Biological Applications of Thermoplasmonics.

Thermoplasmonics has emerged as an extraordinarily versatile tool with profound applications across various biological domains ranging from medical science to cell biology and biophysics. The key feature of nanoscale plasmonic heating involves remote activation of heating by applying laser irradiation to plasmonic nanostructures that are designed to optimally convert light into heat. This unique capability paves the way for a diverse array of applications, facilitating the exploration of critical biological processes such as cell differentiation, repair, signaling, and protein functionality, and the advancement of biosensing techniques. Of particular significance is the rapid heat cycling that can be achieved through thermoplasmonics, which has ushered in remarkable technical innovations such as accelerated amplification of DNA through quantitative reverse transcription polymerase chain reaction. Finally, medical applications of photothermal therapy have recently completed clinical trials with remarkable results in prostate cancer, which will inevitably lead to the implementation of photothermal therapy for a number of diseases in the future. Within this review, we offer a survey of the latest advancements in the burgeoning field of thermoplasmonics, with a keen emphasis on its transformative applications within the realm of biosciences.

Thermoplasmonic Vesicle Fusion Reveals Membrane Phase Segregation of Influenza Spike Proteins.

Many cellular processes involve the lateral organization of integral and peripheral membrane proteins into nanoscale domains. Despite the biological significance, the mechanisms that facilitate membrane protein clustering into nanoscale lipid domains remain enigmatic. In cells, the analysis of membrane protein phase affinity is complicated by the size and temporal nature of ordered and disordered lipid domains. To overcome these limitations, we developed a method for delivering membrane proteins from transfected cells into phase-separated model membranes that combines optical trapping with thermoplasmonic-mediated membrane fusion and confocal imaging. Using this approach, we observed clear phase partitioning into the liquid disordered phase following the transfer of GFP-tagged influenza hemagglutinin and neuraminidase from transfected cell membranes to giant unilamellar vesicles. The generic platform presented here allows investigation of the phase affinity of any plasma membrane protein which can be labeled or tagged with a fluorescent marker.

Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair.

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.

Strength in numbers: effect of protein crowding on the shape of cell membranes.

Continuous reshaping of the plasma membrane into pleomorphic shapes is critical for a plethora of cellular functions. How the cell carries out this enigmatic control of membrane remodeling has remained an active research field for decades and several molecular and biophysical mechanisms have shown to be involved in overcoming the energy barrier associated with membrane bending. The reported mechanisms behind membrane bending have been largely concerned with structural protein features, however, in the last decade, reports on the ability of densely packed proteins to bend membranes by protein-protein crowding, have challenged prevailing mechanistic views. Crowding has now been shown to generate spontaneous vesicle formation and tubular morphologies on cell- and model membranes, demonstrating crowding as a relevant player involved in the bending of membranes. Still, current research is largely based on unnatural overexpression of proteins in non-native domains, and together with efforts in modeling, this has led to questioning the in vivo impact of crowding. In this review, we examine this previously overlooked mechanism by summarizing recent advances in the understanding of protein-protein crowding and its prevalence in cellular membrane-shaping processes.

Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles.

The plasma membrane (PM) of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the PM is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including PM repair. Its binding to membranes is activated by Ca2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant PM vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations, in which we extract molecular information from atomistic scale simulations as input to our macroscopic scale simulations, furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes.

Plasmonic Heating of Nanostructures.

The absorption of light by plasmonic nanostructures and their associated temperature increase are exquisitely sensitive to the shape and composition of the structure and to the wavelength of light. Therefore, much effort is put into synthesizing novel nanostructures for optimized interaction with the incident light. The successful synthesis and characterization of high quality and biocompatible plasmonic colloidal nanoparticles has fostered numerous and expanding applications, especially in biomedical contexts, where such particles are highly promising for general drug delivery and for tomorrow's cancer treatment. We review the thermoplasmonic properties of the most commonly used plasmonic nanoparticles, including solid or composite metallic nanoparticles of various dimensions and geometries. Common methods for synthesizing plasmonic particles are presented with the overall goal of providing the reader with a guide for designing or choosing nanostructures with optimal thermoplasmonic properties for a given application. Finally, the biocompatibility and biological tolerance of structures are critically discussed along with novel applications of plasmonic nanoparticles in the life sciences.

Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells.

The targeted spatial organization (sorting) of Gprotein-coupled receptors (GPCRs) is essential for their biological function and often takes place in highly curved membrane compartments such as filopodia, endocytic pits, trafficking vesicles or endosome tubules. However, the influence of geometrical membrane curvature on GPCR sorting remains unknown. Here we used fluorescence imaging to establish a quantitative correlation between membrane curvature and sorting of three prototypic class A GPCRs (the neuropeptide Y receptor Y2, the ß1 adrenergic receptor and the ß2 adrenergic receptor) in living cells. Fitting of a thermodynamic model to the data enabled us to quantify how sorting is mediated by an energetic drive to match receptor shape and membrane curvature. Curvature-dependent sorting was regulated by ligands in a specific manner. We anticipate that this curvature-dependent biomechanical coupling mechanism contributes to the sorting, trafficking and function of transmembrane proteins in general.

Quantification of Loading and Laser-Assisted Release of RNA from Single Gold Nanoparticles.

Novel RNA-based technologies provide an avenue of possibilities to control the regulation of gene expression in cells. To realize the full potential of small interfering RNA (siRNA)-based therapy, efficient delivery vehicles and novel strategies for triggering release from carrier vehicles have to be developed. Gold nanoparticles (AuNPs) with sizes of ∼50-150 nm have the ability to accumulate in tumor tissue and can be transported across the membrane by endocytosis. Therefore, a laser-controlled oligonucleotide release from such particles is of particular interest. Here, we quantify the loading of specifically attached microRNA oligonucleotides (miRNA) onto single gold nanoparticles with diameters of 80, 100, 150, and 200 nm. We show that AuNPs have a curvature-dependent density of miRNA loading: the higher the curvature, the higher the loading density. Moreover, we demonstrate how one sensing strand of an RNA duplex can be dehybridized and hence released from the AuNP by heating the AuNP by irradiation with a near-infrared (NIR) laser. Laser-induced release is also demonstrated inside living cells. Together, these findings show that plasmonic nanoparticles with high curvatures are ideal carriers of oligonucleotides into cells, and their cargo can be released in a controlled manner by a thermoplasmonic mechanism. Importantly, this remotely controlled release strategy can be applied to any cargo attached to a plasmonic nanocarrier, on either the single particle or ensemble level.

Vesicle Fusion Triggered by Optically Heated Gold Nanoparticles.

Membrane fusion can be accelerated by heating that causes membrane melting and expansion. We locally heated the membranes of two adjacent vesicles by laser irradiating gold nanoparticles, thus causing vesicle fusion with associated membrane and cargo mixing. The mixing time scales were consistent with diffusive mixing of the membrane dyes and the aqueous content. This method is useful for nanoscale reactions as demonstrated here by I-BAR protein-mediated membrane tubulation triggered by fusion.

Interferometric Detection of Single Gold Nanoparticles Calibrated against TEM Size Distributions.

Single nanoparticle analysis: An interferometric optical approach calibrates sizes of gold nanoparticles (AuNPs) from the interference intensities by calibrating their interferometric signals against the corresponding transmission electron microscopy measurements. This method is used to investigate whether size affects the diffusion behavior of AuNPs conjugated to supported lipid bilayer membranes and to multiplex the simultaneous detection of three different AuNP labels.

Heat generation by irradiated complex composite nanostructures.

Heating of irradiated metallic e-beam generated nanostructures was quantified through direct measurements paralleled by novel model-based numerical calculations. By comparing discs, triangles, and stars we showed how particle shape and composition determines the heating. Importantly, our results revealed that substantial heat is generated in the titanium adhesive layer between gold and glass. Even when the Ti layer is as thin as 2 nm it absorbs as much as a 30 nm Au layer and hence should not be ignored.

Filopodia: integrating cellular functions with theoretical models.

Filopodia, widely distributed on cell surfaces, are distinguished by their dynamic extensions, playing pivotal roles in a myriad of biological processes. Their functions span from mechanosensing and guidance to cell-cell communication during cellular organization in the early embryo. Filopodia have significant roles in pathogenic processes, such as cancer invasion and viral dissemination. Molecular mapping of the filopodome has revealed generic components essential for filopodia functions. In parallel, recent insights into biophysical mechanisms governing filopodia dynamics have provided the foundation for broader investigations of filopodia's biological functions. We highlight recent discoveries of engagement of filopodia in various stages of development and pathogenesis and present an overview of intricate molecular and physical features of these cellular structures across a spectrum of cellular activities.

A Thermoplasmonic Approach for Investigating Plasma Membrane Repair in Living Cells and Model Membranes.

The cell membrane is crucial for cell survival, and ensuring its integrity is essential as the cell experiences injuries throughout its entire life cycle. To prevent damage to the membrane, cells have developed efficient plasma membrane repair mechanisms. These repair mechanisms can be studied by combining confocal microscopy and nanoscale thermoplasmonics to identify and investigate the role of key proteins, such as annexins, involved in surface repair in living cells and membrane model systems. The puncturing method employs a laser to induce highly localized heating upon nanoparticle irradiation. The use of near-infrared light minimizes phototoxicity in the biological sample, while the majority of the absorption takes place in the near-infrared resonant plasmonic nanoparticle. This thermoplasmonic method has been exploited for potential photothermal and biophysical research to enhance the understanding of intracellular mechanisms and cellular responses through vesicle and cell fusion studies. The approach has shown to be complementary to existing methods for membrane disruption, such as mechanically, chemically, or optically induced injuries, and provides a high level of control by inflicting extremely localized injuries. The extent of the injury is limited to the vicinity of the spherical nanoparticle, and no detrimental damage occurs along the beam path as opposed to pulsed lasers using different wavelengths. Despite certain limitations, such as the formation of nanobubbles, the thermoplasmonic method offers a unique tool for investigating cellular responses in plasma membrane repair in an almost native environment without compromising cell viability. When integrated with confocal microscopy, the puncturing method can provide a mechanistic understanding of membrane dynamics in model membrane systems as well as quantitative information on protein responses to membrane damage, including protein recruitment and their biophysical function. Overall, the application of this method to reduced model systems can enhance our understanding of the intricate plasma membrane repair machinery in living cells.

Individual vesicle fusion events mediated by lipid-anchored DNA.

Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk. Here, we use a model membrane architecture developed in our lab to directly observe single-vesicle fusion events using fluorescence microscopy. In this system, a planar tethered membrane patch serves as the target membrane for incoming vesicles. This allows us to quantify the kinetics and characteristics of individual fusion events from the perspective of the lipids or the DNA-lipids involved in the process. We find that the fusion pathways are heterogeneous, with an arrested hemi-fusion state predominating, and we quantitate the outcome and rate of fusion events to construct a mechanistic model of DNA-mediated vesicle fusion. The waiting times between docking and fusion are distributed exponentially, suggesting that fusion occurs in a single step. Our analysis indicates that when two lipid bilayers are brought into close proximity, fusion occurs spontaneously, with little or no dependence on the number of DNA hybrids formed.

Close, but not too close: a mesoscopic description of (a)symmetry and membrane shaping mechanisms.

Biomembranes are fundamental to our understanding of the cell, the basic building block of all life. An intriguing aspect of membranes is their ability to assume a variety of shapes, which is crucial for cell function. Here, we review various membrane shaping mechanisms with special focus on the current understanding of how local curvature and local rigidity induced by membrane proteins leads to emerging forces and consequently large-scale membrane deformations. We also argue that describing the interaction of rigid proteins with membranes purely in terms of local membrane curvature is incomplete and that changes in the membrane rigidity moduli must also be considered.

Label-free optical interferometric microscopy to characterize morphodynamics in living plants.

During the last century, fluorescence microscopy has played a pivotal role in a range of scientific discoveries. The success of fluorescence microscopy has prevailed despite several shortcomings like measurement time, photobleaching, temporal resolution, and specific sample preparation. To bypass these obstacles, label-free interferometric methods have been developed. Interferometry exploits the full wavefront information of laser light after interaction with biological material to yield interference patterns that contain information about structure and activity. Here, we review recent studies in interferometric imaging of plant cells and tissues, using techniques such as biospeckle imaging, optical coherence tomography, and digital holography. These methods enable quantification of cell morphology and dynamic intracellular measurements over extended periods of time. Recent investigations have showcased the potential of interferometric techniques for precise identification of seed viability and germination, plant diseases, plant growth and cell texture, intracellular activity and cytoplasmic transport. We envision that further developments of these label-free approaches, will allow for high-resolution, dynamic imaging of plants and their organelles, ranging in scales from sub-cellular to tissue and from milliseconds to hours.

Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer.

Nanometer-scale intermembrane contact areas (CAs) formed between single small unilamellar lipid vesicles (SUVs) and planar supported lipid bilayers are quantified by measuring fluorescence resonance energy transfer (FRET) between a homogenous layer of donor fluorophores labeling the supported bilayer and acceptor fluorophores labeling the SUVs. The smallest CAs detected in our setup between biotinylated SUVs and dense monolayers of streptavidin were approximately 20 nm in radius. Deformation of SUVs is revealed by comparing the quenching of the donors to calculations of FRET between a perfectly spherical shell and a flat surface containing complementary fluorophores. These results confirmed the theoretical prediction that the degree of deformation scales with the SUV diameter. The size of the CA can be controlled experimentally by conjugating polyethylene glycol polymers to the SUV or the surface and thereby modulating the interfacial energy of adhesion. In this manner, we could achieve secure immobilization of SUVs under conditions of minimal deformation. Finally, we demonstrate that kinetic measurements of CA, at constant adhesion, can be used to record in real-time quantitative changes in the bilayer tension of a nano-scale lipid membrane system.

Temperature Controls Onset and Period of NF-κB Oscillations and can Lead to Chaotic Dynamics.

The transcription factor NF-κB plays a vital role in the control of the immune system, and following stimulation with TNF-α its nuclear concentration shows oscillatory behaviour. How environmental factors, in particular temperature, can control the oscillations and thereby affect gene stimulation is still remains to be resolved question. In this work, we reveal that the period of the oscillations decreases with increasing temperature. We investigate this using a mathematical model, and by applying results from statistical physics, we introduce temperature dependency to all rates, resulting in a remarkable correspondence between model and experiments. Our model predicts how temperature affects downstream protein production and find a crossover, where high affinity genes upregulates at high temperatures. Finally, we show how or that oscillatory temperatures can entrain NF-κB oscillations and lead to chaotic dynamics presenting a simple path to chaotic conditions in cellular biology.

Thermoplasmonic nano-rupture of cells reveals annexin V function in plasma membrane repair.

Maintaining the integrity of the cell plasma membrane (PM) is critical for the survival of cells. While an efficient PM repair machinery can aid survival of healthy cells by preventing influx of extracellular calcium, it can also constitute an obstacle in drug delivery and photothermal therapy. We show how nanoscopic holes can be created in a controlled fashion to the cell's plasma membrane, thus allowing identification of molecular components which have a pivotal role in PM repair. Cells are punctured by laser induced local heating of gold nanostructures at the cell surface which causes nano-ruptures in cellular PMs. Recruitment of annexin V near the hole is found to locally reshape the ruptured plasma membrane. Experiments using model membranes, containing recombinant annexin V, provide further biophysical insight into the ability of annexin V to reshape edges surrounding a membrane hole. The thermoplasmonic method provides a general strategy to monitor the response to nanoscopic injuries to the cell surface which offer new insight into how cells respond to photothermal treatment.

Filopodia rotate and coil by actively generating twist in their actin shaft.

Filopodia are actin-rich structures, present on the surface of eukaryotic cells. These structures play a pivotal role by allowing cells to explore their environment, generate mechanical forces or perform chemical signaling. Their complex dynamics includes buckling, pulling, length and shape changes. We show that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling. Importantly, the actin core inside filopodia performs a twisting or spinning motion which is observed for a range of cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist is an emergent phenomenon of active filaments confined in a narrow channel which is supported by measured traction forces and helical buckles that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.