Single-molecule Fluorescence Polarization Research Articles

Mechanical Stress Decreases the Amplitude of Twisting and Bending Fluctuations of Actin Filaments

A variety of biological roles of mechanical forces have been proposed in cell biology, such as cell signaling pathways for survival, development, growth, and differentiation. Mechanical forces alter the mechanical conditions within cells and their environment, which strongly influences the reorganization of the actin cytoskeleton. Single-molecule imaging studies of actin filaments have led to the hypothesis that the actin filament acts as a mechanosensor; e.g., increases in actin filament tension alter their conformation and affinity for regulatory proteins. However, our understanding of the molecular mechanisms underlying how tension modulates the mechanical behavior of a single actin filament is still incomplete. In this study, a direct measurement of the twisting and bending of a fluorescently labeled single actin filament under different tension levels by force application (0.8–3.4 pN) was performed using single-molecule fluorescence polarization (SMFP) microscopy. The results showed that the amplitude of twisting and bending fluctuations of a single actin filament decreased with increasing tension. Electron micrograph analysis of tensed filaments also revealed that the fluctuations in the crossover length of actin filaments decreased with increasing filament tension. Possible molecular mechanisms underlying these results involving the binding of actin-binding proteins, such as cofilin, to the filament are discussed.

Circular orientation fluorescence emitter imaging (COFEI) of rotational motion of motor proteins

Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.

Use of Single Molecule Fluorescence Polarization Microscopy to Study Protein Conformation and Dynamics of Kinesin-Microtubule Complexes.

Single molecule fluorescence polarization microscopy (smFPM) is a technique that enables to monitor changes in the orientation of a single labeled protein domain. Here we describe a smFPM microscope set-up and protocols to investigate conformational changes associated with the movement of motor proteins along cytoskeletal tracks.

Distinct Interaction Modes of the Kinesin-13 Motor Domain with the Microtubule

Kinesins-13s are members of the kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other kinesins, kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct kinesin-13 functionality, we used ensemble and single-molecule fluorescence polarization microscopy to analyze the behavior and conformation of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to the MT lattice. We found that KLP10A interacts with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs we conclude that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile kinesins.

Monitoring Nanoscale Deformations in a Drawn Polymer Melt with Single-Molecule Fluorescence Polarization Microscopy.

Elongating a polymer melt causes polymer segments to align and polymer coils to deform along the drawing direction. Despite the importance of this molecular response for understanding the viscoelastic properties and relaxation behavior of polymeric materials, studies on the single-molecule level are rare and were not performed in real time. Here we use single-molecule fluorescence polarization microscopy for monitoring the position and orientation of single fluorescent perylene diimide molecules embedded in a free-standing thin film of a polymethyl acrylate (PMA) melt with a time resolution of 500 ms during the film drawing and the subsequent stress relaxation period. The orientation distribution of the perylene diimide molecules is quantitatively described with a model of rod-like objects embedded in a uniaxially elongated matrix. The orientation of the fluorescent probe molecules is directly coupled to the local deformation of the PMA melt, which we derive from the distances between individual dye molecules. In turn, the fluorescence polarization monitors the shape deformation of the polymer coils on a length scale of 5 nm. During stress relaxation, the coil shape relaxes four times more slowly than the mechanical stress. This shows that stress relaxation involves processes on length scales smaller than a polymer coil. Our work demonstrates how optical spectroscopy and microscopy can be used to study the coupling of individual fluorescent probe molecules to their embedding polymeric matrix and to an external mechanical stimulus on the single-molecule level.

Exploring the Mechanisms of a Phosphorylation Induced Inhibition of Microtubule Depolymerization in the Kinesin 13 KLP10A

Kinesins 13 are a class of non-motile molecular motors able to regulate microtubule dynamics through their depolymerization activity at microtubule tips. A previous study in our lab on Drosophila Melanogaster KLP10A found a phosphorylation site within the motor domain that reduces microtubule depolymerization activity. This led us to study the inhibition mechanisms related to this phosphorylation site distant from the ATPase site. We have employed an integrated approach to compare the behavior of phosphomutant and wild-type KLP10A constructs with cell biology experiments and a variety of in-vitro biophysical methods. Imaging insect cells having only fluorescently labelled KLP10A variants revealed that the phosphomutant is distributed homogeneously on microtubules (lattice and tips) in contrast with the wild-type or a non-phosphorylatable mutant showing higher kinesin concentration on microtubule tips. Wild-type and phosphomutant neck-motor kinesins constructs have similar properties with isolated structures that mimic these microtubule tips: they can both stabilize curved microtubule protofilaments, an important property for microtubule depolymerization, and have similar affinity and ATPase activity on curved tubulin. However the phosphomutation strongly reduces the microtubule lattice stimulated ATPase activity despite non distinguishable microtubule lattice affinities. Single molecule fluorescence polarization microscopy revealed nucleotide dependent differences in the lattice diffusive behavior between the constructs. EPR and cryo-EM experiments are used to compare the kinesins on the microtubule lattice vs curved protofilaments. The data are compatible with a reduced microtubule depolymerization activity of the phosphomutant caused by a reduction of the lattice stimulated ATPase activity. This reduced activity constrains the kinesin to be in nucleotide states which restrict it from reaching the microtubule ends in comparison to the unphosphorylated kinesin.

Conformational Changes in the β Subunits of F1-Atpase Revealed by Fret Measurements During the Rotation of the γ Subunit

To uncover conformational changes in biomolecules accompanied with biochemical events remains challenging, even though the atomic structures and the kinetics of the biomolecules are revealed. F1-ATPase is a rotary molecular motor in which a central γ subunit rotates against hexagonally arranged subunits α3β3, hydrolyzing ATP sequentially in three β subunits. Previous study using a single-molecule fluorescence polarization method has proposed sets of the β subunit conformations during the rotation (Masaike et al., Nat. Struct. Mol. Biol. 2008). However, further information is indispensable for identifying the ATP-waiting form of which the atomic structure has not been revealed. Here we performed single-pair FRET measurement to detect distance changes between two β subunits. Time trajectories of FRET efficiency showed two-state transitions between high (0.8) and low (0.5). Given the crystal structures, low FRET efficiency indicates one β subunit in the open form but another in the closed form. On the other hand, high FRET efficiency indicates both of the β subunits in the closed form. Next, we performed a simultaneous measurement of FRET between two fluorescently labeled β subunits and the rotation of the γ subunit. High FRET efficiency was occurred in one of the three catalytic dwells. In the remaining five dwells, other two catalytic dwells and three ATP-waiting dwells, FRET efficiency was lower. These results suggest that in the ATP-waiting dwell two of three β subunits would not take the closed form as in the catalytic dwell. We are performing further experiments with a magnetic tweezers.

Breaking the Millisecond Barrier: Single Molecule Motors Wobble to Find their Next Binding Sites

Breaking the Millisecond Barrier: Single Molecule Motors Wobble to Find their Next Binding Sites

Highlights from the Literature

Highlights from the Literature

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V

Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10° accuracy at 33ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90° for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.

MDia1 and formins: screw cap of the actin filament.

Formin homology proteins (formins) are actin nucleation factors which remain bound to the growing barbed end and processively elongate actin filament (F-actin). Recently, we have demonstrated that a mammalian formin mDia1 rotates along the long-pitch helix of F-actin during processive elongation (helical rotation) by single-molecule fluorescence polarization. We have also shown processive depolymerization of mDia1-bound F-actin during which helical rotation was visualized. In the cell where F-actins are highly cross-linked, formins should rotate during filament elongation. Therefore, when formins are tightly anchored to cellular structures, formins may not elongate F-actin. Adversely, helical rotation of formins might affect the twist of F-actin. Formins could thus control actin elongation and regulate stability of cellular actin filaments through helical rotation. On the other hand, ADP-actin elongation at the mDia1-bound barbed end turned out to become decelerated by profilin, in marked contrast to its remarkably positive effect on mDia1-mediated ATP-actin elongation. This deceleration is caused by enhancement of the off-rate of ADP-actin. While mDia1 and profilin enhance the ADP-actin off-rate, they do not apparently increase the ADP-actin on-rate at the barbed end. These results imply that G-actin-bound ATP and its hydrolysis may be part of the acceleration mechanism of formin-mediated actin elongation.

Mechanism of Kinesin-13 Binding to Microtubules as Revealed by Single Molecule Fluorescence Polarization Microscopy

The kinesin superfamily of motor proteins are involved in diverse cellular processes, including intracellular organelle transport, cell division and cytoskeletal dynamics. The widely studied conventional kinesin (or kinesin-1) translocates along the microtubule (MT) by utilizing the energy generated from ATP hydrolysis. In contrast, members of the Kinesin-13 family do not walk along the MT lattice, but use their catalytic core to rapidly target microtubule ends by the process of one-dimensional diffusion (ODD) and promote depolymerization upon reaching there. However, the reason for such a significant difference in the behavior of the structurally conserved motor domain is not clearly understood. In order to reveal the mechanistic details of the kinesin-13 action, fluorescence polarization microscopy (FPM) has been employed to probe the configuration and mobility of BSR-labeled KLP10A (Drosophila m. Kinesin-13) molecules interacting with microtubules in the presence of different nucleotides. Experiments are being performed with KLP10A constructs of variable lengths to identify the potent mediators of diffusive motility. Preliminary results emerging from single-molecule FPM measurements have suggested that the motor core itself can undergo ODD without the assistance from the positively-charged neck domain. In addition, data acquired at both ensemble and single-molecule levels have revealed that the orientation of the KLP10A molecule relative to the MT filament is altered by mutating the crucial residues in the tubulin-binding sites on the motor domain. The structural and functional information extracted from analyses of our experimental findings will be discussed in details.

Investigating the Interaction between Kinesin-13s and Microtubules by Single Molecule Fluorescence Polarization Microscopy

Kinesins are motor proteins that translocate along microtubules (MTs) and assist in transportation of intracellular cargo by utilizing the energy generated from ATP hydrolysis. Interestingly, Kinesin-13s do not walk but depolymerize microtubules at their ends, an activity that is essential for regulating MT dynamics during cell-division. Prior research efforts have shown that Kinesin-13s target MT ends very quickly by the process of one-dimensional diffusion along the MT lattice. In an effort to elucidate the mechanism of such diffusive interactions and MT depolymerization activity, we have investigated the orientation and dynamics of BSR-labeled KLP10A (Drosophila m. Kinesin-13) constructs interacting with MTs in the presence of ATP analogues using fluorescence polarization microscopy (FPM). Unlike conventional kinesins, which show high mobility in nucleotide conditions that induce weak MT binding (presence of ADP), KLP10A shows relatively high fluorescence anisotropy in all nucleotide states investigated, including ADP. Such observations indicate that the nucleotide present has minimal effects on the binding configuration of the majority of KLP10A molecules bound to the MT lattice. However, FPM data acquired at the single-molecule levels suggest that the Kinesin-13 motor domain becomes very mobile (tumbling) when undergoing one-dimensional diffusion. Ongoing experiments with KLP10A constructs of different lengths and mutants will help in identifying the residues in the Kinesin-13 molecule that mediate one-dimensional lattice diffusion.

A mobile kinesin-head intermediate during the ATP-waiting state.

Kinesin1 is a motor protein that uses the energy from ATP hydrolysis to move intracellular cargoes along microtubules. It contains 2 identical motor domains, or heads, that coordinate their mechano-chemical cycles to move processively along microtubules. The molecular mechanism of coordination between head domains remains unclear, partly because of the lack of structural information on critical intermediates of the kinesin1 mechano-chemical cycle. A point of controversy has been whether before ATP binding, in the so called ATP-waiting state, 1 or 2 motor domains are bound to the microtubule. To address this issue, here we use ensemble and single molecule fluorescence polarization microscopy (FPM) to determine the mobility and orientation of the kinesin1 heads at different ATP concentrations and in heterodimeric constructs with microtubule binding impaired in 1 head. We found evidence for a mobile head during the ATP-waiting state. We incorporate our results into a model for kinesin translocation that accounts well for many reported experimental results.

Configuration Of The Kinesin1 Motor Domains In The ATP-waiting State As Revealed By Fluorescence Polarization Microscopy

The molecular mechanism of coordination between kinesin1 heads during processive walking remains unclear, partly due to the lack of structural information on critical intermediates of the kinesin1 mechano-chemical cycle. To address this issue here we used ensemble and single molecule fluorescence polarization microscopy to determine the mobility and orientation of the kinesin motor domains. We first investigated conditions mimicking a state when only one head is bound to the microtubule and the other one is tethered. For this we made heterodimeric constructs with impaired microtubule binding in one head. Our results indicate that the tethered head is very mobile. We then investigated the orientation of the head domains in homodimeric constructs moving processively at saturating or limiting [ATP]. At saturating [ATP] both motor domains are well oriented relative to the microtubule but at limiting [ATP] there is an increase in mobility. This result indicates that before ATP-binding one motor domain is mobile.

Chain-Length Dependent Nematic Ordering of Conjugated Polymers in a Liquid Crystal Solvent

In this communication we demonstrate the dependence of the solute order parameter on the solute molecular weight for polymer solutes dissolved in liquid crystalline solvents. Using ensemble absorption polarization spectroscopy together with single molecule fluorescence polarization measurements, we have determined the order parameter of the conjugated polymer MEH-PPV (poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene]) in the liquid crystal 5CB (4-cyano-4-n-pentylbiphenyl) as a function of polymer chain length. Ensemble absorption polarization measurements agree well with results obtained by single molecule fluorescence polarization spectroscopy, indicating a large-scale ordering of the MEH-PPV solute in 5CB. These results demonstrate that the increasing number of defects for larger polymer weights inherently limits the alignment of the polymer solute.

2P-046 1分子蛍光偏光法による古細菌型シャペロニンの構造変化解析(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)

2P-046 1分子蛍光偏光法による古細菌型シャペロニンの構造変化解析(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)

Fluorescence from Diffusing Single Molecules Illuminates Biomolecular Structure and Dynamics

This review provides an account of single-molecule fluorescence methodologies for freely diffusing molecules applied to a diverse array of biological problems pertaining to biomolecular folding and assembly. We describe the principles of confocal fluorescence microscopy to detect and analyze fluorescence bursts from diffusing single molecules. These methods including single-molecule fluorescence resonance energy transfer, coincidence, correlations and polarization offer a powerful means to uncover hidden information about conformational sub-populations and interconversion dynamics of biomolecular systems in a wide range of timescales. We offer several key examples to illustrate how these methodologies have been extremely useful in teasing out structural and dynamical aspects of many important biomolecular systems.

Redistribution of emitting state population in conjugated polymers probed by single-molecule fluorescence polarization spectroscopy

Fluctuations in the fluorescence polarization degree and direction are reported for the first time for single conjugated polymer molecules embedded in a polystyrene matrix at room temperature. The polymer molecule, a polythiophene derivative, clearly emits as a multi-chromophore ensemble showing that the energy does not funnel to any specific low-energy trap. The fluorescence instead originates from thermally populated exciton states with different relative orientations of the transition dipole moments. The fluctuations in the fluorescence polarization are explained in terms of changes in the relative contributions of the different exciton states to the signal due to conformational fluctuations of the molecule or selective exciton quenching by triplet states.

Functional Studies of Individual Myosin Molecules

The "conventional" isoform of myosin that polymerizes into filaments (myosin II) is the molecular motor powering contraction in all three types of muscle. Considerable attention has been paid to the developmental progression, isoform distribution, and mutations that affect myocardial development, function, and adaptation. Optical trap (laser tweezer) experiments and various types of high-resolution fluorescence microscopy, capable of interrogating individual protein motors, are revealing novel and detailed information about their functionally relevant nanometer motions and pico-Newton forces. Single-molecule laser tweezer studies of cardiac myosin isoforms and their mutants have helped to elucidate the pathogenesis of familial hypertrophic cardiomyopathies. Surprisingly, some disease mutations seem to enhance myosin function. More broadly, the myosin superfamily includes more than 20 nonfilamentous members with myriad cellular functions, including targeted organelle transport, endocytosis, chemotaxis, cytokinesis, modulation of sensory systems, and signal transduction. Widely varying genetic, developmental and functional disorders of the nervous, pigmentation, and immune systems have been described in accordance with these many roles. Compared to the collective nature of myosin II, some myosin family members operate with only a few partners or even alone. Individual myosin V and VI molecules can carry cellular vesicular cargoes much farther distances than their own size. Laser tweezer mechanics, single-molecule fluorescence polarization, and imaging with nanometer precision have elucidated the very different mechano-chemical properties of these isoforms. Critical contributions of nonsarcomeric myosins to myocardial development and adaptation are likely to be discovered in future studies, so these techniques and concepts may become important in cardiovascular research.