Nikolay Buzhynskyy

The supramolecular architecture of junctional microdomains in native lens membranes

Gap junctions formed by connexons and thin junctions formed by lens‐specific aquaporin 0 (AQP0) mediate the tight packing of fibre cells necessary for lens transparency. Gap junctions conduct water, ions and metabolites between cells, whereas junctional AQP0 seems to be involved in cell adhesion. High‐resolution atomic force microscopy (AFM) showed the supramolecular organization of these proteins in native lens core membranes, in which AQP0 forms two‐dimensional arrays that are surrounded by densely packed gap junction channels. These junctional microdomains simultaneously provide adhesion and communication between fibre cells. The AFM topographs also showed that the extracellular loops of AQP0 in junctional microdomains adopt a conformation that closely resembles the structure of junctional AQP0, in which the water pore is thought to be closed. Finally, time‐lapse AFM imaging provided insights into AQP0 array formation. This first high‐resolution view of a multicomponent eukaryotic membrane shows how membrane proteins self‐assemble into functional microdomains.

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes.

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500–2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G‐protein‐coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26 000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein‐free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception. Copyright

Eye lens membrane junctional microdomains: a comparison between healthy and pathological cases

The eye lens is a transparent tissue constituted of tightly packed fiber cells. To maintain homeostasis and transparency of the lens, the circulation of water, ions and metabolites is required. Junctional microdomains connect the lens cells and ensure both tight cell-to-cell adhesion and intercellular flow of fluids through a microcirculation system. Here, we overview membrane morphology and tissue functional requirements of the mammalian lens. Atomic force microscopy (AFM) has opened up the possibility of visualizing the junctional microdomains at unprecedented submolecular resolution, revealing the supramolecular assembly of lens-specific aquaporin-0 (AQP0) and connexins (Cx). We compare the membrane protein assembly in healthy lenses with senile and diabetes-II cataract cases and novel data of the lens membranes from a congenital cataract. In the healthy case, AQP0s form characteristic square arrays confined by connexons. In the cases of senile and diabetes-II cataract patients, connexons were degraded, leading to malformation of AQP0 arrays and breakdown of the microcirculation system. In the congenital cataract, connexons are present, indicating probable non-membranous grounds for lens opacification. Further, we discuss the energetic aspects of the membrane organization in junctional microdomains. The AFM hence becomes a biomedical nano-imaging tool for the analysis of single-membrane protein supramolecular association in healthy and pathological membranes.

Nanomechanical Characterization of the Stiffness of Eye Lens Cells: A Pilot Study

PURPOSE The purpose of this study is to probe the mechanical properties of individual eye lens cells isolated from nucleus and cortex of adult sheep eye lens, and to characterize the effect of cytoskeletal drugs. METHODS We used atomic force microscopy (AFM), featuring a spherical tip at the end of a soft cantilever, to indent single lens cells, and measure the Youngs modulus of isolated nuclear and cortical lens cells. Measurements were performed under basal conditions, and after addition of drugs that disrupt actin filaments and microtubules. RESULTS We found that single lens cells were able to maintain their shape and mechanical properties after being isolated from the lens tissue. The median Youngs modulus value for nuclear lens cells (4.83 kPa) was ~ 20-fold higher than for cortical lens cells (0.22 kPa). Surprisingly, disruption of actin filaments and microtubules did not affect the measured Youngs moduli. CONCLUSIONS We found that single cells from the lens nucleus and cortex can be distinguished unambiguously using the elastic modulus as a criterion. The uncommon maintenance of shape and elastic properties after cell isolation together with the null effect of actin filaments and microtubules targeting drugs suggest that the mechanical stability of fiber cells is provided by cellular elements other than the usual cytoskeletal proteins.