of photoluminescence spectra to a theoretical model, we have deduced the longitudinal-optical-phonon emission time for each electron is to be on the order of 100 fs. We have also measured the decay time constant for LO phonons to be about 4.2 ps. An electric field present in a GaN/AlN heterostructure can bring both the first-order and second-order Raman scattering processes into strong resonances. The resonant Stokes and anti-Stokes Raman scattering results in the increase and decrease of non-equilibrium longitudinal-optical phonon temperatures, respectively. Moreover, the phonon temperature measured from the Raman scattering is increased with an applied electric field at a much higher rate than the lattice temperature due to the presence of field-induced non-equilibrium longitudinal-optical phonons.

We investigate coherences between σ + and σ - exciton states in a 10 nm thick ZnSe/ZnMgSe single quantum well by spectrally resolved three-pulse degenerate four-wave mixing using circularly polarized 100 fs pulses. Intensity dependent measurements are performed to study the influence of exciton-exciton scattering on their dephasing rates. The experiments reveal nearly equal zero-density dephasing rates for coherences between σ + and σ - heavy- and/or light-hole exciton states as compared to the dephasing rate of the interband coherence between these states and the ground state. The measurements also demonstrate that exciton-exciton scattering destroys the coherence between heavy- and light-hole excitons about three times more effectively than the coherence between σ + and σ - heavy-hole excitons.

The kinesin family proteins are often studied as prototypical molecular motors; a deeper understanding of them can illuminate regulation of intracellular transport. It is typically assumed that they function identically. Here we find that this assumption of homogeneous function appears incorrect: variation among motors’ velocities in vivo and in vitro is larger than the stochastic variation expected for an ensemble of “identical” motors. When moving on microtubules, slow and fast motors are persistently slow, and fast, respectively. We develop theory that provides quantitative criteria to determine whether the observed single-molecule variation is too large to be generated from an ensemble of identical molecules. To analyze such heterogeneity, we group traces into homogeneous sub-ensembles. Motility studies varying the temperature, pH and glycerol concentration suggest at least 2 distinct functional states that are independently affected by external conditions. We end by investigating the functional ramifications of such heterogeneity through Monte-Carlo multi-motor simulations.

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Intracellular transport via the microtubule motors kinesin and dynein plays an important role in maintaining cell structure and function. Often, multiple kinesin or dynein motors move the same cargo. Their collective function depends critically on the single motors’ detachment kinetics under load, which we experimentally measure here. This experimental constraint—combined with other experimentally determined parameters—is then incorporated into theoretical stochastic and mean-field models. Comparison of modeling results and in vitro data shows good agreement for the stochastic, but not mean-field, model. Many cargos in vivo move bidirectionally, frequently reversing course. Because both kinesin and dynein are present on the cargos, one popular hypothesis explaining the frequent reversals is that the opposite-polarity motors engage in unregulated stochastic tugs-of-war. Then, the cargos’ motion can be explained entirely by the outcome of these opposite-motor competitions. Here, we use fully calibrated stochastic and mean-field models to test the tug-of-war hypothesis. Neither model agrees well with our in vivo data, suggesting that, in addition to inevitable tugs-of-war between opposite motors, there is an additional level of regulation not included in the models.

Mechanisms for anti-Stokes photoluminescence observed at room temperature from n-type free-standing GaN have been attributed by us to the competition between two-photon absorption and phonon-assisted absorption.

Phonon-assisted anti-Stokes fluorescence has been observed in GaN film grown on Si (111) substrate. The donor-acceptor pairs and bound excitons have played primary roles in the generation of anti-Stokes fluorescence.

We have observed that the temperature of the electrons drifting under a relatively-high electric field in an AlN/GaN-based high-electron-mobility transistor is significantly higher than the lattice temperature (i.e. the hot electrons are generated). These hot electrons are produced through the Frohlich interaction between the drifting electrons and long-lived longitudinal-optical phonons. By fitting electric field vs. electron temperature deduced from the measurements of photoluminescence spectra to a theoretical model, we have deduced the longitudinal-optical-phonon emission time for each electron is to be on the order of 100 fs. This value is consistent with the value measured previously from Raman scattering.

We observe coherent exciton-LO-phonon polarons in a $3\phantom{\rule{0.3em}{0ex}}\mathrm{nm}$ ZnSe single quantum well using four-wave mixing with $30\phantom{\rule{0.3em}{0ex}}\mathrm{fs}$ light pulses. The formation of these superposition states is attributed to a strong LO-phonon coupling by the Fr\"ohlich interaction in a system in which the exciton binding exceeds the LO-phonon energy and the $2s$ exciton is nearly resonant with the LO-phonon energy. The rapid decay of these coherent quasiparticles is mainly attributed to the disintegration into zero-phonon excitons and free LO phonons as well as to the inhomogeneous broadening of LO-phonon energies due to disorder and $\mathbf{k}$-space dispersion.

We report on the decoherence of excitons in ZnSe single quantum wells by intensity dependent four‐wave‐mixing (FWM) experiments using 30 fs pulses. The FWM traces at the 11h exciton transition reveal marked quantum beats and a fast decay at pulse overlap that is attributed to a destructive interference of the polarization wavepacket and to an effective diffraction of the exciton polarization at an electron‐hole‐pair density grating. Three‐pulse FWM experiments are performed to study the formation of excitons from this electron‐hole‐pair grating.

We report on an exciton resonant phase coherent photorefractive effect in ZnMgSe/ZnSe single quantum wells using 30 fs light pulses. The experiments are performed in a four‐wave‐mixing configuration using two or three pulses. The PCP effect is attributed to the formation of an electron grating within the QW that is caused by the interference of excitons in the ZnMgSe barrier.

We observe a phase coherent all‐optical photorefractive effect in a ZnSe single quantum well in four‐wave‐mixing configuration using ultrashort pulses. The microscopic origin of the observed effect is attributed to the coherent formation of an electron grating by interfering excitons.

We investigate interband coherences as well as coherences between δ + and δ - exciton states in a 10 nm thick ZnSe/ZnMgSe single quantum well by spectrally resolved three-pulse degenerate four-wave mixing using circularlypolarized 100 fs pulses. Polarization and intensity dependent measurements are performed to identify the coherent processes responsible for the nonlinear signal into the k 3 +k 2 -k 1 direction and to study the influence of Coulomb scattering on the dephasing rates. The experiments reveal nearly equal zero-density dephasing rates for coherences between δ + and δ - heavy- and/or light-hole exciton states as compared to the dephasing rate of the interband coherence between these states and the ground state. The measurements also demonstrate that exciton-exciton scattering destroys the phase coherence between heavy- and light-hole excitons about three times more effectively than the coherence between δ + and δ - heavy-hole excitons. The characteristic features of the experimental results are explained and reproduced by numerical calculations of the optical Bloch equations for a ten-level model including excitation induced dephasing.

We have observed anti-Stokes photoluminescence from n-type free-standing GaN at room temperature. Such a process is caused by phonon-assisted absorption. When the excitation photon energy is sufficiently below the donor–acceptor transition energy, however, two-photon absorption becomes the dominant mechanism for anti-Stokes photoluminescence. By measuring the dependences of the photoluminescence spectra on temperature, excitation power and excitation photon energy, we have demonstrated that the donor–acceptor pair transition plays an important role in anti-Stokes photoluminescence. Our study could result in efficient laser cooling of semiconductors.

Phonon-assisted anti-Stokes photoluminescence (ASPL) in the ultraviolet region has been observed in the GaN film grown on a Si (111) substrate. The ASPL peaks are observable only at sufficiently low temperatures. In addition, even if the photon energy is ≈318meV below the transition energy for bound excitons, the ASPL peaks can be still observed. Based on our analysis, the donor-acceptor pairs and bound excitons have played primary roles in the generation of ASPL. Upon the absorption of photons, the ionizations of the neutral donors and neutral acceptors are assisted by longitudinal-optical phonons.

An electric field present in a GaN/AlN heterostructure can bring both the first-order and second-order Raman scattering processes into strong resonances. The resonant Stokes and anti-Stokes Raman scatterings result in the increase and decrease in nonequilibrium longitudinal-optical phonon temperatures, respectively. Moreover, the phonon temperature measured from the Raman scattering is increased with an applied electric field at a much higher rate than the lattice temperature due to the presence of field-induced nonequilibrium longitudinal-optical phonons.

We have observed that the temperature of the electrons drifting under a relatively high electric field in an AlN∕GaN-based high-electron-mobility transistor is significantly higher than the lattice temperature (i.e., the hot electrons are generated). These hot electrons are produced through the Frohlich interaction between the drifting electrons and long-lived longitudinal-optical phonons. By fitting electric field versus electron temperature deduced from the measurements of photoluminescence spectra to a theoretical model, we have deduced the longitudinal-optical-phonon emission time for each electron is to be on the order of 100fs.

We have observed resonance-enhanced Stokes and anti-Stokes Raman scattering of coherent picosecond pulses by one as well as two longitudinal-optical phonons in GaN film grown on Si (111) substrate.

We have observed blue and red Stark shifts of two excitonic transition peaks in multiple GaN/AlN asymmetric coupled quantum wells due to increases in electric fields originating from spatial separation of photogenerated electrons and holes.

Intra-cellular transport via the microtubule motors kinesin and dynein plays an important role in maintaining cell structure and function. Often, multiple kinesin or dynein motors move the same cargo. Their collective function depends critically on the single motors’ detachment kinetics under load. Single Kinesin's and Dynein's super-force off rates have been measured using an optical-trap based method. We rapidly increased the force on a moving bead and measured the time to detachment. From such events, detachment time distributions for specific super-force values have been measured. In contrast to a possible constant off-rate kinesin has an off-rate increasing with force. At low loads, dynein is sensitive to load; detaching easily but at higher load it exhibited a catch-bond type behavior, with off rate decreasing with load. The super-force experiments also allowed us to determine the probability of backward stepping for the motors. Kinesin and dynein can back-step under load, but this was relatively rare in both directions (

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signalling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (approximately fourfold) without altering kinesin's single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results provide the first direct evidence of a protein kinase upregulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin. © Macmillan Publishers Limited. All rights reserved.

Targeted transport by intracellular motors can be regulated by posttranslational modifications of polymerized tubulins in the microtubule tracks, but little is known about such effects for motors that drive chromosome motions during mitosis. Microtubules that form mitotic spindle are differentially modified at the C-terminal residue of α-tubulin: polymers that point to the spindle equator, but not the astral microtubules, are preferentially detyrosinated. Here we examine the influence of tubulin detyrosination on CENP-E, the kinetochore-localized kinesin-7 that transports pole-proximal chromosomes to the spindle equator. We polymerized purified human tubulins that were fully tyrosinated or detyrosinated, and examined the suitability of these tracks for motility of recombinant GFP-tagged CENP-E motor. Using fluorescence microscopy we show that single molecules of CENP-E walk faster and more processively on detyrosinated microtubules. Moreover, on these tracks the CENP-E motor can generate larger force than on the tyrosinated microtubules, as determined using stationary optical trap. On both types of microtubules CENP-E took 8-nm steps, exhibited similar dwell times and frequencies of backward stepping. However, motor's detachment increased with resisting force faster when CENP-E was walking on tyrosinated microtubules, leading to the detachment from these polymers at on average smaller load, 4.5 pN vs. 6.4 pN for detyrosinated microtubules. The enhanced motility of CENP-E motor on detyrosinated microtubules, most notably its ability to carry a larger load, could potentially explain the targeted transport of mitotic chromosomes toward the spindle equator.