Journal Club on Monday, January 7

For our Journal Club on January 7, Yidi Sun presented the following paper:

PtdIns4P synthesis by PI4KIIIα at the plasma membrane and its impact on plasma membrane identity.  Nakatsu F, Baskin JM, Chung J, Tanner LB, Shui G, Lee SY, Pirruccello M, Hao M, Ingolia NT, Wenk MR, De Camilli P.  J Cell Biol. 2012 Dec 10;199(6):1003-16.  PMID:  23229899.

PtdIns(4,5)P2-positive intracellular vesicles, visualized with GFP- or RFP-PHPLCδ, colocalize with endocytic clathrin machinery components (endogenous AP-2, epsin 1, and dynamin 2–GFP) but not with markers of endosomes (endogenous EEA1, GFP-Rab5, and GFP-Rab9), the endosomal phosphoinositide PtdIns3P (GFP-2×FYVEHrs), and Golgi-localized PtdIns4P (GFP-PHOSBP). For clarity, PHPLCδ is false colored green, and the second marker is false colored red in all images.

Congratulations to Yidi Sun on her new paper!

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.

 

Journal Club on Monday, November 5

For Journal Club on November 5, Nate Krefman presented the following papers on Golden Gate Cloning:

A one pot, one step, precision cloning method with high throughput capability. Engler C, Kandzia R, Marillonnet S. PLoS One. 2008;3(11):e3647. PMID: 18985154.

Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. Engler C, Gruetzner R, Kandzia R, Marillonnet S. PLoS One. 2009;4(5):e5553. PMID: 19436741.

A modular cloning system for standardized assembly of multigene constructs. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. PLoS One. 2011 Feb 18;6(2):e16765. PMID: 21364738.

Maps of entry clone pE-GFP, expression cloning vector pX-lacZ and expression construct pX-GFP. Black arrows show the position of the restriction sites for the enzymes BseRI and HindIII, and the numbers next to these indicate the sizes of the restriction fragments obtained. The grey triangle represents a Streptomyces phage C31 attB recombination site (this site is not used for the cloning procedure described here). Z, LacZ alpha fragment; N, Viral 3′ Non-translated region; T, Nos terminator; RB/LB, T-DNA right/left borders; S1–S2, selectable markers 1 and 2 (resistance to carbenicillin and kanamycin, respectively).

Journal Club on Monday, October 15

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

Journal Club on Monday, October 1

For our next Journal Club, Padmini Rangamani will present the following paper:

Phase transitions in the assembly of multivalent signalling proteins. Li P, Banjade S, Cheng HC, Kim S, Chen B, Guo L, Llaguno M, Hollingsworth JV, King DS, Banani SF, Russo PS, Jiang QX, Nixon BT, Rosen MK. Nature. 2012. PMID: 22398450

Phase transition correlates to biochemical activity transition in the nephrin/Nck/N-WASP system

Lighting Up Live Cells with Fluorescence (Genetic Engineering & Biotechnology News)

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]

Journal Club on Thursday, July 26

For our next Journal Club, our summer undergraduate researcher Josh Johnson will present the following paper:

Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient.  Wang E, Ballister ER, Lampson MA.  J Cell Biol. 2011 Aug 22;194(4):539-49.  PMID: 21844210.

Centromeres (CB-mCherry) and chromosomes (YFP emission) for a single time point. (bottom) Color-coded YFP/CFP emission ratio at different time points. The timestamp (minutes and seconds) is relative to ZM washout at t = 0. Bar, 5 µm.

Journal Club on Monday, July 09

For our next Journal Club, Adrianne Pigula will present the following paper:

A link between mitotic entry and membrane growth suggests a novel model for cell size control.  Anastasia SD, Nguyen DL, Thai V, Meloy M, MacDonough T, Kellogg DR.  J Cell Biol. 2012 Apr 2;197(1):89-104. Epub 2012 Mar 26.  PMID: 22451696.

A model for signals that link mitotic entry to membrane growth.

Journal Club on Monday, June 25

For Journal Club on Monday, June 25, Connie Peng presented the following paper:

Functional Repurposing Revealed by Comparing S. pombe and S. cerevisiae Genetic Interactions.  Frost A, Elgort MG, Brandman O, Ives C, Collins SR, Miller-Vedam L, Weibezahn J, Hein MY, Poser I, Mann M, Hyman AA, Weissman JS.  Cell. 2012 Jun 8;149(6):1339-52.  PMID: 22682253.

Scatter plot of correlation coefficients comparing GI profiles for Sp versus Sc. Highlighted examples of pairwise relationships that are correlated in Sp but not Sc are listed below the scatter plot. Bold indicates functional relationships explored in this study. See Table S1 for additional examples of correlated pairwise relationships conserved between Sp and Sc (green), and pairwise relationships that are correlated in Sc but not Sp (cyan) or Sp but not Sc (red).

An Interview with David Drubin in Biowire

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