A new tool for quantifying the nanoscale dynamics of liquids at the interface with fluid biological membranes

Lead Research Organisation: Durham University
Department Name: Physics

Abstract

Biological membranes (biomembranes) are highly sophisticated structures that play a major role in the cell function. Aside from acting as a physical barrier between the inside from the outside of the cell, biomembranes control countless biological processes ranging from molecular trafficking to cell signalling, bioenergetics and biochemical function, endocytosis, surface compartmentalisation, and membrane shape regulation. Biomembranes have hence evolved into dynamical entities that can actively re-adjust their local composition and physical properties so as to best support the cell's biological function. This regulation is known to involve specialised proteins and markers, but increasing evidence also points to well controlled local variations in the membrane's mechanical properties, in particular stiffness and fluidity.
The liquid (water and hydrated ions) adjacent to the membrane is also believed to play an important role in shaping the membrane, regulating biomolecules' dynamics and controlling interactions and diffusion along the membrane. This interfacial liquid can strongly interact with the biomembrane and locally alter its mechanical properties.
To date, most results addressing the properties of the interfacial liquid or of the membrane fluidity at the nanoscale are either indirect or derived from theoretical consideration. This is largely due to the lack of experimental techniques able to operate locally with sufficient spatial and temporal resolution.
Recent advances in the field of atomic force microscopy (AFM) have made it possible to probe the interfacial liquid, including at the surface of biomembranes. Significantly, results are acquired locally, in seconds, and can provide sub-nanometre precision maps of the equilibrium structure adopted by the liquid adjacent to the membrane.
At the present time, however, it is not possible to deduce any information about the interfacial liquid dynamics on that scale. It is, for example, not possible to determine the directional flow patterns adopted by the liquid at the interface. Part of the problem comes from the impossibility to quantify the nanoscale fluidity of the biomembranes, a factor that influence the dynamics of the contacting liquid. Furthermore, the mutual influence that the interfacial liquid and the biomembrane mechanical properties have on each other has never been explored at that scale

This proposal aims at developing a new tool, the vortex dissipation microscope (VDM) that can quantify both the equilibrium solvation structure and the lateral dynamics of water at biointerfaces, locally and with nanometre precision. The VDM builds on recent AFM developments and will be able to provide nanoscale charts of the flow patterns naturally adopted by the interfacial water near singularities such as edges of membrane domains or protein assemblies. Significantly, the technique will also be able to quantify the lateral fluidity within the membranes, over the same area and with the same resolution.
The VDM will subsequently be applied to the study of bovine eye lens membranes. It will be used to quantify the flow patterns adjacent to the protein channels aquaporin 0 and connexins that are responsible for the transport of water and small molecules through lens cell membranes. Aside from providing a natural test platform for the VDM's capabilities, the results will bring new insight into the problem of lens membrane aging. Aging is known to be coupled with changes of the membrane mechanical properties, but the underlying molecular mechanisms are still poorly understood. This problem has important medical and hence societal consequences.

Technical Summary

At biointerfaces, liquid molecules tend to be more ordered and exhibit slower dynamics than in bulk liquid, due to their interactions with the biological surface. The properties of this interfacial liquid strongly depend on the biomaterial's nanoscale topography and chemistry. Reciprocally, the interfacial liquid can influence the shape and behaviour of soft biomaterial. Results have evidenced this mutual effect on single molecules, but phenomena on the 1-100nm scale are largely unexplored due to a lack of suitable technique.
The goal of this proposal is to help close this gap by creating an experimental tool that can quantify both the equilibrium solvation structure and the lateral dynamics of water and ions at the surface of biomembranes, locally and with nanometre precision. This tool, called the vortex dissipation microscope (VDM), is based on recent findings in the field of atomic force microscopy (AFM): when operated dynamically (vibrating tip) and under appropriate conditions, it is possible to quantify the local affinity of the liquid for a sample with sub-nanometre precision. This affinity can in turn be related to the liquid slippage along the interface, effectively relating dynamical properties of the interfacial liquid with a quantity measurable by AFM at the nanoscale.
However, AFM cannot capture any directional information about interfacial liquid dynamics, nor about any associated fluidity of the sample: slippage is assumed identical in all directions. This problem is solved in VDM by adding a controlled lateral component to the tip vertical vibration. The lateral motion (typically >5kHz, <10nm) is produced by a specially built scanner. Appropriate software control allows simultaneous mapping of the topography, equilibrium hydration structure and directional liquid slippage on each point of the sample. The VDM will be calibrated on lipid bilayers and subsequently applied to the study of natural bovine eye lens membranes.

Planned Impact

The proposed research will impact different groups of people in the UK and internationally. The main potential beneficiaries are academic researchers, British industry, the scientists directly involved into the project, and, on a longer term, society as a whole.

(i) Academic researchers (immediate impact)
The proposal should rapidly impact across membrane biology, molecular biology and biophysics, physiology, medical sciences, and bio-nanotechnology. The main impact will come from the novel information available through VDM and will contribute towards a better understanding of any nanoscale biological process occurring at interfaces, and where interfacial water plays a role. VDM is likely to open new research avenues and will provide experimental basis for the development of mesoscopic scale (1nm-100nm) computer simulations.
VDM will also impact medical research, although likely on a longer timescale (>5 years). Most drugs target membrane proteins and VDM results could provide a more accurate and functional description of biointerfaces, and a tool for in-vitro nanoscale testing of potential therapeutics.
The VDM findings will impact research in bio-nanotechnology, in particular the design and development of bio-inspired structures such as lipid-based assemblies with a set of desired properties, either structural or interfacial.
The UK has a strong tradition of AFM innovations aimed at supporting biological research. This project contributes to these advances and could open new research directions in the field.
High-profile publications as well as multiple presentations at international conference in the UK and abroad have been planned in order to best disseminate the project's results. All results will be made freely available through the Durham University (DU) online repository. Additionally, a workshop has been planned during the second year of the project on the theme of water at biointerfaces. The workshop will gather leading specialists from the UK and will provide and excellent opportunity to discuss the project's findings as well as potential challenges.

(ii) Industry (3-10 years)
The widespread adoption of the VDM would strongly benefit from its commercialisation as a package that can be retrofitted on existing AFM. To this end, the PI has contacted a British company (Oxford Instruments) who have expressed their interest in the product and will contribute to the project in kind (support letter). The impact of this strategy would hence benefit both research and industry.
Pharmaceutical companies would also benefit from VDM results, as mentioned previously.

(iii) People directly involved in the proposal (immediate)
The project will be conducted by two postdoctoral research assistants (PDRAs) with the help of the PI. Given the large potential impact of VDM on the scientific community, this project should strongly benefit their careers. The PDRAs will be working on a highly interdisciplinary project and will be able to broaden their knowledge in each different field. They will also be presenting their results are international conferences, and will be eligible for a large selection of postgraduate courses available at DU. The training and interdisciplinary experience that the PDRAs will have during the project will equip them with a highly desirable set of skills in research and industry: one PDRA will combine a background in biological sciences with experience in the operation and development of AFM and VDM. The second PDRA will combine a background in engineering and computing with a good knowledge of some important biological systems and challenges.

(iv) Society as a whole (> 10 years)
In the long term, society will benefit from the proposed research and its consequences mainly through improved medical and pharmaceutical research. A better understanding of biointerfaces may also make its way to the education system, for example in biochemistry textbooks.
 
Description Our results have evidenced a previously unknown water landscape near the surface of aquaporin channels and that is believed to support the efficient flow properties of the channel. More work is however needed to assess the dynamical properties of this unique landscape and its ability to support the activity of eye lens membranes.
Development of a novel type of microscopy (ongoing, but proof of principle validated) that can map liquid dynamics at biointerfaces with nanometre precision. The microscope is now working and able to probe simple systems such as model lipid bilayers
Exploitation Route Investigation of other types of membranes using the same approach, and replication of the results under physiological conditions (transmembrane potential). Some of the main findings are being currently written up as publication
Sectors Aerospace, Defence and Marine,Agriculture, Food and Drink,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Dynamic Interactions at Cell Membrane Interfaces
Amount £15,000 (GBP)
Organisation Durham University 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 03/2019
 
Title Calibration method for arbitrarily shaped cantilevers 
Description Atomic Force Microscopy (AFM) is a key tool for nanoscale investigations in biological systems. AFM can quantitatively measure the interaction force between single molecules, but this requires accurate calibration of the flexural spring constant of a the microcantilever used by AFM. Existing methods rely on precise knowledge of cantilever geometry, make significant simplifications, or require potentially damaging contact with the sample. Here, we develop a simple equation to calculate the flexural spring constants of arbitrarily shaped cantilevers in fluid. Our approach only requires the measurement of two resonance frequencies of the cantilever in air and in a liquid, with no need for additional input or knowledge about the system. We validate the method with cantilevers of different shapes and compare its predictions with existing models. We also show how the method's accuracy can be considerably improved, especially in more viscous liquids, if the effective width of the cantilever is known. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact The method has generated a significant interest from the scientific community with more than 800 downloads of the published methodology over the last year. 
URL https://aip.scitation.org/doi/10.1063/1.5009071
 
Title vortex dissipation microscopy 
Description A novel type of microscopy based on the atomic force microscope to map the liquid behaviour in close vicinity with biological membranes 
Type Of Material Biological samples 
Provided To Others? No  
Impact This is still early days but the methodology, when fully developed, will be made available to all interested researchers 
 
Description ACSIN Sendai 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited presentation at a major conference in the field (in Japan). The talk covered both general and technical aspects about the field, aiming at bringing together research, students and companies across disciplines
Year(s) Of Engagement Activity 2018
URL https://dora.bk.tsukuba.ac.jp/event/acsin14/
 
Description Conference presentation (Tempe) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of ongoing research and developments at a large international conference. The presentation generated significant feedback and discussions that helped further develop the project. Additionally, it enabled networking activities that concluded with one the PDRA working on the project being offered a position after the project.
Year(s) Of Engagement Activity 2018
 
Description EuroForum 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation and subsequent discussion of scientific developments to industrialists and academics. Stimulated discussions and possible collaborations
Year(s) Of Engagement Activity 2016
 
Description Intergroup meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Presentation a the project (Mapping nanoscale flow charts at solid-liquid interfaces) in lay person's term to a broad audience of students in view of stimulating a discussion related to the broader impact of the project.
Year(s) Of Engagement Activity 2018
 
Description Launch of WPI NanoLife Science Institute - Kanazawa University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Presentation at the launch of a national centre in Japan (£10 millions/year for 7 years budget). The topic is 'Nano Life Science' and the event invovled presentations and discussions with students, scientists and third party organisations
Year(s) Of Engagement Activity 2017
 
Description Linz Winter School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presentation of the latest developments on the new tool (vortex dissipation microscope) to experts and industry. This triggered many questions and significant interest.
Year(s) Of Engagement Activity 2018
 
Description NanoTribology 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Specialist conference in the field of nanoscale tribology. The presentations stimulated discussions and strategic networks
Year(s) Of Engagement Activity 2017
 
Description Nanofluidics in biological systems 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 3-days workshop aimed at bringing together research and non experts around the emerging topic of nanofluidicis in biological systems. The main outputs are the set up of an active group, collaborations and new sense of research community
Year(s) Of Engagement Activity 2017
 
Description Nanofluidics-Physics for life presentation 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation discussing the principles of Vortex Dissipation microscopy for nanoscale mapping of interfacial liquid dynamics. This lead to useful feedback and advice from the expert audience.
Year(s) Of Engagement Activity 2017
 
Description Organisation of a workshop on nanoscale fluidics 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A 2-day workshop was organised on the theme of nanofluidics in science and industry. The workshop was open to the general public and made accessible (content) to a broad, non-specialist audience. Many students attended and created lasting links with the speakers from all across the UK
Year(s) Of Engagement Activity 2016
 
Description Poster NCAFM 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation and discussion of the project's goals and research with the public using a poster as support. Sparked questions, discussions and contacts
Year(s) Of Engagement Activity 2016
 
Description Seminar Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation of cutting edge results to a group of scientists at Cambridge university. This sparked discussions and debates
Year(s) Of Engagement Activity 2016
 
Description Summer course Harbin 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Summer course given at Harbin Institute of Technology to explain basics aspects of soft matter and its implications in everyday life
Year(s) Of Engagement Activity 2016
 
Description TRNMX Levi 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of the latest project developments and exciting new results to an interdisciplinary audience. This initiated lively discussions and requests for new collaborations
Year(s) Of Engagement Activity 2018