Capillary Refill-The Key to Assessing Dermal Capillary Capacity and Pathology in Optical Coherence Tomography Angiography.

BACKGROUND/OBJECTIVES: Standard optical coherence tomography angiography (OCTA) has been limited to imaging blood vessels actively undergoing perfusion, providing a temporary picture of surface microvasculature. Capillary perfusion in the skin is dynamic and changes in response to the surrounding tissue's respiratory, nutritional, and thermoregulatory needs. Hence, OCTA often represents a given perfusion state without depicting the actual extent of the vascular network. Here we present a method for obtaining a more accurate anatomic representation of the surface capillary network in human skin using OCTA, along with proposing a new parameter, the Relative Capillary Capacity (RCC), a quantifiable proxy for assessing capillary dilation potential and permeability. METHODS: OCTA images were captured at baseline and after compression of the skin. Baseline images display ambient capillary perfusion, while images taken upon capillary refill display the network of existing capillaries at full capacity. An optimization-based automated vessel segmentation method was used to automatically analyze and compare OCTA image sequences obtained from two volunteers. RCC was then compared with visual impressions of capillary viability. RESULTS: Our OCTA imaging sequence provides a method for mapping cutaneous capillary networks independent of ambient perfusion. Differences between baseline and refill images clearly demonstrate the shortcomings of standard OCTA imaging and produce the RCC biometric as a quantifiable proxy for assessing capillary dilation potential and permeability. CONCLUSION: Future dermatological OCTA diagnostic studies should implement the Capillary Refill Methods over standard imaging techniques and further explore the relevance of RCC to differential diagnosis and dermatopathology. Lasers Surg. Med. © The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals, Inc.

Pink-beam serial crystallography.

Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, "pink", beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized for very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.

The prevalence and risk factors of post-inflammatory hyperpigmentation after fractional resurfacing in Asians.

BACKGROUND: Ablative laser resurfacing is considered to be the main therapeutic option for the treatment of wrinkles and acne scarring. However, in Asians, post-inflammatory hyperpigmentation (PIH) is a common adverse effect of laser resurfacing. Fractional resurfacing is a new concept of skin rejuvenation whereby zones of micro thermal injury are generated in the skin with the use of a 1,540-nm laser. The risk and prevalence of hyperpigmentation in dark-skinned patients using this approach have not been studied. OBJECTIVE: To assess the prevalence and risk factors of PIH that is associated with the use of fractional resurfacing in Asians. METHOD: A retrospective study of 37 Chinese patients who were treated with fractional resurfacing for acne scarring, skin rejuvenation, and pigmentation was carried out. In all of the cases, pre- and post-treatment clinical photographs (from standardized and cross-polarized views) were taken using the Canfield CR system. Two independent observers assessed the photographs. A prospective study of treatments of nine different density and energy levels that were applied to the forearms of 18 volunteers was also performed. Clinical photographs were assessed pre- and post-treatment for evidence of PIH. RESULT: In the retrospective study, 119 treatment sessions were performed. Sixty-eight treatment sessions were high energy, low density; 51 sessions were low energy, high density. Patients who underwent a high energy but low-density treatment (range of energy 7-20 mJ; average energy 16.3 mJ, 1,000 MTZ) were associated with a lower prevalence of generalized PIH (7.1% vs. 12.4%) than those who underwent a low energy but high-density (range of energy 6-12 mJ; average energy 8.2 mJ, 2,000 MTZ) treatment. However, the difference was not statistically significant. Localized PIH occurred in the peri-oral area among patients who did not receive air cooling as an adjunctive therapy. CONCLUSION: Both the density and energy of the treatment determines the risk of PIH in dark-skinned patients. Density may be of more important but further studies are necessary to determine this. Cooling to prevent bulk tissue heating is also important, especially in small anatomical areas. By using adequate parameters, the risk of PIH in dark-skinned patients can be significantly reduced.

[Fractional photothermolysis]. / Fraktionierte Photothermolyse.

Fractional photothermolysis (FP) has been recently introduced as a new concept in dermatologic laser medicine. FP employs an array of small laser beams to create many microscopic areas of thermal necrosis within the skin called microscopic treatment zones (MTZ). Even though FP completely destroys the epidermis and dermis within these MTZ, the 3-dimensional pattern of damage heals quickly and with few side effects. FP is currently used to treat fine wrinkles, photodamaged skin, acne scars, and melasma. Due to its clinical efficacy and limited side effects FP has established itself in the past two years as an alternative treatment modality to the conventional ablative and non ablative laser therapy.

Molecular mechanism of actomyosin-based motility.

Sophisticated molecular genetic, biochemical and biophysical studies have been used to probe the molecular mechanism of actomyosin-based motility. Recent solution measurements, high-resolution structures of recombinant myosin motor domains, and lower resolution structures of the complex formed by filamentous actin and the myosin motor domain provide detailed insights into the mechanism of chemomechanical coupling in the actomyosin system. They show how small conformational changes are amplified by a lever-arm mechanism to a working stroke of several nanometres, explain the mechanism that governs the directionality of actin-based movement, and reveal a communication pathway between the nucleotide binding pocket and the actin-binding region that explains the reciprocal relationship between actin and nucleotide affinity. Here we focus on the interacting elements in the actomyosin system and the communication pathways in the myosin motor domain that respond to actin binding.

Interaction between PEVK-titin and actin filaments: origin of a viscous force component in cardiac myofibrils.

The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B-cardiac PEVK was expressed in Escherichia coli and tested-along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains-for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca(2+)] reversed the PEVK effect. PEVK concentrations >/=10 microgram/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0 degrees C, but more strongly at 30 degrees C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca(2+)-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.

Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.

Lighting up the cell surface with evanescent wave microscopy.

Evanescent wave microscopy, also termed total internal reflection fluorescence microscopy (TIR-FM), has shed new light on important cellular processes taking place near the plasma membrane. For example, this technique can enable the direct observation of membrane fusion of synaptic vesicles and the movement of single molecules during signal transduction. There has been a recent surge in the popularity of this technique with the advent of green-fluorescent protein (GFP) as a fluorescent marker and new technical developments. These technical developments and some of the latest applications of TIR-FM are the subject of this review.

The Dictyostelium Bcr/Abr-related protein DRG regulates both Rac- and Rab-dependent pathways.

Dictyostelium discoideum DdRacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it shares with mammalian Bcr and Abr. To elucidate the physiological role of this multifunctional protein, we characterized the enzymatic activity of recombinant DRG fragments in vitro, created DRG-null cells, and studied the function of the protein in vivo by analysing the phenotypic changes displayed by DRG-depleted cells and DRG-null cells complemented with DRG or DRG fragments. Our results show that DRG-GEF modulates F-actin dynamics and cAMP-induced F-actin formation via Rac1-dependent signalling pathways. DRG's RacE-GAP activity is required for proper cytokinesis to occur. Additionally, we provide evidence that the specificity of DRG is not limited to members of the Rho family of small GTPases. A recombinant DRG-GAP accelerates the GTP hydrolysis of RabD 30-fold in vitro and our complementation studies show that DRG-GAP activity is required for the RabD-dependent regulation of the contractile vacuole system in Dictyostelium.

Single-molecule tracking of myosins with genetically engineered amplifier domains.

We combined protein engineering and single molecule measurements to directly record the step size of a series of myosin constructs with shortened and elongated artificial neck domains. Our results show that the step size has a clear linear dependence on the length of the neck domain and we also established that mechanical amplification in the myosin motor is based on a rotation of the neck domain relative to the actin-bound head. For all our constructs, including those with artificial necks, the magnitude of the neck rotation concurrent with the displacement step was approximately 30 degrees. The engineered change in the step size of myosin marks a significant advance in our ability to selectively modify the functional properties of molecular motors.

Structure of a genetically engineered molecular motor.

Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. They achieve this by amplifying small conformational changes in the nucleotide-binding region into force-generating movements of larger protein domains. We present the 2.8 A resolution crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 130 A conformational amplifier consisting of repeats 1 and 2 of alpha-actinin, we demonstrate that it is possible to genetically engineer single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. Furthermore, our structure shows the consequences of mutating a conserved salt bridge in the nucleotide-binding region. Disruption of this salt bridge, which is known to severely inhibit ATP hydrolysis activity, appears to interfere with formation of myosin's catalytically active 'closed' conformation. Finally, we describe the structure of alpha-actinin repeats 1 and 2 as being composed of two rigid, triple-helical bundles linked by an uninterrupted alpha-helix. This fold is very similar to the previously described structures of alpha-actinin repeats 2 and 3, and alpha-spectrin repeats 16 and 17.

Extended theory of selective photothermolysis.

BACKGROUND AND OBJECTIVE: We present a new theory of selective thermal damage of non-uniformly pigmented structures in biological tissues. Spatial separation of the heavily pigmented areas and the target requires limitation of the pigment temperature and heat diffusion from the pigmented to the targeted areas. STUDY DESIGNS/MATERIALS AND METHODS: A concept of selective target damage by heat diffusion is presented for three target geometries: planar, cylindrical, and spherical. An in vitro experiment is described in which the dependence of thermal damage on pulsewidth at constant fluence was evaluated. RESULTS: The in vitro experiment showed that the size of the damage zone for similar hair follicles was pulsewidth-independent over a very broad range of pulsewidths (30-400 ms). We formulated a new theory (extended theory of photothermolysis) to interpret the experimental results. CONCLUSIONS: Based on this new theory, the treatment pulsewidth for non-uniformly pigmented targets is significantly longer than the target thermal relaxation time (TRT). The theory provides new recommendations for photoepilation and photosclerotherapy parameters.

Stabilization of the actomyosin complex by negative charges on myosin.

Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideummyosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemistry 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K(A)) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K(A)() stem almost exclusively from variations in the dissociation rate constant (k(-A)), with the introduction of a single negative charge at position 532 having the same effect on k(-A) as the introduction of 12 positive charges in the loop 2 region.

Charge changes in loop 2 affect the thermal unfolding of the myosin motor domain bound to F-actin.

The thermal unfolding of Dictyostelium discoideum myosin head fragments with alterations in the actin-binding surface loop 2 was studied by differential scanning calorimetry. Lengthening of loop 2 without concomitant charge changes led to decreases in the transition temperature of not more than 1.8 degrees C. Insertions with multiple positive or negative charges had a stronger destabilizing effect and led to reductions in the thermal transition temperature of up to 3.7 degrees C. In the presence of nucleotide, most mutants displayed similar or higher transition temperatures than M765. Only constructs M765(11/+6) and M765(20/+12) with long positively charged inserts showed transition temperatures that were more than 2 degrees C below the values measured for M765 in the presence of ADP, ADP-V(i), and ADP-BeF(3). Interaction with F-actin in the presence of ADP shifted the thermal transition of M765 by 6 degrees C, from 49.1 to 55.1 degrees C. The actin-induced increase in thermal stability varied between 1.2 and 9.1 degrees C and showed a strong correlation with the mutant constructs' affinity for actin. Our results show that length and charge changes in loop 2 do not significantly affect nucleotide-induced structural changes in the myosin motor domain, but they affect structural changes that occur when the motor domain is strongly bound to actin and affect the coupling between the actin- and nucleotide-binding sites.

Functional characterization of the secondary actin binding site of myosin II.

The role of the interaction between actin and the secondary actin binding site of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred to as loop 3 in this work) has been studied with proteolytically generated smooth and skeletal muscle myosin subfragment 1 and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or nonmuscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e., in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and nonmuscle myosin chimera showed a 4-6-fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k(-)(A)) with no significant change in the rate for actin binding (k(+A)). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actin-myosin interface or for the modulation of actomyosin ATPase activity.

Functional characterisation of Dictyostelium myosin II with conserved tryptophanyl residue 501 mutated to tyrosine.

We created a Dictyostelium discoideum myosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in a Dictyostelium strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin in Dictyostelium. Additionally, we expressed the W501 Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased, but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission.

Role of the salt-bridge between switch-1 and switch-2 of Dictyostelium myosin.

Motifs N2 and N3, also referred to as switch-1 and switch-2, form part of the active site of molecular motors such as myosins and kinesins. In the case of myosin, N3 is thought to act as a gamma-phosphate sensor and moves almost 6 A relative to N2 during the catalysed turnover of ATP, opening and closing the active site surrounding the gamma-phosphate. The closed form seems to be necessary for hydrolysis and is stabilised by the formation of a salt-bridge between an arginine residue in N2 and a glutamate residue in N3. We examined the role of this salt-bridge in Dictyostelium discoideum myosin. Myosin motor domains with mutations E459R or R238E, that block salt-bridge formation, show defects in nucleotide-binding, reduced rates of ATP hydrolysis and a tenfold reduction in actin affinity. Inversion of the salt-bridge in double-mutant M765-IS eliminates most of the defects observed for the single mutants. With the exception of a 2,500-fold higher KMvalue for ATP, the double-mutant displayed enzymatic and functional properties very similar to those of the wild-type protein. Our results reveal that, independent of its orientation, the salt-bridge is required to support efficient ATP hydrolysis, normal communication between different functional regions of the myosin head, and motor function.

Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction.

The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a similar extent for both mutant and wild-type constructs. The addition of 0.5 microM actin decreased ADP affinity from 0.6 to 34 microM for M765NL and from 1.6 to 39 microM for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to phosphate release: thus, basal ATPase activity for M765NL (0.19 s-1) was 3-fold larger than for M765 (0.06 s-1), and the stimulation of ATPase activity by actin was 5-fold lower for M765NL. These results indicate different paths of communication between the actin- and nucleotide-binding sites, in regard to ADP and Pi release, and they confirm that loop 2 is involved in high affinity actin binding.

Kinetic analysis of Dictyostelium discoideum myosin motor domains with glycine-to-alanine mutations in the reactive thiol region.

Three conserved glycine residues in the reactive thiol region of Dictyostelium discoideummyosin II were replaced by alanine residues. The resulting mutants G680A, G684A, and G691A were expressed in the soluble myosin head fragment M761-2R [Anson, M., Geeves, M. A., Kurzawa, S. E., and Manstein, D. J. (1996) EMBO J. 15, 6069-6074] and characterized using transient kinetic methods. Mutant G691A showed no major alterations except for a marked increase in basal Mg2+-ATPase activity. Phosphate release seemed to be facilitated by this mutation, and the addition of actin to G691A stimulated ATP turnover not more than 3-fold. In comparison to M761-2R, mutant constructs G691A and G684A showed a 4-fold reduction in the rate of the ATP cleavage step. Most other changes in the kinetic properties of G684A were small ( approximately 2-fold). In contrast, substitution of G680 by an alanine residue led to large changes in nucleotide binding. Compared to M761-2R, rates of nucleotide binding were 20-30-fold slower and the affinity for mantADP was approximately 10-fold increased due to a 200-fold reduction in the dissociation rate constant of mantADP. The ATP-induced dissociation of actin from the acto.680A complex was normal, but the communication between ADP and actin binding was altered such that the two sites are thermodynamically uncoupled but kinetically actin still accelerates ADP release.

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.