Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots.

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 µg protein, with yield of ∼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.

Thermal nanoimprint lithography for drift correction in super-resolution fluorescence microscopy.

Localization-based super-resolution microscopy enables imaging of biological structures with sub-diffraction-limited accuracy, but generally requires extended acquisition time. Consequently, stage drift often limits the spatial precision. Previously, we reported a simple method to correct for this by creating an array of 1 µm3 fiducial markers, every ~8 µm, on the coverslip, using UV-nanoimprint lithography (UV-NIL). While this allowed reliable and accurate 3D drift correction, it suffered high autofluorescence background with shorter wavelength illumination, unstable adsorption to the substrate glass surface, and suboptimal biocompatibility. Here, we present an improved fiducial micro-pattern prepared by thermal nanoimprint lithography (T-NIL). The new pattern is made of a thermal plastic material with low fluorescence backgrounds across the wide excitation range, particularly in the blue-region; robust structural stability under cell culturing condition; and a high bio-compatibility in terms of cell viability and adhesion. We demonstrate drift precision to 1.5 nm for lateral (x, y) and 6.1 nm axial (z) axes every 0.2 seconds for a total of 1 min long image acquisition. As a proof of principle, we acquired 4-color wide-field fluorescence images of live mammalian cells; we also acquired super-resolution images of fixed hippocampal neurons, and super-resolution images of live glutamate receptors and postsynaptic density proteins.

Genetic evidence for a microtubule-capture mechanism during polarised growth of Aspergillus nidulans.

The cellular switch from symmetry to polarity in eukaryotes depends on the microtubule (MT) and actin cytoskeletons. In fungi such as Schizosaccharomyces pombe or Aspergillus nidulans, the MT cytoskeleton determines the sites of actin polymerization through cortical cell-end marker proteins. Here we describe A. nidulans MT guidance protein A (MigA) as the first ortholog of the karyogamy protein Kar9 from Saccharomyces cerevisiae in filamentous fungi. A. nidulans MigA interacts with the cortical ApsA protein and is involved in spindle positioning during mitosis. MigA is also associated with septal and nuclear MT organizing centers (MTOCs). Super-resolution photoactivated localization microscopy (PALM) analyses revealed that MigA is recruited to assembling and retracting MT plus ends in an EbA-dependent manner. MigA is required for MT convergence in hyphal tips and plays a role in correct localization of the cell-end markers TeaA and TeaR. In addition, MigA interacts with a class-V myosin, suggesting that an active mechanism exists to capture MTs and to pull the ends along actin filaments. Hence, the organization of MTs and actin depend on each other, and positive feedback loops ensure robust polar growth.

Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.

Fusion pore formation and expansion induced by Ca2+ and synaptotagmin 1.

Fusion pore formation and expansion, crucial steps for neurotransmitter release and vesicle recycling in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicle fusion, have not been well studied in vitro due to the lack of a reliable content-mixing fusion assay. Using methods detecting the intervesicular mixing of small and large cargoes at a single-vesicle level, we found that the neuronal SNARE complexes have the capacity to drive membrane hemifusion. However, efficient fusion pore formation and expansion require synaptotagmin 1 and Ca(2+). Real-time measurements show that pore expansion detected by content mixing of large DNA cargoes occurs much slower than initial pore formation that transmits small cargoes. Slow pore expansion perhaps provides a time window for vesicles to escape the full collapse fusion pathway via alternative mechanisms such as kiss-and-run. The results also show that complexin 1 stimulates pore expansion significantly, which could put bias between two pathways of vesicle recycling.

The cell-end marker TeaA and the microtubule polymerase AlpA contribute to microtubule guidance at the hyphal tip cortex of Aspergillus nidulans to provide polarity maintenance.

In the absence of landmark proteins, hyphae of Aspergillus nidulans lose their direction of growth and show a zigzag growth pattern. Here, we show that the cell-end marker protein TeaA is important for localizing the growth machinery at hyphal tips. The central position of TeaA at the tip correlated with the convergence of the microtubule (MT) ends to a single point. Conversely, in the absence of TeaA, the MTs often failed to converge to a single point at the cortex. Further analysis suggested a functional connection between TeaA and AlpA (an ortholog of the MT polymerase Dis1/CKAP5/XMAP215) for proper regulation of MT growth at hyphal tips. AlpA localized at MT plus-ends, and bimolecular fluorescence complementation assays suggested that it interacted with TeaA after MT plus-ends reached the tip cortex. In vitro MT polymerization assays showed that AlpA promoted MT growth up to sevenfold. Addition of the C-terminal region of TeaA increased the catastrophe frequency of the MTs. Thus, the control of the AlpA activity through TeaA might be a novel principle for MT growth regulation after reaching the cortex. In addition, we present evidence that the curvature of hyphal tips also could be involved in the control of MT growth at hyphal tips.

Reversible reconfiguration of DNA origami nanochambers monitored by single-molecule FRET.

Today, DNA nanotechnology is one of the methods of choice to achieve spatiotemporal control of matter at the nanoscale. By combining the peculiar spatial addressability of DNA origami structures with the switchable mechanical movement of small DNA motifs, we constructed reconfigurable DNA nanochambers as dynamic compartmentalization systems. The reversible extension and contraction of the inner cavity of the structures was used to control the distance-dependent energy transfer between two preloaded fluorophores. Interestingly, single-molecule FRET studies revealed that the kinetics of the process are strongly affected by the choice of the switchable motifs and/or actuator sequences, thus offering a valid method for fine-tuning the dynamic properties of large DNA nanostructures. We envisage that the proposed DNA nanochambers may function as model structures for artificial biomimetic compartments and transport systems.

Temperature-independent porous nanocontainers for single-molecule fluorescence studies.

In this work, we demonstrate the capability of using lipid vesicles biofunctionalized with protein channels to perform single-molecule fluorescence measurements over a biologically relevant temperature range. Lipid vesicles can serve as an ideal nanocontainer for single-molecule fluorescence measurements of biomacromolecules. One serious limitation of the vesicle encapsulation method has been that the lipid membrane is practically impermeable to most ions and small molecules, limiting its application to observing reactions in equilibrium with the initial buffer condition. To permeabilize the barrier, Staphylococcus aureus toxin α-hemolysin (aHL) channels have been incorporated into the membrane. These aHL channels have been characterized using single-molecule fluorescence resonance energy transfer signals from vesicle-encapsulated guanine-rich DNA that folds in a G-quadruplex motif as well as from the Rep helicase-DNA system. We show that these aHL channels are permeable to monovalent ions and small molecules, such as ATP, over the biologically relevant temperature range (17-37 °C). Ions can efficiently pass through preformed aHL channels to initiate DNA folding without any detectable delay. With addition of the cholesterol to the membrane, we also report a 35-fold improvement in the aHL channel formation efficiency, making this approach more practical for wider applications. Finally, the temperature-dependent single-molecule enzymatic study inside these nanocontainers is demonstrated by measuring the Rep helicase repetitive shuttling dynamics along a single-stranded DNA at various temperatures. The permeability of the biofriendly nanocontainer over a wide range of temperature would be effectively applied to other surface-based high-throughput measurements and sensors beyond the single-molecule fluorescence measurements.

The SNARE complex from yeast is partially unstructured on the membrane.

Molecular recognition between cognate SNAREs leads to the formation of a four-helix bundle, which facilitates vesicle docking and membrane fusion. For a SNARE system involved in trafficking in yeast, target membrane (t-) SNARE Sso1p and vesicle associated (v-) SNARE Snc2p contribute one SNARE motif each, whereas another t-SNARE (Sec9) donates two N-terminal and C-terminal SNARE motifs (SN1 and SN2) to the helical bundle. By use of EPR, it is found that SN2 has a tendency to be uncoiled, leaving a significant population of the SNARE complexes to be partially unstructured on the membrane. In sharp contrast, SN2 is fully engaged in the four-helix bundle when removed from the membrane, showing that the membrane is the main destabilizing factor. Helix-breaking proline mutations in SN2 did not affect the rate of docking but reduced the rate of lipid mixing significantly, indicating that SN2 plays an essential role in activating the transition from docking to fusion.

Photon-separation to enhance the spatial resolution of pulsed STED microscopy.

Stimulated emission depletion microscopy (STED) is one of the pivotal super-resolution techniques. It overcomes the spatial resolution limit imposed by the diffraction by using an additional laser beam, the STED beam, intensity of which is directly related to the achievable resolution. Despite reaching nanometer resolution, much effort in recent years has been devoted to reducing the STED beam intensity because it may lead to photo-damaging effects. Accessing the spatial information encoded in the temporal dynamics of the detected fluorescent photons has been proved to be a powerful strategy and has contributed to the separation by lifetime tuning (SPLIT) technique. The SPLIT method uses the phasor analysis to efficiently distinguish photons emitted from the center and the periphery of the excitation spot. It thus improves the resolution without increasing the STED beam intensity. This method was proposed for architectures based on the STED beam running in continuous waves (CW-STED microscopy). Here, we extend it to pulsed STED beam implementations (pSTED microscopy). We show, through simulated and experimental data, that the pSTED-SPLIT method reduces the detection volume of the pSTED microscope without significantly decreasing the signal-to-noise ratio of the final image, thus effectively improving the resolution without increasing the STED beam intensity.

Protegrin interaction with lipid monolayers: Grazing incidence X-ray diffraction and X-ray reflectivity study.

Interactions of the antimicrobial peptide protegrin-1 (PG-1) with phospholipid monolayers have been investigated by using grazing incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). The structure of a PG-1 film at the air-aqueous interface was also investigated by XR for the first time. Lipid A, dipalmitoyl-phosphatidylglycerol (DPPG) and dipalmitoyl-phosphatidylcholine (DPPC) monolayers were formed at the air-aqueous interface to mimic the surface of the bacterial cell wall and the outer leaflet of the erythrocyte cell membrane, respectively. Experiments were carried out under constant area conditions where the pressure changes upon insertion of peptide into the monolayer. GIXD data suggest that the greatest monolayer disruption produced by PG-1 is seen with the DPPG system at 20 mN/m since the Bragg peaks completely disappear after introduction of PG-1 to the system. PG-1 shows greater insertion into the lipid A system compared to the DPPC system when both films are held at the same initial surface pressure of 20 mN/m. The degree of insertion lessens at 30 mN/m with both DPPC and DPPG monolayer systems. XR data further reveal that PG-1 inserts primarily in the head group region of lipid monolayers. However, only the XR data of the anionic lipids suggest the existence of an additional adsorbed peptide layer below the head group of the monolayer. Overall the data show that the extent of peptide/lipid interaction and lipid monolayer disruption depends not only on the lipid composition of the monolayer, but the packing density of the lipids in the monolayer prior to the introduction of peptide to the subphase.

Superresolution and pulse-chase imaging reveal the role of vesicle transport in polar growth of fungal cells.

Polarized growth of filamentous fungi requires continuous transport of biomolecules to the hyphal tip. To this end, construction materials are packaged in vesicles and transported by motor proteins along microtubules and actin filaments. We have studied these processes with quantitative superresolution localization microscopy of live Aspergillus nidulans cells expressing the photoconvertible protein mEosFPthermo fused to the chitin synthase ChsB. ChsB is mainly located at the Spitzenkörper near the hyphal tip and produces chitin, a key component of the cell wall. We have visualized the pulsatory dynamics of the Spitzenkörper, reflecting vesicle accumulation before exocytosis and their subsequent fusion with the apical plasma membrane. Furthermore, high-speed pulse-chase imaging after photoconversion of mEosFPthermo in a tightly focused spot revealed that ChsB is transported with two different speeds from the cell body to the hyphal tip and vice versa. Comparative analysis using motor protein deletion mutants allowed us to assign the fast movements (7 to 10 µm s-1) to transport of secretory vesicles by kinesin-1, and the slower ones (2 to 7 µm s-1) to transport by kinesin-3 on early endosomes. Our results show how motor proteins ensure the supply of vesicles to the hyphal tip, where temporally regulated exocytosis results in stepwise tip extension.

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: effect of head group electrostatics and tail group packing.

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a beta-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.

Monitoring x-ray beam damage on lipid films by an integrated Brewster angle microscope/x-ray diffractometer.

We describe an integrated Brewster angle microscope (BAM), Langmuir trough, and grazing incidence x-ray diffraction assembly. The integration of these three techniques allows for the direct observation of radiative beam damage to a lipid monolayer at the air-water interface. Although beam damage has been seen in x-ray measurements, it has not been directly observed in situ at the micron scale. Using this integrated assembly, we examined the effects of radiative beam damage on Langmuir monolayers of 1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] (DMPS), 1:1 DMPS:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1:1 DMPS:1,2-dioleoyl-sn-glycero-3-phosphocholine held at a constant surface pressure. For constant surface pressure experiments, we observed a marked decrease in the surface area of the film upon exposure to the beam due to photodissociation. For a condensed lipid film, a change in refractive index of the film was observed post-beam-exposure, indicating areas of damage. For DMPS in an oxygenated environment, the Bragg peak intensity decreased with beam exposure. In mixed monolayer systems, with saturated and unsaturated lipids, an increase in the number of small saturated lipid domains was seen as the unsaturated lipid was preferentially damaged and lost from the monolayer. We show that BAM is a highly effective technique for in situ observation of the effects of radiative damage at the air/water interface during a synchrotron experiment.

Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.

Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 min or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5-10% of bQD-AMPARs were in PSDs and 90-95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD 'slots', our findings suggest that AMPARs rapidly enter stable 'nanodomains' in PSDs with lifetime >15 min, and do not accumulate in extrasynaptic membranes.

Mechanism of supported membrane disruption by antimicrobial peptide protegrin-1.

While pore formation has been suggested as an important step in the membrane disruption process induced by antimicrobial peptides, membrane pore formation has never been directly visualized. We report on the dynamics of membrane disruption by antimicrobial peptide protegrin-1 (PG-1) on dimyristoyl-sn-glycero-phosphocholine-supported bilayer patches obtained via atomic force microscopy. The action of PG-1 is found to be concentration-dependent. At low PG-1 concentrations (1 < [PG-1] < 4 microg/mL), the peptide destabilizes the edge of the membrane to form fingerlike structures. At higher concentrations, PG-1 induces the formation of a sievelike nanoporous structure in the membrane. The highest degree of disruption is attained at concentrations >or=20 microg/mL, at which PG-1 disrupts the entire membrane, transforming it into stripelike structures with a well-defined and uniform stripe width. This first direct visualization of these membrane structural transformations helps elucidate the PG-1-induced membrane disruption mechanism.

Dynamics of Actin Cables in Polarized Growth of the Filamentous Fungus Aspergillus nidulans.

Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 µm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules.

Labeling proteins inside living cells using external fluorophores for microscopy.

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging.

The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M(-1)cm(-1), mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths.

Evaluation of Genetically Encoded Chemical Tags as Orthogonal Fluorophore Labeling Tools for Single-Molecule FRET Applications.

Fluorescence resonance energy transfer (FRET) is a superb technique for measuring conformational changes of proteins on the single molecule level (smFRET) in real time. It requires introducing a donor and acceptor fluorophore pair at specific locations on the protein molecule of interest, which has often been a challenging task. By using two different self-labeling chemical tags, such as Halo-, TMP-, SNAP- and CLIP-tags, orthogonal labeling may be achieved rapidly and reliably. However, these comparatively large tags add extra distance and flexibility between the desired labeling location on the protein and the fluorophore position, which may affect the results. To systematically characterize chemical tags for smFRET measurement applications, we took the SNAP-tag/CLIP-tag combination as a model system and fused a flexible unstructured peptide, rigid polyproline peptides of various lengths, and the calcium sensor protein calmodulin between the tags. We could reliably identify length variations as small as four residues in the polyproline peptide. In the calmodulin system, the added length introduced by these tags was even beneficial for revealing subtle conformational changes upon variation of the buffer conditions. This approach opens up new possibilities for studying conformational dynamics, especially in large protein systems that are difficult to specifically conjugate with fluorophores.