Cells as the fundamental units of life, exhibit significant complexity, hosting intricate networks of biochemical processes and structural components which enable them to perform a vast array of functions necessary for the survival and adaptation of living organisms. The dynamic and diverse nature of cells causes them to constantly interact and adapt to environmental cues. Cell types and morphologies vary widely among organisms and tissues, reflecting the discrete functions and specialized roles in maintaining the viability of the biosystems involved. Abnormalities in cell size and shape can be associated with pathological conditions, which can be monitored via imaging techniques and used for diagnostic purposes. During the processes of endocytosis and exocytosis, the plasma membrane can form invaginations and exhibit shape remodeling events. The ability of the plasma membrane to undergo dynamic morphological alterations allows cells to control cellular communication, recognition processes, adhesion and immune responses as well as the transport of substances between the cell interior and exterior environment. The plasma membrane composed of a diverse arrangement of lipids, proteins and carbohydrates, plays an important role in maintaining the structural integrity and metabolic activities of cells thus taking part in the regulation of the cellular homeostasis. However, the diverse components of the plasma membrane and their various roles in the extracellular and intracellular activities make them very challenging and complex to characterize.
The structural and functional complexity of the plasma membrane has required the construction of simpler membrane models for the investigation of membrane dynamics. These models can also characterize the individual effects and properties of membrane components during cellular interactions and transport processes. Model membranes can be manipulated with external stimuli. Among them, light irradiation offers several advantages as a non-invasive, reversible, biocompatible and facile tool to provide high spatiotemporal control in biomimicry of cellular events. One of the efficient approaches to utilize light for the manipulation of membrane models is to prepare biomimetic platforms with photoswitchable lipids which can reversibly change their molecular conformation upon light irradiation. In this doctoral thesis, we have developed a light-triggered, multifunctional, and smart biomimetic platform by using phosphatidylcholine (referred as POPC here and in the main text), one of the most abundant phospholipids in animal cells, combined with a photoswitchable azobenzene lipid analog (referred as azo-PC). This platform was designed to provide an optical control of the membrane properties, shape, and molecular transport in the biomimetic system through the photoisomerization of azo-PC under UV and blue light.
First, we comprehensively investigated the reversibility, kinetics and effects of photoswitching on the material properties of varying compositions of azo-PC containing minimalistic membrane models including giant unilamellar vesicles (GUVs) as minimal cell-size models, Langmuir monolayers, large unilamellar vesicles and supported lipid bilayers, and combined the results from a variety of experimental approaches to those obtained from molecular dynamics simulations. These investigations showed that azo-PC photoisomerization induces dynamic alterations in membrane properties, affecting bilayer packing, elasticity, and interleaflet interactions. Using a method based on vesicle electrodeformation and optical microscopy, we revealed how the photolipid, introduced at various fractions, alters the membrane area upon isomerization and found excellent agreement with simulations. UV illumination of azo-PC GUVs triggered trans-to-cis photoisomerization, resulting in a significant increase in membrane area and a ten-fold decrease in bending rigidity. Trans azo-PC bilayers were found to be thicker than POPC bilayers but exhibited higher specific membrane capacitance and dielectric constant. This suggests an enhanced ability to store electrical charges across the membrane. Furthermore, incubation of preformed POPC GUVs with azo-PC rendered them photoresponsive, suggesting therapeutic potential for optical control of cellular activities. By using a wide range of experimental and computational approaches, we collected accurate results about the characterization of the light-induced modifications in azo-PC containing membranes, which also allowed us to discuss the discrepancies in the previously reported values in the literature and explain the origins of these discrepancies.
Next, we demonstrated the application of photoswitching of azo-PC doped GUVs for the transport of protein-rich droplets by performing light-triggered endocytosis of biomolecular condensates. Protein-rich condensates are phase separated membraneless organelles acting as vessels for biochemical reactions in cells during important cellular processes including signal transduction and gene expression. In our studies, condensates were prepared from the plant protein glycinin, which is a prominent storage protein in soybeans. UV-light-induced trans-to-cis photoisomerization of azo-PC results in an instantaneous increase in vesicle area, which promotes the wetting of GUVs by condensates and their rapid endocytosis for an appropriate condensate-vesicle size ratio. The process is fully reversible by exposure to blue light, allowing precise spatiotemporal control of the condensate-membrane interaction. The affinity of the protein condensates to the membrane, and the kinetics, reversibility and degree (whether partial or complete) of the engulfment processes were quantified from confocal microscopy images. Theoretical estimations confirmed that the adhesion of protein condensates to azo-PC vesicles in cis conformation under UV irradiation is energetically favorable. Experimental results, in good agreement with theoretical estimations, demonstrated that light and azo-PC photoisomerization can be employed as a versatile system to modify membrane-condensate interactions in a fast and reversible manner. To the best of our knowledge, this is the first study in the literature to utilize photoisomerization to control the delivery process of a biomacromolecule across a minimalistic artificial cell, providing a promising approach for further exploration in the control of cellular transport of biomacromolecules.
Lastly, employing azo-PC photoisomerization we established optical control of the activity of mechanosensitive ion channels reconstituted in GUV membrane, which enabled the transport of small molecules across the membrane. These pore-forming transmembrane proteins can open and close in response to changes in membrane properties and tension. As a model protein, we used the bacterial mechanosensitive ion channel of large conductance (MscL) and reconstituted it into azo-PC containing GUVs. Labeling MscL with a dye allowed us to monitor the reconstitution process through confocal microscopy, determine critical parameters of the reconstitution process and develop a protocol for the reconstitution of MscL into azo-PC containing GUVs. In order to understand the initial conformation of the incorporated MscL in the GUV membrane, the vesicles were subjected to a permeability test by adding a water-soluble, membrane-impermeable sulforhodamine dye to the external medium of the GUVs. Most of the GUVs remained impermeable, indicating that the majority pf MscL in the membrane stayed in the closed state after reconstitution. This trend changed when UV/blue illumination was applied to the MscL-reconstituted azo-PC vesicles. UV/blue illumination altered the membrane properties of azo-PC doped GUVs and triggered the opening of the MscL channel, as monitored by the permeation of the sulforhodamine molecules across the GUV membrane. Our preliminary results showed that light can be used as an efficient tool to catalytically activate the MscL channel. Further studies should focus extensively on the optimization and characterization of MscL reconstitution and gating processes.
Overall, our findings in this doctoral thesis provide an essential background for understanding and optimizing light-triggered drug delivery platforms and photoregulation of shape-dependent cellular processes such as endocytosis, exocytosis and intercellular trafficking through azo-PC photoisomerization. Considering our results on the control of membrane shape, mechanics and trafficking by light irradiation, photoisomerization may be used as a promising biomedical alternative for cell repair processes. Photoswitchable biomimetic platforms can be further developed as light-triggered smart activators for high-precision regulation of cellular mechanisms.
