Reversible solidification of fission yeast cytoplasm after prolonged nutrient starvation.

Cells depend on a highly ordered organisation of their content and must develop strategies to maintain the anisotropic distribution of organelles during periods of nutrient shortage. One of these strategies is to solidify the cytoplasm, which was observed in bacteria and yeast cells with acutely interrupted energy production. Here, we describe a different type of cytoplasm solidification fission yeast cells switch to, after having run out of nutrients during multiple days in culture. It provides the most profound reversible cytoplasmic solidification of yeast cells described to date. Our data exclude the previously proposed mechanisms for cytoplasm solidification in yeasts and suggest a mechanism that immobilises cellular components in a size-dependent manner. We provide experimental evidence that, in addition to time, cells use intrinsic nutrients and energy sources to reach this state. Such cytoplasmic solidification may provide a robust means to protect cellular architecture in dormant cells.

Mechanical Contact Spectroscopy: Characterizing Nanoscale Adhesive Contacts via Thermal Forces.

The adhesion of micro- and nanoparticles to solid substrates immersed in liquids is a problem of great scientific and technological importance. However, the quantitative characterization of such nanoscale adhesive contacts without rupturing them still presents a major experimental challenge. In this article, we introduce mechanical contact spectroscopy (MCS), an experimental technique for the nondestructive probing of particle adhesion in liquid environments. With MCS, the strength of adhesive contacts is inferred from residual position fluctuations of adherent particles excited by thermal forces. In particular, the strength of adhesion is correlated with the standard deviation of the particle lateral position x, with smaller position standard deviations [Formula: see text] indicating higher adhesive strength. For a given combination of particles, substrate, and immersion medium, the adhesion is characterized by the mechanical contact spectrum, which is a histogram of ξ values obtained from tracking an ensemble of adherent particles. Because the energy of thermal excitation at room temperature is very small in comparison to the typical total energy of adhesive contacts, the studied contacts remain in equilibrium during the measurement. Using MCS, we study the adhesion of micrometer-sized particles to planar solid substrates under a wide range of environmental conditions, including liquid immersion media of varying ionic strength and adhesion substrates with different chemical functionality of their surfaces. These experiments provide evidence that MCS is capable of reproducibly detecting minute changes in the particle-substrate work of adhesion while at the same time covering the range of adhesive contact strength relevant in the context of surface chemistry, biology, and microfabrication.

Glucose starvation triggers filamentous septin assemblies in an S. pombe septin-2 deletion mutant.

Using correlative light and electron microscopy (CLEM), we studied the intracellular organization by of glucose-starved fission yeast cells (Schizosaccharomyces pombe) with regards to the localization of septin proteins throughout the cytoplasm. Thereby, we found that for cells carrying a deletion of the gene encoding septin-2 (spn2Δ), starvation causes a GFP-tagged version of septin-3 (spn3-GFP) and family members, to assemble into a single, prominent filamentous structure. It was previously shown that during exponential growth, spn2Δ cells form septin-3 polymers. However, the polymers we observed during exponential growth are different from the spn3p-GFP structure we observed in starved cells. Using CLEM, in combination with anti-GFP immunolabeling on plastic-sections, we could assign spn3p-GFP to the filaments we have found in EM pictures. Besides septin-3, these filamentous assemblies most likely also contain septin-1 as an RFP-tagged version of this protein forms a very similar structure in starved spn2Δ cells. Our data correlate phase-contrast and fluorescence microscopy with electron micrographs of plastic-embedded cells, and further on with detailed views of tomographic 3D reconstructions. Cryo-electron microscopy of spn2Δ cells in vitrified sections revealed a very distinct overall morphology of the spn3p-GFP assembly. The fine-structured, regular density pattern suggests the presence of assembled septin-3 filaments that are clearly different from F-actin bundles. Furthermore, we found that starvation causes substantial mitochondria fission, together with massive decoration of their outer membrane by ribosomes.

Opto-thermoelectric nanotweezers.

Optical manipulation of plasmonic nanoparticles provides opportunities for fundamental and technical innovation in nanophotonics. Optical heating arising from the photon-to-phonon conversion is considered as an intrinsic loss in metal nanoparticles, which limits their applications. We show here that this drawback can be turned into an advantage, by developing an extremely low-power optical tweezing technique, termed opto-thermoelectric nanotweezers (OTENT). Through optically heating a thermoplasmonic substrate, alight-directed thermoelectric field can be generated due to spatial separation of dissolved ions within the heating laser spot, which allows us to manipulate metal nanoparticles of a wide range of materials, sizes and shapes with single-particle resolution. In combination with dark-field optical imaging, nanoparticles can be selectively trapped and their spectroscopic response can be resolved in-situ. With its simple optics, versatile low-power operation, applicability to diverse nanoparticles, and tuneable working wavelength, OTENT will become a powerful tool in colloid science and nanotechnology.

Nanoscopic imaging of thick heterogeneous soft-matter structures in aqueous solution.

Precise nanometre-scale imaging of soft structures at room temperature poses a major challenge to any type of microscopy because fast thermal fluctuations lead to significant motion blur if the position of the structure is measured with insufficient bandwidth. Moreover, precise localization is also affected by optical heterogeneities, which lead to deformations in the imaged local geometry, the severity depending on the sample and its thickness. Here we introduce quantitative thermal noise imaging, a three-dimensional scanning probe technique, as a method for imaging soft, optically heterogeneous and porous matter with submicroscopic spatial resolution in aqueous solution. By imaging both individual microtubules and collagen fibrils in a network, we demonstrate that structures can be localized with a precision of ∼10 nm and that their local dynamics can be quantified with 50 kHz bandwidth and subnanometre amplitudes. Furthermore, we show how image distortions caused by optically dense structures can be corrected for.

Direct observation of intermediate states in model membrane fusion.

We introduce a novel assay for membrane fusion of solid supported membranes on silica beads and on coverslips. Fusion of the lipid bilayers is induced by bringing an optically trapped bead in contact with the coverslip surface while observing the bead's thermal motion with microsecond temporal and nanometer spatial resolution using a three-dimensional position detector. The probability of fusion is controlled by the membrane tension on the particle. We show that the progression of fusion can be monitored by changes in the three-dimensional position histograms of the bead and in its rate of diffusion. We were able to observe all fusion intermediates including transient fusion, formation of a stalk, hemifusion and the completion of a fusion pore. Fusion intermediates are characterized by axial but not lateral confinement of the motion of the bead and independently by the change of its rate of diffusion due to the additional drag from the stalk-like connection between the two membranes. The detailed information provided by this assay makes it ideally suited for studies of early events in pure lipid bilayer fusion or fusion assisted by fusogenic molecules.

Lipid droplets purified from Drosophila embryos as an endogenous handle for precise motor transport measurements.

Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.

Ultrastrong optical binding of metallic nanoparticles.

We demonstrate nanometer precision manipulation of multiple nanoparticles at room temperature. This is achieved using the optical binding force, which has been assumed to be weak compared to the optical gradient and scattering forces. We show that trapping by the optical binding force can be over 20 times stronger than by the gradient force and leads to ultrastable, rigid configurations of multiple nanoparticles free in solution - a realization of "optical matter." In addition, we demonstrate a novel trapping scheme where even smaller nanoparticles are trapped between larger "anchor" particles. Optical binding opens the door for the observation of collective phenomena of nanoparticles and the design of new materials and devices made from optical matter.

High precision and continuous optical transport using a standing wave optical line trap.

We introduce the Standing Wave Optical Line Trap (SWOLT) as a novel tool for precise optical manipulation and long-range transport of nano-scale objects at low laser power. We show that positioning and transport along the trap can be achieved by controlling the lateral component of the scattering force while the confinement of the particles by the gradient force remains unaffected. Multiple gold nanoparticles with a diameter of 100 nm were trapped at a power density 3 times smaller than previously reported while their transverse fluctuations remained sufficiently small (±36 nm) to maintain the order of the particles. The SWOLT opens new doors for sorting, mixing, and assembly of synthetic and biological nanoparticles.

Extracting the mechanical properties of microtubules from thermal fluctuation measurements on an attached tracer particle.

The mechanical properties of microtubules have been the subject of intense study during recent decades because of their importance to the many cell functions that they are involved in. Observations of microtubule thermal fluctuations have proven to be a reliable method to extract mechanical properties because they provide intrinsic calibration. While analysis of the entire microtubule shape is limited by spatial resolution to very long microtubules, we show that even for short microtubules, one can obtain high-precision fluctuation information from one point along the contour by the use of tracer particles attached to the microtubule. The information is sufficient to extract key mechanical parameters such as stiffness and first mode relaxation time. In this article, we discuss sample preparation as well as measurements and data analysis.

Direct measurement of the nonconservative force field generated by optical tweezers.

The force field of optical tweezers is commonly assumed to be conservative, neglecting the complex action of the scattering force. Using a novel method that extracts local forces from trajectories of an optically trapped particle, we measure the three-dimensional force field experienced by a Rayleigh particle with 10 nm spatial resolution and femtonewton precision in force. We find that the force field is nonconservative with the nonconservative component increasing radially away from the optical axis, in agreement with the Gaussian beam model of the optical trap.

Detecting sequential bond formation using three-dimensional thermal fluctuation analysis.

We present a novel experimental method that solves two key problems in nondestructive mechanical studies of small biomolecules at the single-molecule level, namely the confirmation of single-molecule conditions and the discrimination against nonspecific binding. A biotin-avidin ligand-receptor couple is spanned between a glass slide and a 1 microm latex particle using short linker molecules. Optical tweezers are used to initiate bond formation and to follow the particle's thermal position fluctuations with nanometer spatial and microsecond temporal resolution. Here we show that each step in the specific binding process leads to an abrupt change in the magnitude of the particle's thermal position fluctuations, allowing us to count the number of bonds formed one by one. Moreover, three-dimensional position histograms calculated from the particle's fluctuations can be separated into well-defined categories reflecting different binding conditions (single specific, multiple specific, nonspecific). Our method brings quantitative mechanical single-molecule studies to the majority of proteins, paving the way for the investigation of a wide range of phenomena at the single-molecule level.

Development of a fast position-sensitive laser beam detector.

We report the development of a fast position-sensitive laser beam detector. The detector uses a fiber-optic bundle that spatially splits the incident beam, followed by a fast balanced photodetector. The detector is applied to the study of Brownian motion of particles on fast time scales with 1 A spatial resolution. Future applications include the study of molecule motors, protein folding, as well as cellular processes.

Microtubule architecture: inspiration for novel carbon nanotube-based biomimetic materials.

Microtubules are self-assembling biological nanotubes that are essential for cell motility, cell division and intracellular trafficking. Microtubules have outstanding mechanical properties, combining high resilience and stiffness. Such a combination allows microtubules to accomplish multiple cellular functions and makes them interesting for material sciences. We review recent experiments that elucidate the relationship between molecular architecture and mechanics in microtubules and examine analogies and differences between microtubules and carbon nanotubes, which are their closest equivalent in nanotechnology. We suggest that a long-term goal in bionanotechnology should be mimicking the properties of microtubules and microtubule bundles to produce new functional nanomaterials.

Docking of liposomes to planar surfaces mediated by trans-SNARE complexes.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a key role in membrane fusion in the secretory pathway. In vitro, SNAREs spontaneously assemble into helical SNARE complexes with the transmembrane domains at the C-terminal end. During fusion, SNAREs are thought to bridge the two membranes and assemble in a zipper-like fashion, pulling the membranes together and initiating fusion. However, it is not clear to what extent SNARE assembly contributes to membrane attachment and membrane fusion. Using the neuronal SNAREs synaptobrevin (VAMP), SNAP-25, and syntaxin as examples, we show here that liposomes containing synaptobrevin firmly attach to planar surfaces containing immobilized syntaxin. Attachment requires the formation of SNARE complexes because it is dependent on the presence of SNAP-25. Binding is competed for by soluble SNARE fragments, with noncognate SNAREs such as endobrevin (VAMP8), VAMP4, and VAMP7 (Ti-VAMP) being effective but less potent in some cases. Furthermore, although SNAP-23 is unable to substitute for SNAP-25 in the attachment assay, it forms complexes of comparable stability and is capable of substituting in liposome fusion assays. Vesicle attachment is initiated by SNARE assembly at the N-terminal end of the helix bundle. We conclude that SNAREs can indeed form stable trans-complexes that result in vesicle attachment if progression to fusion is prevented, further supporting the zipper model of SNARE function.

Microtubule dynamics depart from the wormlike chain model.

Thermal shape fluctuations of grafted microtubules were studied using high resolution particle tracking of attached fluorescent beads. First mode relaxation times were extracted from the mean square displacement in the transverse coordinate. For microtubules shorter than approximately 10 microm, the relaxation times were found to follow an L2 dependence instead of L4 as expected from the standard wormlike chain model. This length dependence is shown to result from a complex length dependence of the bending stiffness which can be understood as a result of the molecular architecture of microtubules. For microtubules shorter than approximately 5 microm, high drag coefficients indicate contributions from internal friction to the fluctuation dynamics.

Motion of a colloidal particle in an optical trap.

Thermal position fluctuations of a colloidal particle in an optical trap are measured with microsecond resolution using back-focal-plane interferometry. The mean-square displacement and power spectral density are in excellent agreement with the theory for a Brownian particle in a harmonic potential that accounts for hydrodynamic memory effects. The motion of a particle is dominated at short times by memory effects and at longer times by the potential. We identify the time below which the particle's motion is not influenced by the potential, and find it to be approximately tau(k)/20 , where tau(k) is the relaxation time of the restoring force of the potential. This allows us to exclude the existence of free diffusive motion, proportional to t, even for a sphere with a radius as small as 0.27 microm in a potential as weak as 1.5 microN/m. As the physics of Brownian motion can be used to calibrate an optical trap, we show that neglecting memory effects leads to an underestimation of more than 10% in the detector sensitivity and the trap stiffness for an experiment with a micrometer-sized particle and a sampling frequency above 200kHz . Furthermore, these calibration errors increase in a nontrivial fashion with particle size, trap stiffness, and sampling frequency. Finally, we present a method to evaluate calibration errors caused by memory effects for typical optical trapping experiments.

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length.

Microtubules are hollow cylindrical structures that constitute one of the three major classes of cytoskeletal filaments. On the mesoscopic length scale of a cell, their material properties are characterized by a single stiffness parameter, the persistence length l(p). Its value, in general, depends on the microscopic interactions between the constituent tubulin dimers and the architecture of the microtubule. Here, we use single-particle tracking methods combined with a fluctuation analysis to systematically study the dependence of l(p) on the total filament length L. Microtubules are grafted to a substrate with one end free to fluctuate in three dimensions. A fluorescent bead is attached proximally to the free tip and is used to record the thermal fluctuations of the microtubule's end. The position distribution functions obtained with this assay allow the precise measurement of l(p) for microtubules of different contour length L. Upon varying L between 2.6 and 47.5 mum, we find a systematic increase of l(p) from 110 to 5,035 mum. At the same time we verify that, for a given filament length, the persistence length is constant over the filament within the experimental accuracy. We interpret this length dependence as a consequence of a nonnegligible shear deflection determined by subnanometer relative displacement of adjacent protofilaments. Our results may shine new light on the function of microtubules as sophisticated nanometer-sized molecular machines and give a unified explanation of seemingly uncorrelated spreading of microtubules' stiffness previously reported in literature.

Mechanical properties of single motor molecules studied by three-dimensional thermal force probing in optical tweezers.

A new method combining three-dimensional (3D) force measurements in an optical trap with the analysis of thermally induced (Brownian) position fluctuations of a trapped probe was used to investigate the mechanical properties of a single molecule, the molecular motor kinesin. One kinesin molecule attached to the probe was bound in a rigorlike state to one microtubule. The optical trap was kept weak to measure the thermal forces acting on the probe, which were mainly counterbalanced by the kinesin tether. The stiffness of kinesin during stretching and compression with respect to its backbone axis were measured. Our results indicate that a section of kinesin close to the motor domain is the dominating element in the flexibility of the motor structure. The experiments demonstrate the power of 3D thermal fluctuation analysis to characterize mechanical properties of individual motor proteins and indicate its usefulness to study single molecule in general

Determination and correction of position detection nonlinearity in single particle tracking and three-dimensional scanning probe microscopy.

A general method is presented for determining and correcting nonlinear position detector responses in single particle tracking as used in three-dimensional scanning probe microscopy based on optical tweezers. The method uses locally calculated mean square displacements of a Brownian particle to detect spatial changes in the sensitivity of the detector. The method is applied to an optical tweezers setup, where the position fluctuations of a microsphere within the optical trap are measured by an interferometric detection scheme with nanometer precision and microsecond temporal resolution. Detector sensitivity profiles were measured at arbitrary positions in solution with a resolution of approximately 6 nm and 20 nm in the lateral and axial directions, respectively. Local detector sensitivities are used to reconstruct the real positions of the particle from the measured position signals.