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Devising an In Vitro Cell Surface Model

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    Scientists have long been fascinated by the dichotomy that constitutes the cell surface: On one hand it is dynamically fluid, because it is dotted with diverse amounts of proteins, lipids, and carbohydrates. Yet the cell surface also acts as a stalwart rampart against the extracellular environment—setting up checkpoints that selectively allow entrance to molecules the cell needs. A greater understanding of cell surface mechanics is critical for researchers to gain insight into daily cellular processes and how it can be exploited for disease intervention. However, the study of surface dynamics has been problematic for researchers over the years.

    Now, researchers at the National Centre for Biological Sciences (NCBS) in Bangalore, India, and University of California San Francisco (UCSF) have managed to construct a simplified cell surface from its constituent parts, namely, a mixture of fats and proteins. This reconstruction creates a crucial new tool that researchers can use to test theories on cell surface dynamics.

    Movement of molecules at the cell surface is a nonrandom, incredibly complex event that does not seem to follow simple thermodynamic rules, and until recently there were few experimental tools available to study such phenomena to understand better how the cell surface functioned. However, this newly devised minimal model of the cell surface constructed from its primary components—purified fats and proteins—could be the key to understanding how the surface of a living cell works.

    “This is just a beginning but an important one,” explained senior study author Satyajit Mayor, Ph.D., director of the NCBS and the Institute for Stem Cell Biology and Regenerative Medicine (inStem) in Bangalore. “Important because it allows one to test ideas that have come from theory built around providing an explanation for the active organization at the surface of a living cell. It’s an exciting beginning since the feasibility of this simple, minimal system opens up huge possibilities to explore the world of a living cell in a test tube system where every element is under our control.”

    Dr. Mayor added, “This work is inspired by the adage ‘what we understand we should be able to build’ and this is in trying to understand the principles behind how a living material, the cell surface, works.”

    The “active composite model” of the cell surface is one of the latest theories that attempts to explain the behavior of cell surface molecules. This model visualizes the cell surface as not just the cell membrane, but as an amalgamation of two elements—the cell membrane, made of fats and an interwoven mesh of the protein actin that forms a thin layer just below the cell membrane.

    To recapitulate the active composite model, the investigators decided to recreate a cell surface as an assembly of a fat-based membrane and an actin meshwork. This artificial cell surface was therefore constructed using a fat bilayer, actin, and a fluorescent protein specially designed to be embedded in the membrane while also being linked to actin. Using various microscopy techniques, the group was able to study the behavior of the construct via the patterns formed by the fluorescent proteins.

    As predicted by the active composite model, the dynamics of actin-bound fluorescent proteins were found to be dependent on the dynamics of the actin meshwork. When the molecular motor, myosin, was added and chemical energy provided, the forces generated by actin–myosin interactions drove the movements of these proteins. When the chemical energy was exhausted, the actin-bound proteins aggregated to form distinct bundles based on the organization of the actin meshwork.

    The findings from this study were published recently in PNAS in an article entitled “Actomyosin Dynamics Drive Local Membrane Component Organization in an In Vitro Active Composite Layer.”

    “The importance of active or energy consuming processes in understanding biological phenomena is becoming more and more evident,” noted lead study author Darius Köster, Ph.D., postdoctoral researcher in Dr. Mayor’s laboratory. “This is an emerging field in biology called ‘active mechanics.’ Often, the emerging organization of biological molecules is not clear, and theoretical explanations for such observations are also far from complete. This makes it important to have proper experimental tools that go hand in hand with the theory to test and improve our understanding of such systems. Our current study describes the creation of an experimental system that will serve us in this.”

    “The motivation behind this work is to analyze mechanisms influencing the dynamics and organization of molecules on the cell surface,” added co-author Kabir Husain, a doctoral candidate studying in Dr. Mayor’s laboratory. Now, with the recreation of the cell surface in a test tube, scientists have gained a solid experimental footing in the race to comprehend the mechanics of cell surface organization.

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