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Table of 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.
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.