membrane puncture of human embryonic kidney cells and giant unilamellar vesicles using gold nanoparticles

Laser Induced Thermoplasmonic Puncture for the Study of Plasma Membrane Repair

The cell membrane, serving both as a physical barrier and a signaling platform, is vital for cell survival1. Throughout its entire cell cycle, the plasma membrane (PM) is subjected to damage, such as mechanical2,3,4,5 and chemical6 stress-induced injuries. To maintain membrane integrity and ensure cell survival, the cell has developed robust plasma membrane repair (PMR) mechanisms. These mechanisms depend on various strategies, such as cytoskeleton reorganization, membrane fusion, and membrane replacement strategies7,8,9,10,11, all of which rely on the recruitment of specific proteins. Notably, members of the annexin protein family have been identified as key proteins associated with the processes of PMR1,9,12,13,14,15,16. Following PM injury, the cell experiences an influx of calcium ions (Ca2+), which poses an immediate threat to the cell&#39;s survival17. In response to Ca2+ influx, annexin proteins, which are predominantly located in the cytosol, bind to the inner leaflet of the damaged plasma membrane as a part of the PMR strategies18. Annexin A2 (ANXA2) was one of the first members of the annexin family to be associated with PMR in dysferlin-deficient muscular dystrophy and was suggested to mediate repair by fusing intracellular vesicles to the PM near the injury site5,19,20,21. Subsequently, several functions have been attributed to annexins22, and their role in PMR has garnered increased attention over the past 20 years. However, the exact role of annexins in PMR remains not fully understood15,18,21,22.
This article proposes a method for investigating protein-membrane interaction and membrane dynamics in a controlled and highly localized manner, utilizing a combination of confocal microscopy, optical tweezers, and gold nanoparticles (AuNPs). This method enables the quantitative study of protein, lipid, and small molecule interactions in response to membrane damage and Ca2+ influx. Despite the complexity and multiplicity of components involved in the process of membrane repair, simplified membrane systems that mimic the plasma membrane have been employed to gain a deeper mechanistic understanding of membrane dynamics and the response of annexin proteins to membrane disruption16. Giant unilamellar lipid vesicles (GUVs) were chosen as the model membrane system with a specified lipid composition. The GUVs were generated using the gel-assisted hydration method, specifically polyvinyl alcohol gel hydration, as described by Weinberger et al.23, which allowed for efficient encapsulation of annexins into GUVs.
The utilization of near-infrared (NIR) laser irradiation on metallic nanoparticles (NPs) induces significant heating of the NP, making it an effective method to establish a local heat source exploited in biomedical applications24. The method was initially used to directly measure the temperature surrounding a single AuNP in both 2D and 3D biomimetic assays. In these assays25,26, the plasmonic nanoparticles were irradiated on a supported lipid bilayer or optically trapped near GUVs undergoing a local thermal phase transition upon local heating, enabling quantification and control of the exact temperature profile around the particle. This reference temperature profile has been utilized when investigating or manipulating biological specimens. Further advancements in the method have facilitated the induction of nanoscopic pores in membranes27, allowing for vesicle and cell fusion28,29. Other studies have investigated the behavior of peripheral membrane proteins in GUVs29 and transmembrane proteins30 by creating novel hybrid vesicles, while cell-specific drug delivery has also been explored to control and study cellular responses or gene expression28,29,31,32,33. Recently, the method has been used to investigate protein responses to membrane damage32,34,35.
Several methods exist for disrupting the plasma membrane to explore cellular responses and membrane repair. These include microneedle punctures, microbead shaking, and cell scraping, all of which can disrupt the cell membrane mechanically14,36,37. Chemically induced damage can be achieved by adding detergents5,38&#160;or bacterial toxins39,40 that destabilize the lipid bilayer and generate membrane pores across the plasma membrane. Furthermore, optically induced injuries by continuous wave and pulsed lasers have been used to study PMR components, such as annexin proteins5,14,21,41, in combination with plasmonic nanoparticles42,43,44,45. Despite the efficiency of high-power pulsed lasers, they can cause significant injuries and damage to the cell&#39;s interior along the beam path. Moreover, the detailed changes that occur in the biological matter upon pulsed laser irradiation and whether it creates a well-defined pore remain to be further investigated. An alternative method is presented in this article, employing thermoplasmonics to induce nanoscopic holes in the PM in a controlled manner34,35 without causing significant harm to the internal structures. This is accomplished by exposing plasmonic NPs to a highly focused NIR laser, resulting in an extremely localized temperature increase that can easily reach temperatures exceeding 200 &#176;C, which can lead to small nanoscopic explosions25,46,47. This process can be controlled by adjusting the laser intensity as well as the size, shape, and composition of the NPs48. By employing this technique, researchers can explore the role of proteins in PM repair in living cells, which could help address some of the unanswered questions regarding the involvement of annexin proteins in membrane repair without compromising cell viability.
The optical trapping of plasmonic nanoparticles has been well established by previous studies25,49,50,51,52; however, additional insights regarding the thermoplasmonic properties of the nanoparticles53,54,55&#160;can be obtained in the supplementary materials (Supplementary File 1). The thermoplasmonic method can be used to create nanoscopic holes in the PM for the purpose of studying the cellular response and repair mechanisms. More precisely, the puncture can be achieved through the optical heating of AuNPs in close proximity to the membrane, as shown in Figures 1A and B. This localized puncture allows for Ca2+ influx, which was verified by a calcium sensor, thus activating the PMR machinery. For live cell experiments, AuNPs with a diameter of 200 nm were immobilized on the surface beneath the cell to monitor the role of ANXA2 in PMR via confocal microscopy. The NIR laser (Figure 1A,B), with a wavelength of 1064 nm, irradiates the AuNP, exploiting its plasmonic properties (Figure 1C), resulting in substantial local heating (Figure 1D) in the biological transparency window49 while causing minimal damage to the cell itself. The high-temperature region surrounding the AuNP rapidly decreases by 30-40% at a distance corresponding to the radius of the NP, as depicted in Figure 1E, allowing for an extremely confined injury in all three dimensions.
Supplementary File 1.&#160;<a href="https://www.jove.com/files/ftp_upload/65776/Supplementary File 1_updated.zip" target="_blank">Please click here to download this File.</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65776/65776fig01.jpg" /> 
Figure 1: Schematic outline of the experimental method. (A) ANXA-transfected cells are situated on top of immobilized gold nanoparticles (AuNPs) on the surface, or (B) giant unilamellar vesicles (GUVs)&#160;with encapsulated ANXA are suspended in a medium containing AuNPs. (C) A single AuNP is irradiated by the NIR optical trap, where the interaction between the incoming electromagnetic field and the conduction electrons leads to the collective oscillation of electrons within the NP. (D) This process results in a highly confined yet significant increase in temperature. To estimate the temperature at the NP surface, Mie theory is employed, and a (E) temperature profile is calculated for an AuNP with a diameter of 200 nm and laser intensity I = 6.36 x 108 W/cm2. <a href="https://www.jove.com/files/ftp_upload/65776/65776fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To minimize the thermal effect on the cell membrane, the AuNPs are only irradiated for ~1 second. This causes a transient and local burst of heating, which reduces the damage to proteins that typically require more time to unfold. Upon membrane puncture, annexin proteins are recruited in a fraction of a second, and within a few seconds, an annexin ring-like scaffold is formed around the site of injury (Figure 2). This approach has also been applied to explore the involvement of ANXA5 in both living cells and model membranes16 in an effort to shed light on the complete scheme of the repair processes. While the primary focus has been on the correlating recruitment of various annexin proteins, the biophysical aspects of the repair mechanism have yet to be elucidated.
To fully implement the proposed method, three key components are required: confocal microscopy, optical tweezers, and metal nanoparticles. Optical tweezers are used to trap AuNPs, and their construction can be achieved by following the procedure outlined by Neuman et al.49. However, if building an optical tweezer proves to be too challenging, a tightly focused NIR laser can be used to irradiate AuNPs immobilized beneath the cells. While spherical AuNPs were chosen for this protocol, a variety of plasmonic particles with tunable absorption spectra could also be utilized to achieve a highly localized temperature gradient within the NIR region48.
Fluorescence imaging is necessary for observing the role of the fluorescently labeled proteins, and therefore, total internal reflection microscopy (TIRF)56 could be considered as an alternative to confocal imaging. However, this technique only allows for surface imaging and would not be compatible with the model membrane vesicle experiments. Consequently, both the optical tweezers and confocal microscope are essential for the precise localization of the nanoparticle and detailed investigation of the local area surrounding the cell injury. To effectively irradiate the nanoparticle with a diffraction-limited laser focus, it is necessary to visualize the nanoparticle. This can be optimally achieved by reflection microscopy, which is a standard imaging feature of Leica confocal microscopes. However, if reflection or scattering imaging is not available, alternative methods, such as the less efficient fluorescent AuNP labeling, may be considered.
In summary, the highly controllable and localized thermoplasmonic method presented in this study has the potential to serve as an excellent platform for investigating the molecular components involved in cellular responses and PM repair mechanisms in living cells. In addition to studying the protein response upon PM damage, this approach can also be utilized for locally puncturing vesicles, thereby enabling an investigation of the protein response in both protein-protein and protein-membrane dynamics. Moreover, this method allows for a quantitative analysis of the interactions between proteins, lipids, and small molecules when membranes are disrupted. Collectively, these advances have the potential to shed light on some of the unresolved questions regarding the intricate and complex plasma membrane repair machinery.

Author Spotlight: Exploring Plasma Membrane Repair Mechanisms with Innovative Thermoplasmonic Puncturing 

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

Our research is focused on investigation of plasma membrane repair mechanisms, in both living cells, but also in biomimetic systems, called the giant unilamellar vesicles. We are particularly interested in understanding the role of proteins like annexins and facilitating surface repair. These intricate processes are explored using our innovative thermoplasmonic puncture technique.Currently, cell repair is being investigated using pulse lasers, combined with molecular biology, to identify proteins that are recruited to the site of injury. The drawback of this approach is that it's hard to control the extent of damage caused by using pulse lasers. Currently, our experiments come with a few challenges.We're working on fine tuning the alignment of our laser focus with a nanoparticle, which is crucial for precision, and although it can be a bit tricky, we're also actively working on minimizing the formation of nanobubbles during the heating process. Our research demonstrated that annexin proteins swiftly respond to calcium influx, playing a key role in membrane repair, and reveal diverse behaviors of individual annexins. To better understand the mechanisms involved, we turned by biomimetic systems, allowing us to precisely measure how annexins influence membrane bending near a membrane hole.Our protocol meets the need for precise localized membrane injuries in healthy cells. It provides valuable insight into live cell membrane repair mechanisms. Furthermore, it enabled the study of the biophysical role of membrane proteins recruited to the annular region near upper head in biomimetic membranes.

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