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.

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.

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.

Interdisciplinary Synergy to Reveal Mechanisms of Annexin-Mediated Plasma Membrane Shaping and Repair.

The plasma membrane surrounds every single cell and essentially shapes cell life by separating the interior from the external environment. Thus, maintenance of cell membrane integrity is essential to prevent death caused by disruption of the plasma membrane. To counteract plasma membrane injuries, eukaryotic cells have developed efficient repair tools that depend on Ca2+- and phospholipid-binding annexin proteins. Upon membrane damage, annexin family members are activated by a Ca2+ influx, enabling them to quickly bind at the damaged membrane and facilitate wound healing. Our recent studies, based on interdisciplinary research synergy across molecular cell biology, experimental membrane physics, and computational simulations show that annexins have additional biophysical functions in the repair response besides enabling membrane fusion. Annexins possess different membrane-shaping properties, allowing for a tailored response that involves rapid bending, constriction, and fusion of membrane edges for resealing. Moreover, some annexins have high affinity for highly curved membranes that appear at free edges near rupture sites, a property that might accelerate their recruitment for rapid repair. Here, we discuss the mechanisms of annexin-mediated membrane shaping and curvature sensing in the light of our interdisciplinary approach to study plasma membrane repair.

Ultrasensitive optical biosensor for detection of miRNA-155 using positively charged Au nanoparticles.

An ultrasensitive optical biosensor for microRNA-155 (miR-155) was developed to diagnose breast cancer at early stages. At first, the probe DNA covalently bind to the negatively charged gold nanoparticles (citrate-capped AuNPs). Then, the target miR-155 electrostatically adsorb onto the positively charged gold nanoparticles (polyethylenimine-capped AuNP) surface. Finally, by mixing citrate-capped AuNP/probe and polyethylenimine-capped AuNP/miR-155, hybridization occurs and the optical signal of the mixture give a measure to quantify the miR-155 content. The proposed biosensor is able to specify 3-base-pair mismatches and genomic DNA from target miR-155. The novelty of this biosensor is in its ability to trap the label-free target by its branched positively charged polyethylenimine. This method increases loading the target on the polyethylenimine-capped AuNPs' surface. So, proposed sensor enables miR-155 detection at very low concentrations with the detection limit of 100 aM and a wide linear range from 100 aM to 100 fM.