Time-resolved Phosphorescence Anisotropy Research Articles

Human Cardiac Myosin-Binding Protein C N-Terminal Domains Cooperatively Impact Actin Structural Dynamics

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein that modulates contractility by influencing interactions between myosin and actin. Despite being a leading cause of familial hypertrophic cardiomyopathy, the mechanism by which cMyBP-C modulates contractility is not well understood. N-terminal domains of cMyBP-C (C0 through C2) have been shown to interact with the actin-thin filament; however, the cMyBP-C regions involved in the interaction and how cMyBP-C influences actin's dynamic structure remain unclear. Here, we have used cosedimentation assays for measuring binding kinetics and time-resolved phosphorescence anisotropy (TPA) for measuring actin rotational dynamics. We examined the effects of actin interactions with human fragments C0-C1 and C0-C2 of cMyBP-C. We also evaluated roles of cMyBP-C phosphorylation sites known to influence contractility. We find that C0-C2 binds actin with ∼4-fold increase in apparent affinity compared to C0-C1. TPA of labeled-actin showed marked differences in the potency of effects between C0-C2, C0-C1, and phosphorylated cMyBP-C fragments with respect to decreasing the amplitude of torsional motions, increasing the rate of monomer rotation, and increasing relative resilience of the filament. A Hill equation was used to determine the level of cooperativity of the TPA effects for each fragment. Our results demonstrate that C0-C2 exhibits stronger cooperativity in propagating its effects on amplitude, rate, and resilience to more distant actin monomers than C0-C1. Assuming a thin filament cooperative unit of ∼7 actin monomers (length of tropomyosin), our TPA analyses suggest that one molecule of C0-C2 bound to actin propagated effects 2-3 cooperative units (14-21 monomers), whereas C0-C1 effects were confined to one unit, and phosphorylation reduced cooperativity. These findings provide new insight into the molecular basis for cMyBP-C effects on actin structure and function. This work was supported by an NIH R00HL122397 to B.A.C.

Time-Tagged Single Photon Counting Examination of Rotation of Receptor-Bound Quantum Dots

Rotation of single membrane receptors can be observed by examination of polarized optical signals from receptor-bound asymmetric nanoparticles such as fluorescent quantum dots (QD) or nanogold. For example, we have examined the slow, hindered rotation of the Type I Fce receptor (FceRI) on 2H3 RBL cells using polarized fluorescence imaging of receptor-bound Qdot655. With imaging methods, only receptor rotational correlation times (RCT) slower than the camera frame time can be examined and suitable low-light cameras typically require 1ms or longer per frame. However, time-resolved phosphorescence anisotropy shows the hydrodynamic RCT of FcεRI to be about 40 μs at 25°C. To examine such rapid reorientation, an alternate approach is to illuminate individual QD on the cell surface with a focused laser beam, collect fluorescence using a confocal detector and direct signals polarized parallel and perpendicular to the laser polarization into separate APD detectors. A time-tagged single photon counter records the channel of each detected photon and its arrival time with a precision of 165 ps. The auto- and cross-correlations of the two signals are calculated directly from arrival times without binning, combined using adjustable constants such as the g-factor, and these constants optimized to obtain the maximum statistical independence between the anisotropy and intensity time-autocorrelation functions (TAC). While RCTs below 1 ns are theoretically accessible, useful rotational information exists only when RCT∗photon count rate ≥ 1. Thus, given photon count rates, our data potentially provide information on RCTs ≥ 20 μs. However, intensity changes due to QD blinking may feed through to some extent into calculated anisotropies and thus slightly distort apparent anisotropy TAC decay shapes. Efforts aimed at removing this possible complication are underway.

Continuous Fluorescence Depletion Anisotropy Measurement of Protein Rotation.

Protein rotation in viscous environments can be measured by fluorescence depletion anisotropy (FDA) which combines long lifetimes of chromophore triplet states with the sensitivity of fluorescence excitation and detection. FDA achieves sensitivity well beyond that attainable by the more common technique of time-resolved phosphorescence anisotropy (TPA). We have now combined benefits of both time-domain and frequency-domain FDA into a single continuous technique (CFDA). Intensity and polarization of a single laser beam are modulated continuously according to a complex, repeating waveform. Fluorescence signals excited from triplet-forming fluorescent probes are digitized over recurring waveform periods by a high-speed signal averager. CFDA experiments typically involve substantial ground state depletion. Thus signals, unlike those of TPA, are not linear in the exciting light intensity and simple data analysis based on such linearity is not appropriate. An exact solution of the coupled diffusion and triplet production/decay equation describing CFDA within individual data points has been combined with simulated annealing optimization to extract triplet and anisotropy decay kinetics from experimental data. Related calculations compare possible excitation waveforms with respect to rotational information provided per fluorescence photon. We present CFDA results for the model system of eosin conjugates of carbonic anhydrase, BSA and immunoglobulin G in 90% glycerol at various temperatures and initial cellular results on eosin-IgE bound to 2H3 cell Type I Fcε receptors. We explore how CFDA reflects rotational parameters of heterogeneous systems and discuss challenges of extending this method to single cell microscopic measurements.

Phosphomimetic S3D cofilin binds but only weakly severs actin filaments

Many biological processes, including cell division, growth, and motility, rely on rapid remodeling of the actin cytoskeleton and on actin filament severing by the regulatory protein cofilin. Phosphorylation of vertebrate cofilin at Ser-3 regulates both actin binding and severing. Substitution of serine with aspartate at position 3 (S3D) is widely used to mimic cofilin phosphorylation in cells and in vitro The S3D substitution weakens cofilin binding to filaments, and it is presumed that subsequent reduction in cofilin occupancy inhibits filament severing, but this hypothesis has remained untested. Here, using time-resolved phosphorescence anisotropy, electron cryomicroscopy, and all-atom molecular dynamics simulations, we show that S3D cofilin indeed binds filaments with lower affinity, but also with a higher cooperativity than wild-type cofilin, and severs actin weakly across a broad range of occupancies. We found that three factors contribute to the severing deficiency of S3D cofilin. First, the high cooperativity of S3D cofilin generates fewer boundaries between bare and decorated actin segments where severing occurs preferentially. Second, S3D cofilin only weakly alters filament bending and twisting dynamics and therefore does not introduce the mechanical discontinuities required for efficient filament severing at boundaries. Third, Ser-3 modification (i.e. substitution with Asp or phosphorylation) "undocks" and repositions the cofilin N terminus away from the filament axis, which compromises S3D cofilin's ability to weaken longitudinal filament subunit interactions. Collectively, our results demonstrate that, in addition to inhibiting actin binding, Ser-3 modification favors formation of a cofilin-binding mode that is unable to sufficiently alter filament mechanical properties and promote severing.

Rotation of Single Cell Surface Molecules Examined via Polarized FCS Measurements using Quantum Dot Probes

Rotational motions of cell surface molecules are of particular interest given the sensitivity of such motions to molecular size and aggregation state. The time-autocorrelation function (TACF) of fluctuations in fluorescence polarization from asymmetric quantum dots (QD) such as Molecular Probes’ Qdot655 labeling cell surface molecules indicates the rotational correlation times (RCT) of the QD-containing complexes. Type I Fce receptors (FceRI) and insulin receptors (IR) on 2H3 cells, subjected to various treatments potentially affecting receptor rotation, including polyvalent DNP-BSA, methyl-β-cyclodextrin, cytochalasin D or paraformaldehyde, were labeled by Qdot655s. Over 700 individual QD were examined. Side-by-side vertically(v)- and horizontally(h)-polarized fluorescence images of QD-labeled cells were obtained simultaneously as image stacks at 10 ms per frame by means of a Princeton Instrument Dual-View. To minimize apparent polarization TACF arising from QD lateral diffusion, v- and h-substacks were aligned to sub-pixel accuracy. From fluorescence in identical regions around each QD in each stack, intensity and polarization fluctuation TACF were calculated. Given the blinking of QD, experimental parameters such as g-factor, camera background, etc. were carefully optimized for each QD to minimize cross-correlation between polarization and intensity fluctuations. This could otherwise appear as intensity fluctuation TACF contributing to the apparent polarization fluctuation TACF. Using these techniques, the initial polarization fluctuation TACF for FceRI averages about 0.0015 and the geometrically-averaged RCT is about 55-85 ms, both independent of cell treatment. More limited results on IR appear similar. Absence of treatment effects on magnitudes or decay rates of polarization TACF is puzzling. Previous time-resolved phosphorescence anisotropy studies on FceRI suggest limited presence of orientational relaxations slower than 1 ms. However, the absence of treatment effects here suggests such slow reorientation may be a property of the membrane itself, perhaps reflecting large-scale fluctuations of mesoscale membrane regions. Supported by NSF grant MCB-1024668 and NIH grant CA175937 to BGB.

Hydrodynamic Radii of Ranibizumab, Aflibercept and Bevacizumab Measured by Time-Resolved Phosphorescence Anisotropy.

PurposeTo measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with μs time-resolved phosphorescence anisotropy.MethodsRuthenium-based dye Ru(bpy)2(mcbpy − O − Su − ester)(PF6)2, whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)2(mcbpy − O − Su − ester)(PF6)2-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity.ResultsThe measured radii of 2.76±0.04 nm for ranibizumab, 3.70±0.03 nm for aflibercept and 4.58±0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements.ConclusionsTime-resolved phosphorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of the hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-016-1940-2) contains supplementary material, which is available to authorized users.

The N-Terminal Domains of MyBPC3 Restrict Actin Dynamics and Increase Resilience in a Phosphorylation-Dependent Manner

We have determined the effects of myosin binding protein-C (MyBP-C) N-terminal domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C modulates muscle contractility and is capable of interacting with thin filaments. Protein kinase A (PKA) phosphorylates cardiac and slow skeletal MyBP-C, altering myofilament structure and function. We previously used TPA to show that full-length MyBP-C restricts actin torsional dynamics, and effects by cardiac and slow skeletal MyBP-C (cMyBP-C; ssMyBP-C) are relieved by PKA phosphorylation. To determine the effects of MyBP-C N-terminal domains on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin flexibility at saturation of the TPA effect. PKA phosphorylation of ssMyBP-C domains C1-C2 and cMyBP-C domains C0-C2 relieved the restriction of torsional amplitude but also increased the rate of torsional motion, thus increasing actin resilience (rate/amplitude; less brittle). Moreover, effects of cardiac C0-C2 on actin resilience were also PKA-dependent, whereas slow skeletal C1-C2 effects which promote actin resilience persisted independently of phosphorylation. In the case of fast skeletal C1-C2, actin resilience was unaffected and its effect to restrict actin dynamics was unchanged by phosphorylation. The N-terminal domains of cMyBP-C (C0-C2) and skeletal MyBP-C's (C1-C2) had effects on actin dynamics similar to those determined for the full-length MyBP-C isoforms. Effects of phosphomimetic mutations were also investigated. These unique isoform-dependent MyBP-C-induced changes in actin dynamics may play a role in the functional effects of MyBP-C on contraction. This work was supported by NIH grants to DDT (R01 AR032961), to SS (R01 AR067279), and to BC (R00 HL122397).

Blinking of Quantum Dot Probes in Single Membrane Molecule Rotation Measurements

Asymmetric quantum dots (QD) provide non-bleaching probes of the rotation of individual cell surface proteins. We have conducted imaging measurements of individual 2H3 cell Type I Fce receptor rotation on timescales down to 10 ms per frame. We have also used time-tagged single photon counting measurements of individual QD to examine μs timescales, though such rapid timescales are limited by QD emission rates. In both approaches we calculate the time-autocorrelation functions (TACF) for fluorescence polarization fluctuations. Decays of these fluctuations extend into the ms timescale, as implied by time-resolved phosphorescence anisotropy results. Effects of cell treatments such as receptor crosslinking suggest that this slow decay may be a property of the membrane itself, perhaps reflecting large-scale fluctuations of mesoscale membrane regions. However, depending on instrumental parameters used in data analysis, polarization fluctuation TACFs can contain a contribution from the intensity fluctuation TACF arising from QD blinking. Such QD blinking feed-through is extremely sensitive to these analysis parameters which effectively change slightly from one measurement to another. We discuss approaches based on the necessary statistical independence of polarization and intensity fluctuations to guarantee removal of a blinking-based component from rotation measurements. Supported by NSF grant MCB-1024668.

Nanoparticle Probes of Cell Surface Molecule Rotation

Rotation of membrane proteins is a sensitive measure of their aggregation state and their interactions with other membrane species. We have used nanoparticles including asymmetric quantum dots as non-bleaching probes of the rotation of individual cell surface proteins. Invitrogen QD655s conjugated to A2 DNP-specific IgE allow examination of slow rotation of the Type I Fce receptor (FceRI) on RBL-2H3 cells. Fluorescence from cell-bound quantum dots is excited by illumination polarized at 45 deg and a Princeton Instruments Dual View equipped with a polarizing beam splitter allows recording image sequences containing simultaneous x- and y-polarized sub-images in each frame. For individual quantum dots, we calculate the time-autocorrelation functions for fluorescence polarization fluctuations. Decays of these fluctuations extend down to the ms timescale, as implied by time-resolved phosphorescence anisotropy results. Treatment effects suggest that such slow decay may be a property of the membrane itself, perhaps reflecting large-scale fluctuations of mesoscale membrane regions. To examine rotational correlation function decays faster than imaging measurements permit, we have applied time-tagged single photon counting to individual quantum dot fluorescence and analyzed data down to correlation times below 1μs. These measurements should thus include the 80μs hydrodynamic rotation of single FceRI molecules, but significance of current data is limited by quantum dot emission rates. Other probes are therefore being explored as are the consequences of the two-dimensional transition dipoles exhibited by quantum dots. Supported by NSF grant MCB-1024668.

The Structural Dynamics of Actin during Active Interaction with Myosin Depends on the Isoform of the Essential Light Chain

We have used time-resolved phosphorescence anisotropy to investigate the effects of essential light chain (ELC) isoforms (A1 and A2) on the interaction of skeletal muscle myosin with actin, to relate structural dynamics to previously reported functional effects. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes S1A1 and S1A2 by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin concentrations but substantially higher apparent actin affinity, resulting in a higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin's final anisotropy cooperatively and to a similar extent, indicating a similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (S) state of actomyosin. In contrast, in the presence of a saturating level of ATP, S1A1 increased actin anisotropy much more than S1A2 and with greater cooperativity, indicating that S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin. We conclude that during the active interaction of actin and ATP with myosin, S1A1 is more effective at stabilizing the S state (probably the force-generating state) of actomyosin, while S1A2 tends to stabilize the weak-binding (non-force-generating) W state. When a mixture of isoenzymes is present, S1A1 is dominant in its effects on actin dynamics. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, with significant implications for the contractile function of actomyosin.

Structural Dynamics of Actin during Active Interaction with Myosin depends on the Isoform of the Essential Light Chain

Muscle contraction results from cyclic interaction of myosin and actin, driven by ATP hydrolysis, where the actomyosin complex undergoes transitions between weakly and strongly bound structural states. Rabbit skeletal muscle myosin has two isoforms of the essential light chain (ELC), designated A1 and A2. These isoforms have significant effects on myosin's interaction with actin, resulting in higher catalytic efficiency for A1 but higher in vitro motility and muscle shortening velocity for A2. In order to understand the structural basis of these differences, we have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of ELC isoforms on the microsecond dynamics of actin during interaction with skeletal muscle myosin in the absence and presence of saturating ATP. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes containing A1 (S1A1) and A2 (S1A2) by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin's final anisotropy similarly, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (rigor) state of actomyosin. In contrast, in the presence of saturating ATP, the final anisotropy of actin was substantially higher during interaction with S1A1 than with S1A2. Thus, S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin, suggesting greater population of the strong-binding state of actomyosin. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, and thus modulates the contractile function of actomyosin.

Advancements in laboratory diagnostics: an invaluable tool for assessing quality of blood transfusions.

The interesting article by Willy A. Flegel that recently appeared in this journal provides valuable insights into an ongoing debate on the use of fresher material for blood transfusions1. Regardless of important considerations about the delicate balance between selection of units and blood supply, an innovative approach for defining the quality of transfused components is increasingly supported by recent advancements in laboratory diagnostics, which would enable to determine whether leucocytes, platelets and red blood cells are suitable for transfusion regardless of the ageing of the stored material. Several examples can be given, whereby routine, automatic flow haemocytometry combined with additional technical tools provides valuable information. As regards platelets, the loss of function during storage is mirrored by disc-to-sphere transformation, extension of pseudopodia, alteration of the canalicular system and granules, up to loss of storage granules2. Some haemocytometers already provide a variety of activation-related information about these blood elements. For example, in Advia 2120 (Siemens Diagnostics, Tarrytown, New York, United States of America), the intensity of light scattered by platelets is assessed at two different angles (i.e., 2 to 3° and 5 to 15°). Mean platelet volume and mean platelet component are then calculated and from these paired measures. The latter parameter, in particular, has been shown to be a reliable index of platelet degranulation, since it is directly related to CD62 expression, which is highly indicative of platelet activation and ageing3. The same instrument, along with others such as the PENTRA 60C (Horiba-ABX Diagnostics, Montpellier, France), provide specific flags or alerts in the presence of degranulation of polymorphonuclear leucocytes4, which is a hallmark of functional impairment5. More specifically, the Advia 2120 also enables reliable quantification of the number of degranulated neutrophils in specific clusters of the peroxidase scattergram based on cell volume and intracellular myeloperoxidase concentration. Additional morphological changes that characterise gradual deterioration of leucocytes in stored blood include a more homogeneous staining of nuclei, the separation of the nuclear lobes or irregular lobulation and vacuolisation, which can be reliably identified in the scattergram of modern haemocytometers such as those of the Sysmex XE series, in which the intensity of side scatter is dependent on the complexity of nuclear lobulation and granules within the cytoplasm6. It is also noteworthy that several changes occur to red blood cells during medium- and long-term storage in blood bags, including reduced deformability, altered adhesiveness and aggregability, and decreased 2,3-diphosphoglycerate and ATP content, to the point of frank injury and spurious haemolysis7. Specific macromolecular remodelling, along with lipid peroxidation and/or oxidative damage to the membrane, are mainly responsible for the onset of irreversible morphological abnormalities and loss of function. Most of these physicochemical changes, usually referred to as the “storage lesion”, can be identified by time-consuming or expensive analytical techniques such as flow cytometry, time-resolved phosphorescence anisotropy and microscopy, which are unsuitable for rapid examination of a large number of blood bags8. Nevertheless, the presence of red blood cell crenation or fragmentation -and finally haemolysis- can now be reliably and rapidly identified by specific erythrocyte indices on automated flow haemocytometers, such as the “immature platelet fraction” on XE-2100 (Sysmex, Kobe, Japan), or “RBC ghosts” on Advia 21209. Another interesting approach is ThromboLUX™ (LightIntegra Technology Inc., Vancouver, British Columbia, Canada), which uses the principle of dynamic light scattering -also known as photon correlation spectroscopy or quasi-elastic light scattering- to identify and quantify the particles that may be present in a platelet concentrate, as well as showing how they respond to temperature stress. The instrument finally generates a quality score on a scale ranging from 0 to 40, with platelet concentrates scoring from 13 to 40 containing mostly discoid and functional platelets. Recent data show that the information from ThromboLUX™ correlates adequately with that from flow cytometry and microscopy and may, therefore, be suitable for quality assessment of platelet concentrate samples10. Several technical advances have occurred in laboratory diagnostics over the past decades. These have not only improved the global quality of testing, but have also enabled reliable application to transfusion medicine for the assessment of the quality of transfused components, as attested by some of the examples previously provided. The modern, fully-automated haemocytometers -which are characterised by high accuracy and precision, notable throughput and full integration with information systems- can also provide a kaleidoscope of cellular indices. Most of these novel parameters are still partially unexploited, but it is conceivable that they may be reliably used to detect artefactual changes in leucocytes, platelets and red blood cells, which may be suggestive of less functional, defective or even apoptotic cells, and finally establish as to whether cellular elements within blood bags are still viable for transfusion. Future studies should be focused on the evaluation of the usefulness of these parameters in the setting of transfusion medicine.

Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner

We have determined the effects of myosin binding protein-C (MyBP-C) and its domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multidomain modulator of striated muscle contraction, interacting with myosin, titin, and possibly actin. Cardiac and slow skeletal MyBP-C are known substrates for protein kinase-A (PKA), and phosphorylation of the cardiac isoform alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin torsional flexibility by 15-20° at saturation of the TPA effect. PKA phosphorylation of slow skeletal and cardiac MyBP-C relieved the restriction of torsional amplitude but also decreased the rate of torsional motion. In the case of fast skeletal MyBP-C, its effect on actin dynamics was unchanged by phosphorylation. The isolated C-terminal half of cardiac MyBP-C (C5-C10) had effects similar to those of the full-length protein, and it bound actin more tightly than the N-terminal half (C0-C4), which had smaller effects on actin dynamics that were independent of PKA phosphorylation. We propose that these MyBP-C-induced changes in actin dynamics play a role in the functional effects of MyBP-C on the actin-myosin interaction.

Impacts of Dystrophin and Utrophin Domains on Actin Structural Dynamics: Implications for Therapeutic Design

We have used time-resolved phosphorescence anisotropy (TPA) of actin to evaluate domains of dystrophin and utrophin, with implications for gene therapy in muscular dystrophy. Dystrophin and its homolog utrophin bind to cytoskeletal actin to form mechanical linkages that prevent muscular damage. Because these proteins are too large for most gene therapy vectors, much effort is currently devoted to smaller constructs. We previously used TPA to show that both dystrophin and utrophin have a paradoxical effect on actin rotational dynamics—restricting amplitude while increasing rate, thus increasing resilience, with utrophin more effective than dystrophin. Here, we have evaluated individual domains of these proteins. We found that a “mini-dystrophin,” lacking one of the two actin-binding domains, is less effective than dystrophin in regulating actin dynamics, correlating with its moderate effectiveness in rescuing the dystrophic phenotype in mice. In contrast, we found that a “micro-utrophin,” with more extensive internal deletions, is as effective as full-length dystrophin in the regulation of actin dynamics. Each of utrophin's actin-binding domains promotes resilience in actin, while dystrophin constructs require the presence of both actin-binding domains and the C-terminal domain for full function. This work supports the use of a utrophin template for gene or protein therapy designs. Resilience of the actin–protein complex, measured by TPA, correlates remarkably well with previous reports of functional rescue by dystrophin and utrophin constructs in mdx mice. We propose the use of TPA as an in vitro method to aid in the design and testing of emerging gene therapy constructs.

Temporal sequence of major biochemical events during blood bank storage of packed red blood cells.

We used sensitive spectroscopic techniques to measure changes in Band 3 oligomeric state during storage of packed red blood cells (RBC); these changes were compared to metabolic changes, RBC morphology, cholesterol and membrane protein loss, phospholipid reorganisation of the RBC membrane, and peroxidation of membrane lipid. The aim of the study was to temporally sequence major biochemical events occurring during cold storage, in order to determine which changes may underlie the structural defects in stored RBC. Fifteen RBC units were collected from normal volunteers and stored under standard blood bank conditions; both metabolic changes and lipid parameters were measured by multiple novel assays including a new mass spectrometric measurement of isoprostane (lipid peroxidation) and flow cytometric assessment of CD47 expression. Band 3 oligomeric state was assessed by time-resolved phosphorescence anisotropy, and RBC morphology by microscopy of glutaraldehyde-fixed RBC. Extracellular pH decreased and extracellular potassium increased rapidly during cold storage. Band 3 on the RBC membrane aggregated into large oligomers early in the storage period and coincident with changes in RBC morphology. Membrane lipid changes, including loss of unesterified cholesterol, lipid peroxidation and expression of CD47, also changed early during the storage period. In contrast loss of acetylcholinesterase activity and haemolysis of RBC occurred late during storage. Our results demonstrate that changes in the macromolecular organisation of membrane proteins on the RBC occur early in storage and suggest that lipid peroxidation and/or oxidative damage to the membrane are responsible for irreversible morphological changes and loss of function during red cell storage.

Effects of Myosin Binding Protein-C Isoform, Phosphorylation, and Domains on the Rotational Dynamics of Actin Filaments

We have determined the effects of Myosin binding protein-C (MyBP-C) and its domains on the microsecond time-scale rotational dynamics of actin, using time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multi-domain thick filament-associated modulator of striated muscle contraction, spanning the interfilament spacing to contact both myosin and actin. Cardiac (c-) and slow skeletal (ss-) MyBP-C are known substrates for Protein kinase-A (PKA), and phosphorylation of cMyBP-C alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin's microsecond structural dynamics, we labeled actin at C374 with erythrosine iodoacetamide and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy (r∞) of the TPA decay in a concentration-dependent manner, indicating restriction of the rotational amplitude of actin dynamics. While PKA phosphorylation had little effect on fast skeletal (fs-) MyBP-C, phosphorylation of cMyBP-C and ssMyBP-C nearly eliminated the effects of these proteins to restrict actin dynamics, despite no change in binding affinity with phosphorylation. Skeletal MyBP-C (C1-C10) affected actin anisotropy at lower concentrations than cMyBP-C (C0-C10), suggesting that skeletal and cardiac N-terminal MyBP-C interactions with actin have distinct properties. The effects of truncated cMyBP-C on actin anisotropy determined that C-terminal domains are important for restricting rotational dynamics, whereas N-terminal domains are important for regulating this effect. These MyBP-C-induced changes in actin dynamics may play a role in the known effects of MyBP-C on the functional actin-myosin interaction. This work was funded by grants from NIH (F32 HL107039-01 to BAC and T32 AR007612 to DDT).

Rotation of Single Cell-Surface Fc Receptors Examined by Quantum Dot Probes

Rotational diffusion, which depends linearly on the in-membrane volume of the rotating molecule, is, relative to lateral diffusion, a more sensitive probe of an individual molecule’s size and local environment. Single-particle tracking has provided new perspectives on lateral compartmentalization of membrane proteins. However, little is known at present about rotational motions of single membrane protein molecules. We discuss correlation analysis of fluctuations in fluorescence polarization from proteins labeled by asymmetric quantum dots. Such analysis provides rotational information on single membrane molecules which can be directly related to that from ensemble measurements of cell surface protein rotation. We have used asymmetric quantum dots to examine individual Type I Fce receptors on 2H3-RBL cells and to conduct imaging measurements of receptor rotational diffusion on timescales down to 10 ms per frame. To achieve removal of blinking-based contributions to rotation measurements, we discuss an approach based on the necessary statistical independence of polarization and intensity fluctuations. Imaging results demonstrate rotational correlation times broadly ranging from 50 to 500 ms among individual molecules of the same type. The magnitude of these orientational fluctuations is comparable to the fraction of molecules which appear rotationally immobile when examined on the microsecond timescale by time-resolved phosphorescence anisotropy. These slow motions, not observable previously, appear to be a property of the membrane rather than the receptor state. Our results suggest that individual mesoscale membrane regions may rotate or liberate with respect to the overall cell surface.

The Carboxy-Terminal Third of Dystrophin Enhances Actin Binding Activity

Dystrophin is an actin binding protein that is thought to stabilize the cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene–actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule [from N-terminal actin binding domain (ABD) 1 through ABD2] bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays than full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction by restricting actin rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin.

Structural Dynamics of the Actin-Binding Domains in Dystrophin and Utrophin

We are using time-resolved phosphorescence anisotropy (TPA) and dipolar electron-electron-resonance (DEER) to determine and compare the structural dynamics of the actin-binding domains in dystrophin and utrophin. Dystrophin and utrophin bind actin in vitro with similar affinities, but with different molecular contacts (Rybakova et al, 2006, J. Biol. Chem). We hypothesize that these differences alter the elasticity of dystrophin-actin and utrophin-actin linkages to the sarcolemma, affecting the cell's response to muscle stretches, with important implications for muscular dystrophy and its therapy. Our previous TPA studies, detecting the microsecond dynamics of phosphorescent-labeled actin, showed that both proteins have novel effects on actin flexibility, with utrophin more effective than dystrophin (Prochniewicz et al., 2009, PNAS). We have now compared the effects of the isolated actin-binding domains of dystrophin, ABD1 and ABD2. TPA shows that the enhanced rate of actin rotational dynamics is induced primarily by ABD1, while both ABD1 and ABD2 contribute to the restriction in rotational amplitude. Disease-causing point mutations in ABD1 decrease the effects on actin's rotational rate. We propose that this in turn causes the dystrophin-actin complex to be less resilient and thus less able to prevent damage to the muscle cytoskeleton during contraction. Finally, we have attached probes directly to ABD1 in dystrophin and utrophin, to detect changes in structure upon actin binding. High-resolution distance measurements, provided by DEER, show that the two lobes (calponin-homology domains) within ABD1 undergo a dramatic opening upon actin binding, helping to resolve a previous controversy (Lin et al., 2011, PNAS). Analogous studies with dystrophin show more subtle changes upon actin binding, providing insight into the structural and functional differences in dystrophin and utrophin.

Changes in Band 3 oligomeric state precede cell membrane phospholipid loss during blood bank storage of red blood cells.

Lipid loss in the form of vesicles contributes to the red blood cell (RBC) storage lesion, and this loss of lipid is correlated with changes in membrane protein function. Sensitive spectroscopic techniques were used to measure changes in Band 3 oligomeric state during storage of RBCs, compared to metabolic changes and phospholipid loss. The aim of the study was to determine whether changes in the macromolecular organization of membrane proteins occur before, coincident with, or after lipid loss during RBC storage. Five RBC units were collected from normal volunteers and stored under standard blood bank conditions, and both metabolic changes and lipid loss were measured by multiple assays. Band 3 oligomeric state was assessed by time-resolved phosphorescence anisotropy and fluorescence resonance energy transfer of eosin-5-maleimide-labeled RBC ghosts. Extracellular pH decreased and extracellular potassium increased rapidly during cold storage of blood. Band 3 on the RBC membrane exhibited a shift from small to large oligomers early in the storage period and before detectable loss of phospholipid from the RBC membrane. The immobilized fraction of Band 3, that which is tethered to the cytoskeletal network via spectrin and ankyrin, did not change during cold storage. Our results demonstrate that changes in the macromolecular organization of membrane proteins on the RBC occur early in storage, and these changes may induce phospholipid loss, irreversible morphologic changes, and loss of function during RBC storage.