Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Boise State University

It is well-studied that the significant factor in cataract formation is the association of α-crystallin, a major eye lens protein, with the fiber cell plasma membrane of the eye lens. The fiber cell plasma membrane of the eye lens consists of four major phospholipids (PLs), i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). Despite several attempts to study the interaction of α-crystallin with PLs of the eye lens membrane, the role of individual PL for the binding with α-crystallin is still unclear. We recently developed the electron paramagnetic resonance (EPR) spin-labeling method to study the binding of α-crystallin to the PC membrane (Mainali et al., 2020a). Here, we use the recently developed EPR method to explicitly measure the binding affinity (K) of α-crystallin to the individual (PE*, PS, and SM) and two-component mixtures (SM/PE, SM/PS, and SM/PC in 70:30 and 50:50 mol%) of PL membranes as well as the physical properties (mobility parameter and maximum splitting) of these membranes upon binding with α-crystallin. One of the key findings of this study was that the K of α-crystallin binding to individual PL membranes followed the trends: K(PC) > K(SM) > K(PS) > K(PE*), indicating PE* inhibits binding the most whereas PC inhibits binding the least. Also, the K of α-crystallin binding to two-component mixtures of PL membranes followed the trends: K(SM/PE) > K(SM/PS) > K(SM/PC), indicating SM/PC inhibits binding the most whereas SM/PE inhibits binding the least. Except for the PE* membrane, for which there was no binding of α-crystallin, the mobility parameter for all other membranes decreased with an increase in α-crystallin concentration. It represents that the membranes become more immobilized near the headgroup regions of the PLs when more and more α-crystallin binds to them. The maximum splitting increased only for the SM and the SM/PE (70:30 mol%) membranes, with an increase in the binding of α-crystallin. It represents that the PL headgroup regions of these membranes become more ordered after binding of α-crystallin to these membranes. Our results showed that α-crystallin binds to PL membranes in a saturable manner. Also, our data suggest that the binding of α-crystallin to PL membranes likely occurs through hydrophobic interaction between α-crystallin and the hydrophobic fatty acid core of the membranes, and such interaction is modulated by the PL headgroup's size and charge, hydrogen bonding between headgroups, and PL curvature. Thus, this study provides an in-depth understanding of α-crystallin interaction with the PL membranes made of individual and two-component mixtures of the four major PLs of the eye lens membranes.

Interaction of alpha-crystallin with four major phospholipids of eye lens membranes.

The concentration of α-crystallin decreases in the eye lens cytoplasm, with a corresponding increase in membrane-bound α-crystallin during cataract formation. The eye lens's fiber cell plasma membrane consists of extremely high cholesterol (Chol) content, forming cholesterol bilayer domains (CBDs) within the membrane. The role of high Chol content in the lens membrane is unclear. Here, we applied the continuous-wave electron paramagnetic resonance spin-labeling method to probe the role of Chol and CBDs on α-crystallin binding to membranes made of four major phospholipids (PLs) of the eye lens, i.e., phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Small unilamellar vesicles (SUVs) of PC, SM*, and PS with 0, 23, 33, 50, and 60 mol% Chol and PE* with 0, 9, and 33 mol% Chol were prepared using the rapid solvent exchange method followed by probe-tip sonication. The 1 mol% CSL spin-labels used during SUVs preparation distribute uniformly within the Chol/PL membrane, enabling the investigation of Chol and CBDs' role on α-crystallin binding to the membrane. For PC, SM*, and PS membranes, the binding affinity (K) and the maximum percentage of membrane surface occupied (MMSO) by α-crystallin decreased with an increase in Chol concentration. The K and MMSO became zero at 50 mol% Chol for PC and 60 mol% Chol for SM* membranes, representing that complete inhibition of α-crystallin binding was possible before the formation of CBDs within the PC membrane but only after the formation of CBDs within the SM* membrane. The K and MMSO did not reach zero even at 60 mol% Chol in the PS membrane, representing CBDs at this Chol concentration were not sufficient for complete inhibition of α-crystallin binding to the PS membrane. Both the K and MMSO were zero at 0, 9, and 33 mol% Chol in the PE* membrane, representing no binding of α-crystallin to the PE* membrane with and without Chol. The mobility parameter profiles decreased with an increase in α-crystallin binding to the membranes; however, the decrease was more pronounced for the membrane with lower Chol concentration. These results imply that the membranes become more immobilized near the headgroup regions with an increase in α-crystallin binding; however, the Chol antagonizes the capacity of α-crystallin to decrease the mobility near the headgroup regions of the membranes. The maximum splitting profiles remained the same with an increase in α-crystallin concentration, but there was an increase in the maximum splitting with an increase in the Chol concentration in the membranes. It implies that membrane order near the headgroup regions does not change with an increase in α-crystallin concentration but increases with an increase in Chol concentration in the membrane. Based on our data, we hypothesize that the Chol and CBDs decrease hydrophobicity (increase polarity) near the membrane surface, inhibiting the hydrophobic binding of α-crystallin to the membranes. Thus, our data suggest that Chol and CBDs play a positive physiological role by preventing α-crystallin binding to lens membranes and possibly protecting against cataract formation and progression.

Cholesterol and cholesterol bilayer domains inhibit binding of alpha-crystallin to the membranes made of the major phospholipids of eye lens fiber cell plasma membranes.

Cholesterol (Chol) content in most cellular membranes does not exceed 50 mol%, only in the eye lens's fiber cell plasma membrane, its content surpasses 50 mol%. At this high concentration, Chol induces the formation of pure cholesterol bilayer domains (CBDs), which coexist with the surrounding phospholipid-cholesterol domain (PCD). Here, we applied atomic force microscopy to study the mechanical properties of Chol/phosphatidylcholine membranes where the Chol content was increased from 0 to 75 mol%, relevant to eye lens membranes. The surface roughness of the membrane decreases with an increase of Chol content until it reaches 60 mol%, and roughness increases with a further increment in Chol content. We propose that the increased roughness at higher Chol content results from the formation of CBDs. Force spectroscopy on the membrane with Chol content of 50 mol% or lesser exhibited single breakthrough events, whereas two distinct puncture events were observed for membranes with the Chol content greater than 50 mol%. We propose that the first puncture force corresponds to the membranes containing coexisting PCD and CBDs. In contrast, the second puncture force corresponds to the "CBD water pocket" formed due to coexisting CBDs and PCD. Membrane area compressibility modulus (K) increases with an increase in Chol content until it reaches 60 mol%, and with further increment in Chol content, CBDs are formed, and K starts to decrease. Our results report the increase in membrane roughness and decrease K at very high Chol content (>60 mol%) relevant to the eye lens membrane.

Mechanical properties of the high cholesterol-containing membrane: An AFM study.

This research aims to probe the interaction of α-crystallin with a model of human, porcine, and mouse lens-lipid membranes.

Alpha-Crystallin Association with the Model of Human and Animal Eye Lens-Lipid Membranes is Modulated by Surface Hydrophobicity of Membranes.

The lens of the eye loses elasticity with age, while α-crystallin association with the lens membrane increases with age. It is unclear whether there is any correlation between α-crystallin association with the lens membrane and loss in lens elasticity. This research investigated α-crystallin membrane association using atomic force microscopy (AFM) for the first time to study topographical images and mechanical properties (breakthrough force and membrane area compressibility modulus (K), as measures of elasticity) of the membrane. α-Crystallin extracted from the bovine lens cortex was incubated with a supported lipid membrane (SLM) prepared on a flat mica surface. The AFM images showed the time-dependent interaction of α-crystallin with the SLM. Force spectroscopy revealed the presence of breakthrough events in the force curves obtained in the membrane regions where no α-crystallin was associated, which suggests that the membrane's elasticity was maintained. The force curves in the α-crystallin submerged region and the close vicinity of the α-crystallin associated region in the membrane showed no breakthrough event within the defined peak force threshold, indicating loss of membrane elasticity. Our results showed that the association of α-crystallin with the membrane deteriorates membrane elasticity, providing new insights into understanding the molecular basis of lens hardening and presbyopia.

An AFM Approach Applied in a Study of α-Crystallin Membrane Association: New Insights into Lens Hardening and Presbyopia Development.

Experimental evidence shows that the eye lens loses its elasticity dramatically with age. It has also been reported that the cholesterol (Chol) content in the eye lens fiber cell plasma membrane increases significantly with age. High Chol content leads to the formation of cholesterol bilayer domains (CBDs) in the lens membrane. The role of high Chol associated with lens elasticity is unclear. The purpose of this research is to investigate the membrane elasticity of the model of porcine lens-lipid (MPLL) membrane with increasing Chol content to elucidate the role of high Chol in lens membrane elasticity. In this study, we used atomic force microscopy (AFM) to study the mechanical properties (breakthrough force and area compressibility modulus (K)) of the MPLL membrane with increasing Chol content where K is the measure of membrane elasticity. We varied Chol concentration in Chol/MPLL membrane from 0 to ∼71 mol%. Supported Chol/MPLL membranes were prepared by fusion of small unilamellar vesicles (SUVs) on top of a flat mica surface. SUVs of the Chol/MPLL lipid mixture were prepared with the rapid solvent exchange method followed by probe-tip sonication. For the Chol/MPLL mixing ratio of 0, AFM image showed the formation of two distinct phases of the membrane, i.e., liquid-disordered phase (l) and solid-ordered phase (s) membrane. However, with Chol/MPLL mixing ratio of 0.5 and above, only liquid-ordered phase (l) membrane was formed. Also, two distinct breakthrough forces corresponding to l and s were observed for Chol/MPLL mixing ratio of 0, whereas only one breakthrough force was observed for membranes with Chol/MPLL mixing ratio of 0.5 and above. No significant difference in the membrane surface roughness was measured with increasing Chol content for these membranes; however, breakthrough force and K for l membrane increased when Chol/MPLL mixing ratio was increased from 0.5 to 1. Interestingly above the Chol/MPLL mixing ratio of 1, both breakthrough force and K decreased, indicating the formation of CBDs. Furthermore, these results showed that membrane elasticity increases at high Chol content, suggesting that high Chol content in lens membrane might be responsible for maintaining lens membrane elasticity.

Nawal K. Khadka

Department of Physics

Boise State University

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

Nawal K. Khadka

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physics

Boise State University

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

Department of Physics