Tomography of Actin Cytoskeletal Networks

Sanford-Burnham Medical Research Institute, La Jolla, California, USA.
Methods in enzymology (Impact Factor: 2.09). 01/2010; 483:203-14. DOI: 10.1016/S0076-6879(10)83010-1
Source: PubMed


Structural biology research is increasingly focusing on unraveling structural variations at the micro-, meso-, and macroscale aiming at interpreting dynamic biological processes and pathways. Toward this goal, high-resolution transmission cryoelectron microscopy (cryo-EM) and cryoelectron tomography (cryo-ET) are indispensable, as these provide the ability to determine 3D structures of large, dynamic macromolecular assemblies in their native, fully hydrated state in situ. Underlying such analyses is the implicit assumption that specific structural states yield specific cellular outputs. The dependence on this structure-function paradigm is not unique to studies pertaining a particular pathway or biological process but it sets the foundation for all cell biological analyses of macromolecular assemblies. Yet, the paradigm still awaits formal proof. The field of high-resolution electron microscopy (HREM) is in dire need of establishing approaches and technologies to systematic and quantitative determining structure-function correlates in physiologically relevant environment. Here, using the actin cytoskeletal networks as an example, we will provide snapshots of current advances in defining the structures of these highly dynamic networks in situ. We will further detail some of the major stumbling blocks on the way to quantitatively correlate the dynamic state to network morphology in the same window of time and space.

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    • ": cryoET for the study of actin–ABP complexes CryoET has already been used extensively for the study of actin filaments and networks, but no studies of actin–ABP interactions were found. The Hanein lab has performed numerous studies of actin using cryoET and written several good reviews on the subject [67] [68] "
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    ABSTRACT: Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescence speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryo-electron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs.
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    ABSTRACT: Adhesions between the cell and the extracellular matrix (ECM) are mechanosensitive multi-protein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to the chemical and mechanical environment. These combined functions in force transduction, signaling and mechanosensing contribute to cellular phenotypes that span development, homeostasis and disease. These adhesions form, mature and disassemble in response to actin organization and physical forces that originate from endogenous myosin activity or external forces by the extracellular matrix. Despite advances in our understanding of the protein composition, interactions and regulation, our understanding of matrix adhesion structure and organization, how forces affect this organization, and how these changes dictate specific signaling events is limited. Insights across multiple structural levels are acutely needed to elucidate adhesion structure and ultimately the molecular basis of signaling and mechanotransduction. Here we describe the challenges and recent advances and prospects for unraveling the structure of cell-matrix adhesions and their response to force.
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