Clathrin-mediated endocytosis (CME) is a ubiquitous internalization process in eukaryotic cells. It consists of the formation of an approximately 50-nm diameter vesicle out of a flat membrane. Genetics, biochemistry, and microscopy experiments performed in the last four decades have been instrumental to discover and characterize major endocytic proteins in yeast and mammals. However, due to the highly dynamic nature of the endocytic assembly and its small size, many questions remain unresolved: how are endocytic proteins organized spatially and dynamically? How are forces produced and how are their directions controlled? How do the biochemical activities of endocytic proteins and the membrane shape and mechanics regulate each other? These questions are virtually impossible to visualize or measure directly with conventional approaches but thanks to new quantitative biology methods, it is now possible to infer the mechanisms of endocytosis in exquisite detail. This book introduces quantitative microscopy and mathematical modeling approaches that have been used to count the copy number of endocytic proteins, infer their localization with nanometer precision, and infer molecular and physical mechanisms that are involved in the robust formation of endocytic vesicles.

The cell interior is another world that we are only beginning to explore. Although there are a number of approaches for examining the inner workings of the cell, the reductionist approach of building up complexity appeals to many with physical science and engineering backgrounds. This volume of Methods in Cell Biology spans a range of spatial scales from single protein molecules to vesicle and cell sized structures capable of complex behaviors. Contributions include; methods for combining different motors and cytoskeletal components in defined ways to produce more complex behaviors; methods to combine cytoskeletal assemblies with fabricated devices such as chambers or pillar arrays; reconstituting membrane fission and fusion; reconstituting important biological processes that normally take place on membrane surfaces; and methods for encapsulating protein machines within vesicles or droplets.

The goal of this book is to collect methods and protocols for studying cilia in a wide range of different cell types, so that researchers from many fields of biology can start exploring the role of cilia in their own system.

For the past decade or more, much of cell biology research has been focused on determining the key molecules involved in different cellular processes, an analytical problem that has been amenable to biochemical and genetic approaches. Now, we face an integrative problem of understanding how all of these molecules work together to produce living cells, a challenge that requires using quantitative approaches to model the complex interactions within a cell, and testing those models with careful quantitative measurements. This book is an introductory overview of the various approaches, methods, techniques, and models employed in quantitative cell biology, which are reviewed in greater detail in the other volumes in this e-book series. Particular emphasis is placed on the goals and purpose of quantitative analysis and modeling, and the special challenges that cell biology holds for understanding life at the physical level.

This new volume of "Methods in Enzymology" continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers cilia and includes chapters on such topics as methods for studying ciliary polarity in Xenopus, analysis of signaling pathways in mammalian spermatozoa, and biochemical and physiological analysis of axonemal dyneins.
Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers cilia Contains chapters on such topics as methods for studying ciliary polarity in Xenopus,
analysis of signaling pathways in mammalian spermatozoa, and biochemical and physiological analysis of axonemal dyneins

This new volume of "Methods in Enzymology" continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers cilia and includes chapters on such topics as electron microscopy of IFT in cilia and flagella, radial spoke isolation and assays, and biomechanical measurements of kinocilium.

Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers ciliaContains chapters on such topics as electron microscopy of IFT in cilia and flagella, radial spoke isolation and assays, and biomechanical measurements of kinocilium

For the past decade or more, much of cell biology research has been focused on determining the key molecules involved in different cellular processes, an analytical problem that has been amenable to biochemical and genetic approaches. Now, we face an integrative problem of understanding how all of these molecules work together to produce living cells, a challenge that requires using quantitative approaches to model the complex interactions within a cell, and testing those models with careful quantitative measurements. This book is an introductory overview of the various approaches, methods, techniques, and models employed in quantitative cell biology, which are reviewed in greater detail in the other volumes in this e-book series. Particular emphasis is placed on the goals and purpose of quantitative analysis and modeling, and the special challenges that cell biology holds for understanding life at the physical level.

Clathrin-mediated endocytosis (CME) is a ubiquitous internalization process in eukaryotic cells. It consists of the formation of an approximately 50-nm diameter vesicle out of a flat membrane. Genetics, biochemistry, and microscopy experiments performed in the last four decades have been instrumental to discover and characterize major endocytic proteins in yeast and mammals. However, due to the highly dynamic nature of the endocytic assembly and its small size, many questions remain unresolved: how are endocytic proteins organized spatially and dynamically? How are forces produced and how are their directions controlled? How do the biochemical activities of endocytic proteins and the membrane shape and mechanics regulate each other? These questions are virtually impossible to visualize or measure directly with conventional approaches but thanks to new quantitative biology methods, it is now possible to infer the mechanisms of endocytosis in exquisite detail. This book introduces quantitative microscopy and mathematical modeling approaches that have been used to count the copy number of endocytic proteins, infer their localization with nanometer precision, and infer molecular and physical mechanisms that are involved in the robust formation of endocytic vesicles.