VOLUME 540. Reconstituting the Cytoskeleton
Available here.
"This book captures the excitement and progress on reconstituting cytoskeletal processes. It begins with new methods of assaying polymer dynamics, including powerful computational methods (Prahl et al.) and ways of modulating experimental conditions using microfluidics (Carlier et al.). In addition to the classic systems of actin and microtubules, the in vitro polymerization of actin-like proteins from bacteria can now be studied as well (Petek and Mullins). The nucleation and dynamics of cytoskeletal filaments are controlled by numerous regulatory proteins that interact with actin and microtubules, and these mechanisms can be dissected by in vitro experimentation. The nucleation step is controlled by large, multi-subunit protein complexes. In this volume, Chen et al. describe how to prepare the WAVE complex (a regulator of Arp2/3 and actin nucleation) using a very clever protein expression method that may prove generally useful for other multi-subunit protein machines as well. Our understanding of tubulin nucleation lags behind that of actin, but Choi et al. have made progress in isolating an active g-tubulin ring complex and assaying its nucleation of tubulin in vitro. Other proteins act as polymerases that add subunits to the filament end, and Mizuno and Watanabe (formin) and Al-Bassam (TOG domain proteins) describe elegant means of assaying these activities by microscopy. Other enzymes posttranslationally modify the filament after it has been assembled, and Vemu et al. describe how tubulin-modifying enzymes can be purified and their activities measured."
"As described earlier, molecular motor proteins have been a focus for single-molecule investigations. A recent trend has been to develop motility assays that reflect greater and more physiological complexity. For example, many cargos contain multiple, rather than single, motor proteins and even can bind different types of motors that pull the cargo in opposite directions (e.g., kinesin and dynein). What are the properties of such complex multimotor systems? To answer such questions, one approach is to use scaffolds where the numbers and types of bound motors can be precisely controlled. To achieve this, Rogers et al. as well as Goodman and Reck-Peterson attach motors to DNA through precisely controlled linkages. To mimic other scenarios in which motors are bound to the cell cortex and pull on microtubules (causing displacement of the centrosome), Roth et al. attached dynein motors to the walls of microfabricated chambers. It is also important to study how ensembles of motors work on natural cargos. Both Hendricks et al. and Barak et al. describe methods for isolating membranous organelles and assaying their movement along microtubules in vitro."
"Another challenge is to reconstitute complex cytoskeletal processes using networks of interacting purified proteins. Both Murrell et al. and Boujemaa-Paterski et al. describe amazing self-organization patterns, motility, and contractile properties of actin networks, assembled with different geometric constrains and protein mixtures. Substructures and reactions in the mitotic spindle are also proving amenable to reconstitution. Driver et al. describe methods for isolating kinetochores and studying their interactions with microtubules, and Fourniol et al. have reconstituted the basic ingredients of the microtubule overlap zone that forms during anaphase. And why not start to bridge the worlds of actin and microtubules? By adding proteins that interact with both actin and microtubules, Preciado Lo´pez et al. have developed assays where the interactions and self-organization of actin and microtubules can be studied in vitro."
"For some activities, it is not yet possible (and perhaps not even desirable, since regulatory mechanism might be lost) to achieve a complete reconstitution with purified proteins. In such a case, reconstitution can be performed in a crude extract, which still enables types of experiments that cannot be easily achieved with intact cells (e.g., immunoprecipitations, certain drug treatments, kinetic experiments, etc). The Xenopus egg extract, because of its very high protein concentration, has been a remarkable source for reconstitution experiments. In this volume, Groen et al. and Field et al. describe very useful tricks for preparing different types of extracts and ways in which these extracts can be used to study the self-organization of microtubules or actin."
"From examining these chapters, it is apparent that new trends are emerging in reconstitution work. First, the reconstitution of cytoskeletal functions is attracting and benefitting from the interdisciplinary research of biologists, chemists, physicists, and engineers. For example, the microfabrication of chambers, lithography, and microfluidics are involved in many of the methods described in this volume. Furthermore, the self-organization and mechanical properties of the cytoskeleton in vitro are attractive experimental and theoretical problems for physicists (many of the authors in this series were originally trained as physicists). A second trend is the use of light microscopy for the vast majority of the assays in this book. Microscopy based assays provide much more detailed information and require less material than bulk population measurements using fluorimeters or spectrophotometers. For such microscopy assays, one has to prepare specific types of chambers, clean and passivate the glass, and attach specific proteins of interest to the surface. Most of the chapters describe such methods, which are often derived from painstaking development work. There is a wealth of information in browsing and comparing the methods for surface preparation in these different chapters."
