Changes in Cell Morphology and Actin Organization in Embryonic Stem Cells Cultured under Different Conditions.

The cellular cytoskeleton provides the cell with a mechanical rigidity that allows mechanical interaction between cells and the extracellular environment. The actin structure plays a key role in mechanical events such as motility or the establishment of cell polarity. From the earliest stages of development, as represented by the ex vivo expansion of naïve embryonic stem cells (ESCs), the critical mechanical role of the actin structure is becoming recognized as a vital cue for correct segregation and lineage control of cells and as a regulatory structure that controls several transcription factors. Naïve ESCs have a characteristic morphology, and the ultrastructure that underlies this condition remains to be further investigated. Here, we investigate the 3D actin cytoskeleton of naïve mouse ESCs using super-resolution optical reconstruction microscopy (STORM). We investigate the morphological, cytoskeletal, and mechanical changes in cells cultured in 2i or Serum/LIF media reflecting, respectively, a homogeneous preimplantation cell state and a state that is closer to embarking on differentiation. STORM imaging showed that the peripheral actin structure undergoes a dramatic change between the two culturing conditions. We also detected micro-rheological differences in the cell periphery between the cells cultured in these two media correlating well with the observed nano-architecture of the ESCs in the two different culture conditions. These results pave the way for linking physical properties and cytoskeletal architecture to cell morphology during early development.

Remotely controlled fusion of selected vesicles and living cells: a key issue review.

Remote control over fusion of single cells and vesicles has a great potential in biological and chemical research allowing both transfer of genetic material between cells and transfer of molecular content between vesicles. Membrane fusion is a critical process in biology that facilitates molecular transport and mixing of cellular cytoplasms with potential formation of hybrid cells. Cells precisely regulate internal membrane fusions with the aid of specialized fusion complexes that physically provide the energy necessary for mediating fusion. Physical factors like membrane curvature, tension and temperature, affect biological membrane fusion by lowering the associated energy barrier. This has inspired the development of physical approaches to harness the fusion process at a single cell level by using remotely controlled electromagnetic fields to trigger membrane fusion. Here, we critically review various approaches, based on lasers or electric pulses, to control fusion between individual cells or between individual lipid vesicles and discuss their potential and limitations for present and future applications within biochemistry, biology and soft matter.

Dynamics of membrane nanotubes coated with I-BAR.

Membrane deformation is a necessary step in a number of cellular processes such as filopodia and invadopodia formation and has been shown to involve membrane shaping proteins containing membrane binding domains from the IRSp53-MIM protein family. In reconstituted membranes the membrane shaping domains can efficiently deform negatively charged membranes into tubules without any other proteins present. Here, we show that the IM domain (also called I-BAR domain) from the protein ABBA, forms semi-flexible nanotubes protruding into Giant Unilamellar lipid Vesicles (GUVs). By simultaneous quantification of tube intensity and tubular shape we find both the diameter and stiffness of the nanotubes. I-BAR decorated tubes were quantified to have a diameter of ~50 nm and exhibit no stiffening relative to protein free tubes of the same diameter. At high protein density the tubes are immobile whereas at lower density the tubes diffuse freely on the surface of the GUV. Bleaching experiments of the fluorescently tagged I-BAR confirmed that the mobility of the tubes correlates with the mobility of the I-BAR on the GUV membrane. Finally, at low density of I-BAR the protein upconcentrates within tubes protruding into the GUVs. This implies that I-BAR exhibits strong preference for negatively curved membranes.

Dynamic buckling of actin within filopodia.

Filopodia are active tubular structures protruding from the cell surface which allow the cell to sense and interact with the surrounding environment through repetitive elongation-retraction cycles. The mechanical behavior of filopodia has been studied by measuring the traction forces exerted on external substrates.(1) These studies have revealed that internal actin flow can transduce a force across the cell surface through transmembrane linkers like integrins. In addition to the elongation-retraction behavior filopodia also exhibit a buckling and rotational behavior. Filopodial buckling in conjunction with rotation enables the cell to explore a much larger 3-dimensional space and allows for more complex, and possibly stronger, interactions with the external environment.(2) Here we focus on how bending of the filopodial actin dynamically correlates with pulling on an optically trapped microsphere which acts like an external substrate attached to the filopodial tip. There is a clear correlation between presence of actin near the tip and exertion of a traction force, thus demonstrating that the traction force is transduced along the actin shaft inside the filopodium. By extending a filopodium and holding it while measuring the cellular response, we also monitor and analyze the waiting times for the first buckle observed in the fluorescently labeled actin shaft.

An updated look at actin dynamics in filopodia.

Cells dynamically interact with and probe their environment by growing finger-like structures named filopodia. The dynamics of filopodia are mainly caused by the actin rich core or shaft which sits inside the filopodial membrane and continuously undergoes changes like growth, shrinking, bending, and rotation. Recent experiments combining advanced imaging and manipulation tools have provided detailed quantitative data on the correlation between mechanical properties of filopodia, their molecular composition, and the dynamic architecture of the actin structure. These experiments have revealed how retrograde flow and twisting of the actin shaft within filopodia can generate traction on external substrates. Previously, the mechanism behind filopodial pulling was mainly attributed to retrograde flow of actin, but recent experiments have shown that rotational dynamics can also contribute to the traction force. Although force measurements have indicated a step-like behavior in filopodial pulling, no direct evidence has been provided to link this behavior to a molecular motor like myosin. Therefore, the underlying biochemical and mechanical mechanisms behind filopodial force generation still remain to be resolved.

Helical buckling of actin inside filopodia generates traction.

Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell-cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.

Nanoscale phase behavior on flat and curved membranes.

The diverse physical properties of membranes play a critical role in many membrane associated biological processes. Proteins responsible for membrane transport can be affected by the lateral membrane order and lateral segregation of proteins is often controlled by the preference of certain membrane anchors for membrane phases having a physically ordered state. The dynamic properties of coexisting membrane phases are often studied by investigating their thermal behavior. Optical trapping of gold nanoparticles is a useful tool to generate local phase transitions in membranes. The high local temperatures surrounding an irradiated gold nanoparticle can be used to melt a part of a giant unilamellar lipid vesicle (GUV) which is then imaged using phase sensitive fluorophores embedded within the bilayer. By local melting of GUVs we reveal how a protein-free, one component lipid bilayer can mediate passive transport of fluorescent molecules by localized and transient pore formation. Also, we show how tubular membrane curvatures can be generated by optical pulling from the melted region on the GUV. This will allow us to measure the effect of membrane curvature on the phase transition temperature.

Fluorescent quantification of size and lamellarity of membrane nanotubes.

Membrane nanotubes, ubiquitous in cellular systems, adopt a spectrum of curvatures and shapes that are dictated by their intrinsic physical characteristics as well as their interactions with the local cellular environment. A high bending flexibility is needed in the crowded cytoplasm where tubes often need to bend significantly in the axial direction at sub-micron length scales. We find the stiffness of spontaneously formed membrane nanotubes by measuring the persistence length of reconstituted membrane nanotubes freely suspended in solution and imaged by fluorescence microscopy. By quantifying the tube diameter we demonstrate for the first time that the persistence length scales linearly with radius. Although most tubes are uni-lamellar, the predicted linear scaling between tube radius and persistence length allows us to identify tubes that spontaneously form as multilamellar structures upon hydration. We provide the first experimental evidence that illumination of lipid fluorophores can have a profound effect on the lipid bilayer which we sensitively detect as a continuous change in the tube persistence length with time. The novel assay and methodology here presented has potential for quantification of the structural reinforcement of membrane tubes by scaffolding proteins.

Local and transient permeation events are associated with local melting of giant liposomes.

We reveal that the gel to fluid phase transition causes spherical membrane vesicles to release a finite number of molecules in several consecutive and localized events. By locally melting Giant Unilamellar lipid Vesicles (GUVs), using an optically trapped gold nanoparticle (AuNP) as a local heat source, we establish a local phase transition on the spherical GUV membrane clearly visualized using a phase sensitive fluorescent marker. We measure transient permeation events through this transition zone visualized as de-quenching of calcein as it escapes the interior of the GUV. Since biological membranes share several features with melting membranes, like nanoscale domain formation and critical density fluctuations, similar passive membrane transport could potentially be abundant in living cells.

Revealing hidden dynamics within living soft matter.

In the study of living soft matter, we often seek to understand the mechanisms underlying the motion of a single molecule, an organelle, or some other tracer. The experimentally observed signature of the tracer is masked by its thermal fluctuations, inherent drift of the system, and instrument noise. In addition, the timing or length scales of the events of interest are often unknown. In the current issue of ACS Nano, Chen et al. present a general method for extracting the underlying dynamics from time series. Here, we provide an easily accessible introduction to the method, put it into perspective with the field, and exemplify how it can be used to answer important out-standing questions within soft matter and living systems.

Mapping 3D focal intensity exposes the stable trapping positions of single nanoparticles.

The photonic interactions between a focused Gaussian laser beam and a nanoscopic particle are highly dependent on exact particle location and focal intensity distribution. So far, the 3D focal intensity distribution and the preferred position of a nanoparticle confined within the focal region were only theoretically predicted. Here, we directly map the three-dimensional focal intensity distribution, quantify stable trapping positions, and prove that certain sizes of nanoparticles stably trap in front of the focus.

Large-scale orientation dependent heating from a single irradiated gold nanorod.

We quantify the extreme heating associated with resonant irradiation of individual gold nanorods by using a novel assay based on partitioning of lipophilic dyes between membrane phases. The temperature increase is sensitively dependent on the angle between the laser polarization and the orientation of the nanorod. A dramatic and irreversible decrease in the heating of a nanorod occurs at high-illumination intensities; this effect is attributed to surface melting of the nanorod causing it to restructure into a more spherical shape and lose its extreme photothermal properties.

Expanding the optical trapping range of lipid vesicles to the nanoscale.

Small unilamellar lipid vesicles with diameters down to 50 nm enclosing high refractive index sucrose cores can be optically trapped individually in three dimensions using a focused laser beam. Combined optical trapping and confocal microscopy allows for simultaneous quantitative measurements of the forces exerted on individual vesicles and of their size and shape. The position of individual vesicles in three dimensions is measured with nanometer spatial and ∼10 µs temporal resolution.

Heat profiling of three-dimensionally optically trapped gold nanoparticles using vesicle cargo release.

Irradiated metallic nanoparticles hold great promise as heat transducers in photothermal applications such as drug delivery assays or photothermal therapy. We quantify the temperature increase of individual gold nanoparticles trapped in three dimensions near lipid vesicles exhibiting temperature sensitive permeability. The surface temperature can increase by hundreds of degrees Celsius even at moderate laser powers. Also, there are significant differences of the heat profiles in two-dimensional and three-dimensional trapping assays.

Direct measurements of heating by electromagnetically trapped gold nanoparticles on supported lipid bilayers.

Absorption of electromagnetic irradiation results in significant heating of metallic nanoparticles, an effect which can be advantageously used in biomedical contexts. Also, metallic nanoparticles are presently finding widespread use as handles, contacts, or markers in nanometer scale systems, and for these purposes it is essential that the temperature increase associated with electromagnetic irradiation is not harmful to the environment. Regardless of whether the heating of metallic nanoparticles is desired or not, it is crucial for nanobio assays to know the exact temperature increase associated with electromagnetic irradiation of metallic nanoparticles. We performed direct measurements of the temperature surrounding single gold nanoparticles optically trapped on a lipid bilayer, a biologically relevant matrix. The lipid bilayer had incorporated fluorescent molecules which have a preference for either fluid or gel phases. The heating associated with electromagnetic radiation was measured by visualizing the melted footprint around the irradiated particle. The effect was measured for individual gold nanoparticles of a variety of sizes and for a variety of laser powers. The temperatures were highly dependent on particle size and laser power, with surface temperature increments ranging from a few to hundreds of degrees Celsius. Our results show that by a careful choice of gold nanoparticle size and strength of irradiating electromagnetic field, one can control the exact particle temperature. The method is easily applicable to any type of nanoparticle for which the photothermal effect is sought to be quantified.

An active biopolymer network controlled by molecular motors.

We describe an active polymer network in which processive molecular motors control network elasticity. This system consists of actin filaments cross-linked by filamin A (FLNa) and contracted by bipolar filaments of muscle myosin II. The myosin motors stiffen the network by more than two orders of magnitude by pulling on actin filaments anchored in the network by FLNa cross-links, thereby generating internal stress. The stiffening response closely mimics the effects of external stress applied by mechanical shear. Both internal and external stresses can drive the network into a highly nonlinear, stiffened regime. The active stress reaches values that are equivalent to an external stress of 14 Pa, consistent with a 1-pN force per myosin head. This active network mimics many mechanical properties of cells and suggests that adherent cells exert mechanical control by operating in a nonlinear regime where cell stiffness is sensitive to changes in motor activity. This design principle may be applicable to engineering novel biologically inspired, active materials that adjust their own stiffness by internal catalytic control.

Efficient optical trapping and visualization of silver nanoparticles.

We performed efficient optical trapping combined with sensitive optical detection of individual silver nanoparticles. The particles ranging in size from 20 to 275 nm in diameter were trapped in three dimensions using low laser power by minimizing spherical aberrations at the focus. The optical forces were quantified, and we found that the larger the particle, the stronger the optical force. The particles were imaged by an additional strongly scattered laser.

A quantitative analysis of contractility in active cytoskeletal protein networks.

Cells actively produce contractile forces for a variety of processes including cytokinesis and motility. Contractility is known to rely on myosin II motors which convert chemical energy from ATP hydrolysis into forces on actin filaments. However, the basic physical principles of cell contractility remain poorly understood. We reconstitute contractility in a simplified model system of purified F-actin, muscle myosin II motors, and alpha-actinin cross-linkers. We show that contractility occurs above a threshold motor concentration and within a window of cross-linker concentrations. We also quantify the pore size of the bundled networks and find contractility to occur at a critical distance between the bundles. We propose a simple mechanism of contraction based on myosin filaments pulling neighboring bundles together into an aggregated structure. Observations of this reconstituted system in both bulk and low-dimensional geometries show that the contracting gels pull on and deform their surface with a contractile force of approximately 1 microN, or approximately 100 pN per F-actin bundle. Cytoplasmic extracts contracting in identical environments show a similar behavior and dependence on myosin as the reconstituted system. Our results suggest that cellular contractility can be sensitively regulated by tuning the (local) activity of molecular motors and the cross-linker density and binding affinity.