抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

/pdf/imaging-intracellular-fluorescent-proteins-at-nanometer-3hryxg5982.pdf

Imaging intracellular fluorescent proteins at nanometer resolution.

We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.

/pdf/sub-diffraction-limit-imaging-by-stochastic-optical-52u8r6g2lp.pdf

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).

Recent developments have greatly improved the sensitivity of optical sensors based on metal nanoparticle arrays and single nanoparticles. We introduce the localized surface plasmon resonance (LSPR) sensor and describe how its exquisite sensitivity to size, shape and environment can be harnessed to detect molecular binding events and changes in molecular conformation. We then describe recent progress in three areas representing the most significant challenges: pushing sensitivity towards the single-molecule detection limit, combining LSPR with complementary molecular identification techniques such as surface-enhanced Raman spectroscopy, and practical development of sensors and instrumentation for routine use and high-throughput detection. This review highlights several exceptionally promising research directions and discusses how diverse applications of plasmonic nanoparticles can be integrated in the near future.

Biosensing with plasmonic nanosensors

Nobel Lecture: Prions

Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of 1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 2x nm and 37 – 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model. Myosin V is a cargo-carrying processive motor

/pdf/myosin-v-walks-hand-over-hand-single-fluorophore-imaging-158ahd2s5m.pdf

Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization

Dynein and kinesin motor proteins transport cellular cargoes toward opposite ends of microtubule tracks. In neurons, microtubules are abundantly decorated with microtubule-associated proteins (MAPs) such as tau. Motor proteins thus encounter MAPs frequently along their path. To determine the effects of tau on dynein and kinesin motility, we conducted single-molecule studies of motor proteins moving along tau-decorated microtubules. Dynein tended to reverse direction, whereas kinesin tended to detach at patches of bound tau. Kinesin was inhibited at about a tenth of the tau concentration that inhibited dynein, and the microtubule-binding domain of tau was sufficient to inhibit motor activity. The differential modulation of dynein and kinesin motility suggests that MAPs can spatially regulate the balance of microtubule-dependent axonal transport.

/pdf/differential-regulation-of-dynein-and-kinesin-motor-proteins-1gszpqafo6.pdf

Differential regulation of dynein and kinesin motor proteins by tau.

The structural change that generates force and motion in actomyosin motility has been proposed to be tilting of the myosin light chain domain, which serves as a lever arm. Several experimental approaches have provided support for the lever arm hypothesis; however, the extent and timing of tilting motions are not well defined in the motor protein complex of functioning actomyosin. Here we report three-dimensional measurements of the structural dynamics of the light chain domain of brain myosin V using a single-molecule fluorescence polarization technique that determines the orientation of individual protein domains with 20-40-ms time resolution. Single fluorescent calmodulin light chains tilted back and forth between two well-defined angles as the myosin molecule processively translocated along actin. The results provide evidence for lever arm rotation of the calmodulin-binding domain in myosin V, and support a 'hand-over-hand' mechanism for the translocation of double-headed myosin V molecules along actin filaments. The technique is applicable to the study of real-time structural changes in other biological systems.

Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

A small-molecule inhibitor of skeletal muscle myosin II.

III. EXCITATION-CONTRACT/ON COUPLING PROCESSES IN THE MAMMALIAN VENTRICLE A. Structure 1. Transverse-tubular system 2. Sarcoplasmic reticulum B. Functional Implications of the Structure of the Mammalian Myocardium C. Action Potential Control of Contraction D. Membrane Potential and Development of Tension in Mammalian Ventricle E. Potassium Chloride Contracture Studies F. Ionic and Drug Dependence of the Two Components of Tension in Mammalian Heart 1. The phasic response 2. The tonic response G. The Mechanism of Development of the Tonic Tension H. The Mechanism of the Triggered Release of the Phasic Store 1. Secondary inward current and activation of contraction 2. Other observations concerning the secondary inward current I. Replenishment of the Phasic Store 1. Depletion of the activator store 2. Beat-dependent kinetics 3. Quantity of activator in the phasic store and the concept of recirculation 4. An estimate of the magnitude of the recirculated fraction of activator 5. Delay in availability of activator 6. Anatomical correlates 7, Plateau and replenishment J. A Model of the E.-C Coupling Mechanism in Mammalian Heart Muscle K. Events Leading to a Normal Contraction L. A Mechanism for the Altered Inotropic State (the Interval Strength Relation) 1. Post-extrasystolic potentiation 2. Treppe 3. Other theories (a) Inotropic mediator hypothesis (b) Sodium-lag hypothesis

Yale E. Goldman

Papers

Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization

Differential regulation of dynein and kinesin motor proteins by tau.

Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization

A small-molecule inhibitor of skeletal muscle myosin II.

Excitation-contraction coupling in heart muscle: Membrane control of development of tension