@article {48416, title = {Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples.}, journal = {Nature Protocols}, volume = {12}, year = {2017}, month = {2017 May}, pages = {916-946}, abstract = {

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (\~{}10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

}, issn = {1750-2799}, doi = {10.1038/nprot.2017.017}, author = {Kopek, Benjamin G and Paez-Segala, Maria G and Shtengel, Gleb and Sochacki, Kem A and Sun, Mei G and Wang, Yalin and Xu, C Shan and Van Engelenburg, Schuyler B and Taraska, Justin W and Looger, Loren L and Hess, Harald F} } @article {6710, title = {Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.}, journal = {Science}, volume = {343}, year = {2014}, month = {2014 Jan 16}, pages = {653-6}, abstract = {

The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.

}, issn = {1095-9203}, doi = {10.1126/science.1247786}, author = {Van Engelenburg, Schuyler B and Shtengel, Gleb and Sengupta, Prabuddha and Waki, Kayoko and Jarnik, Michal and Ablan, Sherimay D and Freed, Eric O and Hess, Harald F and Lippincott-Schwartz, Jennifer} } @article {47631, title = {Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.}, journal = {Science (New York, N.Y.)}, volume = {343}, year = {2014}, month = {2014 Feb 7}, pages = {653-6}, abstract = {

The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.

}, keywords = {Endosomal Sorting Complexes Required for Transport, gag Gene Products, Human Immunodeficiency Virus, HIV Infections, HIV-1, Humans, Imaging, Three-Dimensional, Microscopy, Protein Subunits, Virion, Virus Assembly, Virus Release}, issn = {1095-9203}, doi = {10.1126/science.1247786}, author = {Van Engelenburg, Schuyler B and Shtengel, Gleb and Sengupta, Prabuddha and Waki, Kayoko and Jarnik, Michal and Ablan, Sherimay D and Freed, Eric O and Hess, Harald F and Lippincott-Schwartz, Jennifer} }