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Over the past two decades experimental studies have solidified the int- pretation of the cytoskeleton as a highly dynamic network of microtubules, actin microfilaments, intermediate filaments, and myosin filaments. Rather than a network of disparate fibers, these polymers are often interconnected and display synergy, which is the combined action of two or more cytoskeletal polymers to achieve a specific cellular structure or function. Cross-commu- cation among cytoskeletal polymers is thought to be achieved through cytoskeletal polymer accessory proteins and molecular motors that bind two or more cytoskeletal polymers. Development of the modern concept of the cytoskeleton is a direct o- growth of advances in experimental tools and reagents that are available to cell and molecular biologists. Technological advances and refinements in cell imaging have made it possible to selectively image a single cytoskeletal po- mer and monitor its dynamics through the use of fluorescence probes in vitro and in vivo. Two decades ago, cytoskeletal research was limited to a few perturbation reagents that included colchicine and cytochalasin. Today, the perturbation arsenal has expanded to a highly selective group of reagents that includes Taxol, nocodazole, benomyl, latrunculin, jasplakinolide, and such endogenous proteins as gelsolin. These reagents enable the investigator to selectively perturb or destroy a cytoskeletal polymer while leaving other cytoskeletal polymers intact. Site-specific monoclonal antibodies that target a specific cytoskeletal polymer have proven to be highly selective affinity tools for cytoskeletal research.
This volume details comprehensive state-of-the-art methods on actin microfilaments and microtubules and how they work to achieve different cellular functions in different cellular contexts. Chapters guide readers through protein purification, in vitro reconstitution of several cytoskeleton properties, analyses of microtubule- and actin-based structures, functional dissection of post-translational modifications, and roles in several biological processes. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Cytoskeleton Dynamics: Methods and Protocols aims to provide a wide range of experimental approaches and be an invaluable resource for present and future generations of cytoskeleton researchers. The chapter “Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes” is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
This volume aims to present a large panel of techniques for the study of Plant Cell Division. Plant Cell Division: Methods and Protocols captures basic experimental protocols that are commonly used to study plant cell division processes, as well as more innovative procedures. Chapters are split into five parts covering several different aspect of plant cell division such as, cell cultures for cell division studies, cell cycle progression and mitosis, imaging plant cell division, cell division and morphogenesis, and cytokinesis. Written for the Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Plant Cell Division: Methods and Protocols is a valuable tool for the study of plant cell division at both the cellular and molecular levels, and in the context of plant development.
The third edition of this volume focuses on experimental models that are useful for investigating various aspects of cytoskeleton structure and function. Animal, plant, protist, and fungal models highlight twenty-four chapters that provide detailed protocols for live and fixed-cell imaging, dynamics of cytoskeleton components, cell and organelle motility, and genetics and proteomics. Written in the highly successful Methods in Molecular Biology series format, protocols in each chapter are up-to-date menus organized in a useful step-by-step format appropriate for novice and established investigators. Each chapter is equipped with a valuable notes section that provides a troubleshooting guide and helpful, and often unpublished, technical information aimed at ensuring success with implementation of the protocols. Authoritative and thorough, Cytoskeleton Methods and Protocols, Third Edition helps researchers expand their understanding of cytoskeleton structure and function.
The third edition of this volume focuses on experimental models that are useful for investigating various aspects of cytoskeleton structure and function. Animal, plant, protist, and fungal models highlight twenty-four chapters that provide detailed protocols for live and fixed-cell imaging, dynamics of cytoskeleton components, cell and organelle motility, and genetics and proteomics. Written in the highly successful Methods in Molecular Biology series format, protocols in each chapter are up-to-date menus organized in a useful step-by-step format appropriate for novice and established investigators. Each chapter is equipped with a valuable notes section that provides a troubleshooting guide and helpful, and often unpublished, technical information aimed at ensuring success with implementation of the protocols. Authoritative and thorough, Cytoskeleton Methods and Protocols, Third Edition helps researchers expand their understanding of cytoskeleton structure and function.
In Confocal Microscopy Methods and Protocols, Stephen Paddock and a highly skilled panel of experts lead the researcher using confocal techniques from the bench top, through the imaging process, to the journal page. They concisely describe all the key stages of confocal imaging-from tissue sampling methods, through the staining process, to the manipulation, presentation, and publication of the realized image. Written in a user-friendly, nontechnical style, the methods specifically cover most of the commonly used model organisms: worms, sea urchins, flies, plants, yeast, frogs, and zebrafish. Centered in the many biological applications of the confocal microscope, the book makes possible the successful imaging of both fixed and living specimens using primarily the laser scanning confocal microscope. The powerful hands-on methods collected in Confocal Microscopy Methods and Protocols will help even the novice to produce first-class cover-quality confocal images.
This volume covers broad aspects of cell expansion in three different cell types: root hairs, pollen tubes, and hypocothyl cells. Chapters focus on the cutting-edge methods to study in detail several complex aspects of cell expansion such as secretion, endocytosis and recycling, cellular signaling and trafficking, and protein and polysaccharides cell wall biosynthesis in real time during cell expansion. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Plant Cell Expansion: Methods and Protocols is an essential reference book for plant scientist, molecular, and cell biologist as well as plant biochemists​.
12 The average human body has in the order of 10 circulating platelets. They are crucial for hemostasis, and yet excessive platelet activation is a major cause of m- bidity and mortality in western societies. It is therefore not surprising that platelets have become one of the most extensively investigated biological cell types. We are, however, far from understanding precisely how platelets become activated under physiological and pathophysiological conditions. In addition, there are large gaps in our knowledge of platelet production from their giant precursor cell, the megakar- cyte. Understanding megakaryocyte biology will be crucial for the development of platelet gene targeting. The aim of Platelets and Megakaryocytes is therefore to bring together established and recently developed techniques to provide a comprehensive guide to the study of both the platelet and the megakaryocyte. It consists of five s- tions split between two volumes. The more functional assays appear in Volume 1, whereas Volume 2 includes signaling techniques, postgenomic methods, and a n- ber of key perspectives chapters. Part I of Volume 1, Platelets and Megakaryocytes: Functional Assays, describes many well established approaches to the study of platelet function, including aggregometry, secretion, arachidonic acid metabolism, procoagulant responses, pla- let adhesion under static or flow conditions, flow cytometry, and production of microparticles. Although one would ideally wish to perform experiments with human platelets, studies within the circulation using intravital microscopy require the use of animal models, which are described in Chapter 16, vol. 1.