Quantitative biology of endocytosis / Julien Berro, Michael M. Lacy.

Format:

Book

Author/Creator:

Berro, Julien, author.

Lacy, Michael M., author.

Series:

Colloquium digital library of life sciences

Colloquium series on quantitive cell biology ; 2375-7752 # 4.

Colloquium series on quantitive cell biology, 2375-7752 ; # 4

Language:

English

Subjects (All):

Endocytosis.

Medical Subjects:

Endocytosis.

Physical Description:

1 online resource (xi, 73 pages) : illustrations.

Place of Publication:

[San Rafael, California] : Morgan & Claypool, 2018.

System Details:

Mode of access: World Wide Web.

System requirements: Adobe Acrobat reader.

text file

Summary:

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.

Contents:

1. Introduction to clathrin-mediated endocytosis

1.1 Proteins involved in CME

Membrane coat proteins

Endocytic actin meshwork

Membrane scission and late regulation

1.2 Membrane in CME

1.3 Forces in CME

1.4 Comparison of CME in yeast and mammals

1.5 CME in human health and disease

1.6 Understanding CME requires quantitative approaches

1.7 Conclusions

2. Collecting quantitative data

2.1 Using fluorescence microscopy to obtain quantitative data about endocytosis

Overview of fluorescence microscopes that can be used for quantitative analysis of endocytosis

Comparison of different fluorescence microscopy systems

General considerations about fluorescent tags

2.2 Practical considerations for counting molecules using a fluorescence microscope

Tag a protein of interest at its genomic locus

Limit photobleaching

Limit uneven illumination of the field of view

Ensure the microscopy system is used in conditions where the signal is linear

Calibrate the microscope using flexible settings to avoid calibrating too often

Best practices to limit sources of variability

2.3 Electron microscopy and correlative light and electron microscopy (CLEM)

2.4 Super-resolution microscopy

3. From raw images to quantitative measurements: extracting, correcting, and aligning the fluorescence microscopy data

3.1 Tracking endocytic events

Challenges in tracking endocytic events

Tracking and track curation

Best practice in tracking and curating tracks

3.2 Processing the data to count molecules

Pre-processing of the movies

Correcting for cytoplasmic background

3.3 Aligning the tracking data

Simple but biased alignment methods

Advanced and more accurate alignment methods

3.4 Counting the number of endocytic events

4. Using quantitative microscopy data to infer the molecular mechanisms of endocytosis

4.1 How are endocytic proteins organized?

The protein copy numbers and their stoichiometries constrain possible molecular organizations and mechanisms

High-resolution track alignment can infer the organization of endocytic proteins

The organization of the actin meshwork around the endocytic vesicle can be inferred from the vesicle's movements

4.2 What are the molecular mechanisms of actin dynamics during endocytosis?

Inferring mechanisms by comparing numbers and stoichiometries

Mathematical modeling constrained by quantitative data allows one to test different mechanisms and make testable predictions

4.3 What are the mechanical properties of the membrane and the forces involved in membrane deformations?

Modeling of membrane shapes can elucidate mechanical properties and forces involved during CME

Simulations to test possible force distributions to elongate the CCP and propose new mechanisms

5. Perspectives and future of quantitative biology of endocytosis

5.1 Open questions in CME

5.2 Quantities that are not (yet?) measurable

5.3 Advances in microscopy techniques

5.4 Advances in computational tools

5.5 Applications to other systems

References

Author biographies.

Notes:

Part of: Colloquium digital library of life sciences.

Includes bibliographical references (pages 61-72).

Title from PDF title page (viewed on August 3, 2018).

Other Format:

Print version:

ISBN:

9781615047857

OCLC:

1047605916

Access Restriction:

Restricted for use by site license.