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NIH Director's Seminar: Imaging the nanoscale structure of endocytosis with correlative super-resolution light and electron microscopy

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Air date: Friday, March 8, 2019, 12:00:00 PM
Time displayed is Eastern Time, Washington DC Local
Views: Total views: 115, (79 Live, 36 On-demand)
Category: NIH Director's Seminars
Runtime: 00:58:20
Description: NIH Director's Seminar Series

The plasma membrane separates the cell’s interior from the outside world. The exchange of signals and material across this barrier is regulated by a multitude of channels, transporters, receptors, and trafficking organelles. Mapping the molecular structure and dynamics of the plasma membrane is key to understanding how human cells function in health and malfunction in disease. Electron microscopy can produce high resolution images of the membrane. Historically, it has been challenging to locate and identify proteins within these images. Recently-developed super-resolution localization microscopy, however, can image fluorescently-labeled molecules with better than 20 nm precision. We developed a correlative super-resolution light and EM method (CLEM) that couples these complementary methods to produce images where identified proteins are mapped within the dense native structural environment of the cell at the molecular scale. This correlative method is uniquely suited to determine the nanometer-scale organization of the plasma membrane and associated organelles. Clathrin-mediated endocytosis is the primary mechanism human cells use to internalize receptors, nutrients, hormones, and other cargo at the plasma membrane. It is fundamental to the function of cells and tissues. Using our CLEM method, we studied 19 key proteins involved in clathrin-mediated endocytosis. Our data provides a comprehensive molecular architecture of endocytic vesicles in human cells. We discovered that key regulatory and cargo proteins distribute into distinct nanoscale spatial zones; inside, outside, and at the edge of the clathrin coat in human cells. The presence and amount of many factors within these zones change during vesicle growth. From these data we present a model that the formation and curvature of single endocytic vesicles is driven by the recruitment, re-organization, and loss of proteins within these distinct nanoscale zones. Currently, gaps exists between understanding protein structures and their dynamic cellular contexts. Our studies aim to fill these gaps by developing and using new ultra-high resolution imaging tools to determine the nanoscale structures, organizations, and dynamics of molecules that are important for membrane traffic in neurons, endocrine, and immune cells. As we piece together structures in their specific cellular contexts, a new holistic understanding of how biological processes work will be gained. These studies will help to map the fundamental architecture of cellular machines to better understand how these complex assemblies function in healthy cells and malfunction in disease.
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NLM Title: Imaging the nanoscale structure of endocytosis with correlative super-resolution light and electron microscopy / Justin W. Taraska.
Author: Taraska, Justin.
National Institutes of Health (U.S.),
Publisher:
Abstract: (CIT): NIH Director's Seminar Series The plasma membrane separates the cell's interior from the outside world. The exchange of signals and material across this barrier is regulated by a multitude of channels, transporters, receptors, and trafficking organelles. Mapping the molecular structure and dynamics of the plasma membrane is key to understanding how human cells function in health and malfunction in disease. Electron microscopy can produce high resolution images of the membrane. Historically, it has been challenging to locate and identify proteins within these images. Recently-developed super-resolution localization microscopy, however, can image fluorescently-labeled molecules with better than 20 nm precision. We developed a correlative super-resolution light and EM method (CLEM) that couples these complementary methods to produce images where identified proteins are mapped within the dense native structural environment of the cell at the molecular scale. This correlative method is uniquely suited to determine the nanometer-scale organization of the plasma membrane and associated organelles. Clathrin-mediated endocytosis is the primary mechanism human cells use to internalize receptors, nutrients, hormones, and other cargo at the plasma membrane. It is fundamental to the function of cells and tissues. Using our CLEM method, we studied 19 key proteins involved in clathrin-mediated endocytosis. Our data provides a comprehensive molecular architecture of endocytic vesicles in human cells. We discovered that key regulatory and cargo proteins distribute into distinct nanoscale spatial zones; inside, outside, and at the edge of the clathrin coat in human cells. The presence and amount of many factors within these zones change during vesicle growth. From these data we present a model that the formation and curvature of single endocytic vesicles is driven by the recruitment, re-organization, and loss of proteins within these distinct nanoscale zones. Currently, gaps exists between understanding protein structures and their dynamic cellular contexts. Our studies aim to fill these gaps by developing and using new ultra-high resolution imaging tools to determine the nanoscale structures, organizations, and dynamics of molecules that are important for membrane traffic in neurons, endocrine, and immune cells. As we piece together structures in their specific cellular contexts, a new holistic understanding of how biological processes work will be gained. These studies will help to map the fundamental architecture of cellular machines to better understand how these complex assemblies function in healthy cells and malfunction in disease.
Subjects: Cell Membrane--ultrastructure
Endocytosis
Microscopy, Electron--methods
Publication Types: Webcasts
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NLM Classification: QU 375
NLM ID: 101744880
CIT Live ID: 31609
Permanent link: https://videocast.nih.gov/launch.asp?27371