@article {65678, title = {Actin chromobody imaging reveals sub-organellar actin dynamics.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 Sep 01}, pages = {917-21}, abstract = {

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are challenging to detect with fluorescence microscopy, making it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed fluorescent-protein-tagged actin nanobodies, termed {\textquoteright}actin chromobodies{\textquoteright} (ACs), targeted to organelle membranes to enable high-resolution imaging of sub-organellar actin dynamics.

}, issn = {1548-7105}, doi = {10.1038/s41592-020-0926-5}, author = {Schiavon, Cara R and Zhang, Tong and Zhao, Bing and Moore, Andrew S and Wales, Pauline and Andrade, Leonardo R and Wu, Melissa and Sung, Tsung-Chang and Dayn, Yelena and Feng, Jasmine W and Quintero, Omar A and Shadel, Gerald S and Grosse, Robert and Manor, Uri} } @article {45782, title = {Isolation of mitochondria from animal tissue.}, journal = {Cold Spring Harbor Protocols}, volume = {2014}, year = {2014}, month = {2014 Oct}, pages = {pdb.prot080010}, abstract = {

Rat or mouse liver is the most frequently used tissue for mitochondrial preparations because it is readily available, easy to homogenize, and replete with mitochondria. A motor-driven Teflon and glass Potter-Elvehjem homogenizer is the best choice for homogenizing liver, but if one is not available, this tissue is soft enough that a Dounce homogenizer with a loose (A) pestle can also be used. The yield and purity of the mitochondrial preparation will be influenced by the method and speed of preparation and the age and physiological condition of the animal.

}, issn = {1559-6095}, doi = {10.1101/pdb.prot080010}, author = {Clayton, David A and Shadel, Gerald S} } @article {47884, title = {Isolation of mitochondria from cells and tissues.}, journal = {Cold Spring Harbor Protocols}, volume = {2014}, year = {2014}, month = {2014 Oct}, pages = {pdb.top074542}, abstract = {

Mitochondria are complex organelles at the center of cellular metabolism, apoptosis, and signaling. They continue to be the subject of intense basic investigation to understand their composition and function, but they have also captivated the attention of clinical researchers because of the growing knowledge of the (sometimes unexpected) roles of mitochondria in human diseases and aging. A full understanding of these intriguing organelles often requires their purification from cells or tissues under specific physiological or pathological conditions. Here we provide some introductory considerations for those interested in purifying mitochondria for subsequent downstream biophysical, structural, and functional analysis.

}, keywords = {Animals, Cell Fractionation, Cells, Cultured, Humans, Mitochondria, Tissue Culture Techniques}, issn = {1559-6095}, doi = {10.1101/pdb.top074542}, author = {Clayton, David A and Shadel, Gerald S} } @article {45783, title = {Isolation of mitochondria from tissue culture cells.}, journal = {Cold Spring Harbor Protocols}, volume = {2014}, year = {2014}, month = {2014 Oct}, pages = {pdb.prot080002}, abstract = {

The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from \~{}10$^{9}$ cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation.

}, issn = {1559-6095}, doi = {10.1101/pdb.prot080002}, author = {Clayton, David A and Shadel, Gerald S} } @article {45784, title = {Purification of mitochondria by sucrose step density gradient centrifugation.}, journal = {Cold Spring Harbor Protocols}, volume = {2014}, year = {2014}, month = {2014 Oct}, pages = {pdb.prot080028}, abstract = {

Mitochondrial fractions isolated from tissue culture cells or tissue such as liver after differential centrifugation can be purified further by density gradient centrifugation. Here we describe the use of sucrose for this purpose because it is commonly used and inexpensive and the resulting mitochondria preparations are useful for many purposes.

}, issn = {1559-6095}, doi = {10.1101/pdb.prot080028}, author = {Clayton, David A and Shadel, Gerald S} }