# Mechanisms of Regulation of Clathrin-mediated Endocytosis

> **NIH NIH R01** · UT SOUTHWESTERN MEDICAL CENTER · 2022 · $328,000

## Abstract

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for
uptake of nutrients, hormones and antibodies, for remodeling of plasma membrane (PM) composition in
response to environmental changes, and for the regulation of signaling from cell surface receptor tyrosine
kinases and G-protein coupled receptors. Thus, CME plays a critical role in many aspects of cellular
differentiation, physiology and homeostasis. Consequently, defects in CME impinge on many human diseases,
including cancer, cardiovascular disease, diabetes, neurological defects and others. CME is a multistep process
initiated by the assembly of clathrin and the AP2 adaptor complex to form nascent clathrin-coated pits (CCPs).
Subsequent steps including CCP stabilization, invagination, maturation and their scission from the PM to release
clathrin-coated vesicles (CCVs) are orchestrated by myriad endocytic accessory proteins (EAPs). While EAPs
have been functionally classified based on domain structure, and the timing of their recruitment to CCPs, in most
cases their precise functions remain poorly understood, and in many cases still controversial. Under the auspices
of this grant, we have developed accurate and highly sensitive particle tracking software to quantitatively
measure the dynamics of clathrin-eGFP labeled CCPs imaged by live cell total internal reflection fluorescence
microscopy. We have also developed methods of data analyses that allow us to independently measure multiple
discrete stages CCV formation. Through this multi-PI mechanism, we have assembled interdisciplinary expertise
in biochemistry, cell and molecular biology, quantitative live cell microscopy, biophysicists, statistics and
computational biology. Hence, we are uniquely positioned, as proposed in Aim 1, to conduct the first
comprehensive phenotypic characterization of EAPs following CRISPRi knockdown. Completion of these studies
will define the stage-specific roles of EAPs in CME. A subset of EAPs chosen based on the severity and novelty
of their phenotypes and whether they function at critical nodes for regulation of CME will be analyzed in more
detail in Aim 2 to mechanistically dissect their roles in CME. While the functions of few EAPs have been
unambiguously defined, nonetheless much is known about the molecular machinery driving CME. Thus, this
complex process is ripe for the application of modeling approaches that describe mechanochemical events
driving CCV formation based on first principles from physics. Such a model will then serve in the coming years
as an integrator of data on the contributions of individual EAPs at discrete stages of CME. Thus, in Aim 3, we
will develop a biophysical model of CCP assembly, invagination and maturation, and complement it with a
computational framework to directly calibrate unknown model parameters based on the phenotypic information
from the data acquired in Aims 1 & 2. Completion of these aims will provide an unprec...

## Key facts

- **NIH application ID:** 10469508
- **Project number:** 5R01GM073165-17
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Marcel Bernard Mettlen
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $328,000
- **Award type:** 5
- **Project period:** 2006-03-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10469508

## Citation

> US National Institutes of Health, RePORTER application 10469508, Mechanisms of Regulation of Clathrin-mediated Endocytosis (5R01GM073165-17). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10469508. Licensed CC0.

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