For our Journal Club on June 3, Daphné Dambournet presented the following paper:
Divergent modes for cargo-mediated control of clathrin-coated pit dynamics. Soohoo AL, Puthenveedu MA. Mol Biol Cell. 2013 Jun;24(11):1725-34. PMID: 23536704.
Model for divergent modes of cargo-mediated control of endocytosis by signaling receptors.
For our Journal Club on April 29, Sun Hae Hong presented the following paper:
The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM. Miller SE, Sahlender DA, Graham SC, Höning S, Robinson MS, Peden AA, Owen DJ. Cell. 2011 Nov 23;147(5):1118-31. PMID: 22118466
Schematic representation of the model of VAMP8 trafficking from the plasma membrane.
Yidi Sun‘s new paper is out now as an electronic publication ahead of print in the Journal of Cell Science. Congratulations to Yidi on her great work! The abstract is below. The PDF can be downloaded from JCS here.
The functions of anionic phospholipids during clathrin-mediated endocytosis site initiation and vesicle formation. Sun Y, Drubin DG. J Cell Sci. 2012 Oct 24. PMID: 23097040.
Abstract
Anionic phospholipids PI(4,5)P(2) and phosphatidylserine (PS) are enriched in the cytosolic leaflet of the plasma membrane where endocytic sites form. In this study, we investigated the roles of PI(4,5)P(2) and PS in clathrin-mediated endocytosis (CME) site initiation and vesicle formation in Saccharomyces cerevisiae. Live-cell imaging of endocytic protein dynamics in an mss4(ts) mutant, which has severely reduced PI(4,5)P(2) levels, revealed that PI(4,5)P(2) is required for endocytic membrane invagination but is less important for endocytic site initiation. We also demonstrated that in various deletion mutants of genes encoding components of the Rcy1-Ypt31/32 GTPase pathway, endocytic proteins dynamically assemble not only on the plasma membrane but also on intracellular membrane compartments, which are likely derived from early endosomes. In rcy1Δ cells, fluorescent biosensors indicated that PI(4,5)P(2) only localized to the plasma membrane while PS localized to both the plasma membrane and intracellular membranes. Furthermore, we found that polarized endocytic patch establishment is defective in the PS-deficient cho1Δ mutant. We propose that PS is important for directing endocytic proteins to the plasma membrane and that PI(4,5)P(2) is required to facilitate endocytic membrane invagination.
For our Journal Club on October 15, Jay Ryoo presented the following paper:
The first five seconds in the life of a clathrin-coated pit. Cocucci E, Aguet F, Boulant S, Kirchhausen T. Cell. 2012 Aug 3;150(3):495-507. PMID: 22863004.
Schematic representation of the experimental setup and computational analysis
Genetic Engineering & Biotechnology News is out with a Feature Article this week including some comments from David Drubin about targeted genome modification in mammalian cells for fluorescence microscopy studies.
Lighting Up Live Cells with Fluorescence. Christine Herman. GEN. Sep 1, 2012 (Vol. 15, No. 32)
“The difference between taking snapshots of the process and watching a movie is just night and day,” says David Drubin, Ph.D., professor of cell and developmental biology at the University of California, Berkeley, whose lab uses fluorescence to understand the intricate details underlying clathrin-mediated endocytosis.
Researchers in David Drubin’s lab at the University of California, Berkeley genetically engineered a human cell line to express endogenous levels of RFP-tagged clathrin light chain A (red) and GFP-tagged dynamin 2 (green) for studying clathrin-mediated endocytosis. The above 3D kymograph of the cell surface, with the time dimension in the z-axis, shows the full lifetime of hundreds of clathrin patches on the membrane, which terminate upon recruitment of dynamin. [Aaron T. Cheng]
The May 2012 edition of Biowire, a publication of Sigma-Aldrich, includes an interview with David Drubin about the projects in our lab looking at clathrin-mediated endocytosis (CME) in mammalian cells using zing finger nuclease (ZFN) technology to undertake targeted genome modification. Traditionally, CME has been studied in cells in which fluorescently-tagged components of endocytic machinery are overexpressed using exogenous constructs. Data obtained in many labs using these methods suggested that CME was highly variable. Using ZFN technology, in collaboration with Sangamo Biosciences, our lab recently showed that CME is robust and efficient in mammalian cells. The new results highlight the technical advantages of tagging genes at their endogenous loci, an approach that has been historically limited to genetically tractable organisms, such as the Drubin/Barnes Lab favorite Saccharomyces cerevisiae (budding yeast). Emerging technologies, such as ZFNs and TALENs, however, are now making this sort of precise genomic manipulation possible in animal cells, including human cells, giving us new and powerful ways of studying cellular biology.
Cellular processes should be studied as close to their natural states as possible. I suspect that, as we see more uses of zinc finger nucleases [for tagging endogenous genes], people will find that they have been inadvertently perturbing the processes that they have been studying.
David Drubin (Biowire, May 2012)
Prof. David Drubin, for Biowire
For our next Journal Club, Lillie Cohn will present the following paper:
Reconstitution of clathrin-coated bud and vesicle formation with minimal components. Dannhauser PN, Ungewickell EJ. Nat Cell Biol. 2012 Apr 22;14(6):634-9. PMID: 22522172.
Ultrathin section of H6-ΔANTH-AP180328–896-coated liposomes that were incubated with clathrin at 4 °C (c) or 37 °C
For our next Journal Club, Christa Cortesio will present the following paper:
Distinct and separable activities of the endocytic clathrin-coat components Fcho1/2 and AP-2 in developmental patterning. Umasankar PK, Sanker S, Thieman JR, Chakraborty S, Wendland B, Tsang M, Traub LM. Nat Cell Biol. 2012 Apr 8. doi: 10.1038/ncb2473. PMID: 22484487.
Interaction diagram for FCHO1/2 and select endocytic pioneer coat components. Presumptive contacts are indicated with dotted lines.