Steven Chou determined the structure of actin filaments labeled with pyrene on cysteine 374 at a resolution of 3.0 Å, a record. The new structure shows that polymerization increases the fluorescence of pyrene by trapping it in a hydrophobic pocket between adjacent subunits along the long pitch helix of the filament. The paper is at Chou SZ, Pollard TD. (2020) Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments. Nature Comm. 11:5897. PMID: 33214556.
Helen Sun published a quantitative microscopy paper on how fixation and imaging conditions affect the photoconversion and photobleaching of mEos3.2, the fluorescent protein we use for super resolution microscopy. She found that mEos3.2 survives fixation by formaldehyde, but that it becomes sensitive to the buffer pH. She found conditions where the fluorescence is similar to that in live cells. Her paper is at Sun M, Hu K, Bewersdorf J, Pollard TD. (2021) Sample preparation and imaging conditions affect mEos3.2 photophysics in fission yeast cells. Biophys J. 120:21-34. PMID: 33217381.
Sam Dundon’s paper on microtubules regulating cytokinesis in fission yeast in now online: Microtubule Nucleation Promoters Mto1 and Mto2 Regulate Cytokinesis in Fission Yeast. Dundon SER, Pollard TD. Mol Biol Cell. 2020 Jun 10:mbcE19120686. doi: 10.91/mbc.E19-12-0686. https://www.molbiolcell.org/doi/10.1091/mbc.E19-12-0686
Online ahead of print.
Tom Pollard published an autobiographical account of the lab’s work over five decades, highlighting the contributions of many colleagues, who helped discover the molecular mechanisms of cell motility and cytokinesis. Pollard TD. Cell motility and cytokinesis: from mysteries to molecular mechanisms in five decades. Ann Rev Cell Devel Biol. 35:1-28, 2019. PMID: 31394047.
A new paper in eLife compares actin assembly during endocytosis in fission yeast and budding yeast. Tom Pollard initiated the work in 2017 during a short sabbatical in the laboratory of David Drubin at the University of California, Berkeley. Research scientist Yidi Sun did most of the work. This direct comparison of the two yeasts revealed that process is more conserved than indicated by previous studies, although different nucleation promoting factors are more important to stimulate actin assembly by Arp2/3 complex in the two yeasts: myosin-I takes the lead in budding yeast, while fission yeast relies more on WASp. Sun Y, Schoeneberg J, Chen S, Jiang T, Kaplan C, Xu K, Pollard TD, Drubin DG. Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features. eLife 8:e50749, 2019. PMID: 31829937.
Simulation of the mechanics of actin assembly during endocytosis in yeast, a new paper in collaboration with colleagues at the University of Connecticut Health Center, uses mathematical modeling and computer simulations to verify that actin polymerization produces enough force to invaginate the plasma membrane at the site of endocytosis in fission yeast. https://www.biorxiv.org/content/early/2019/01/11/518423
Molecular Biology of the Cell published the Pollard family paper (Dan, Tom and Katie) “Empowering statistical methods for cellular and molecular biologists.” The paper was downloaded more than 3000 times in the first 12 days. We provided guidelines for using statistical methods to analyze data from experiments in cellular and molecular biology. The aim is to help experimentalists use these methods skillfully, avoid mistakes, and extract the maximum amount of information from their laboratory work.
Biochemistry accepted Moon Chatterjee’s paper entitled “The functionally important N-terminal half of fission yeast Mid1p anillin is intrinsically disordered and undergoes phase separation.” The features explained by the title need to be considered when thinking about how Mid1p organizes other proteins in the cytokinetic nodes of fission yeast.
Former graduate student Shamba Saha has opened his lab at the IMBA-Institute of Molecular Biotechnology in Vienna, Austria. His new email address is firstname.lastname@example.org
Former graduate student Shih-Chien (Jeff) Ti is now Assistant Professor at the University of Hong Kong, School of Biomedical Sciences.
Chou, S.Z., and Pollard, T.D. (2019) Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides. Proc. Nat. Acad. Sci. USA. doi:10.1073/pnas.1807028115. This paper illustrates high-resolution structures of actin filaments with three different nucleotides, answering decades-old questions about the assembly process.
The lab published a new paper by former lab members Ikko Fujiwara (now at the Nagoya Institute of Technology) and Naomi Courtemanche (now at the University of Minnesota).
Fujiwara, I., Zweifel, M.E., Courtemanche, N. and Pollard, T.D. (2018) Latrunculin A accelerates actin filament depolymerisation in addition to sequestering actin monomers. Curr. Biol. 28, 3183-3192. PMID: 30270183
We discovered that Latrunculin A, a natural product used to depolymerize actin filaments in cellular experiments, has a more complicated mechanism of action than had been assumed for 20 years. Not only does it bind and sequester actin monomers, but it also increases the aging of the filaments by promoting phosphate dissociation from terminal subunits and thus the rate of depolymerization.
Two papers published in August 2018:
Dey SK, Pollard TD. Involvement of the sepation initiation network in events during cytokinesis in fission yeast. J Cell Sci. 2018 Aug 23;131(16). pii:jcs216895. Sumit discovered that the septation initiation network (SIN) is required for the mechanical stability of the contractile ring in fission yeast but is not required to trigger the constriction of the contractile ring.
Espinoza-Sanchez S, Metskas LA, Chou SZ, Rhoades E, Pollard TD. Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments. Proc Natl Acad Sci U S A. 2018 Aug 27. pii: 201717594. Sofia and Lauren Ann Metskas used FRET and Steven Chou used electron microscopy to characterize the conformational changes of Arp2/3 complex induced by binding of ATP, nucleation promoting factors and actin filaments. NPFs such as WASp shift the conformation partially toward the active conformation, while binding a mother actin filament extends these conformational changes.
Professor Pollard has been appointed to the Governing Board of the National Research Council of the US National Academy of Sciences. Also, on July 1, 2018, he will take over the leadership of the Yale Institute for Physics-Engineering-Biology as Interim Director.
Friend, J.E., Sayyad, W.A., Arasada, R., McCormick, C.D., Heuser, J.E., and Pollard, T.D. (2017) Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm. Cytoskeleton. In press. This paper shows that single Myo2 molecules with two heads and a long tail diffuse in the cytoplasm of interphase and mitotic cells.
Arasada,R., Sayyad, W.A., Berro, J., and Pollard, T.D. (2017) High-speed super-resolution imaging of the proteins in fission yeast clathrin-mediated endocytic actin patches. Molec. Biol. Cell. In press. This study used FPALM super resolution microscopy with 35 nm spatial resolution and 1 second time resolution to track single protein molecules in fission yeast actin patches. The actin assembles in two zones at sites of clathrin-mediated endocytosis.
Sofia Espinoza defended her thesis on July 17, 2017.
Lab member undergraduate student Frank Chen graduated from Yale College with honors including Phi Beta Kappa and will enter Weill-Cornell Medical School in New York next term.
Alexander Epstein, 2018, is one of three Yale University juniors to be awarded the prestigious Barry Goldwater Scholarship for excellence in science, technology, engineering, and math (STEM) fields and education. These awards are given to sophomores and juniors who also demonstrate a long term commitment to their chosen field of inquiry.
Tom Pollard was elected to a three year term on the Council of the National Academy of Sciences, which will begin this summer.
Tom Pollard will give the keynote address at the Cold Spring Harbor Laboratory meeting on “Cellular Dynamics and Models” April 12, 2017.
Caroline Laplante and colleagues published their analysis of the structure of cytokinesis nodes by super resolution localization microscopy. The article entitled “Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast” appeared in PNAS. See www.pnas.org/cgi/doi/10.1073/pnas.16081252113
Sam Dundon joined the laboratory as a postdoctoral associate coming from Amy Gladfelter’s laboratory at Dartmouth College.
Naomi Courtemanche and Qian Chen published a paper in Nature Cell Biology revealing the adverse effects of Lifeact-GFP on actin assembly in vitro and in cells, but found ways to use Lifeact-GFP to make valuable quantitative measurements of polymerized actin in live cells. Two important finding were that the fission yeast contractile ring consists of about 500 µm of actin filaments and these filaments shorten as the ring constricts.
Tom Pollard will receive the Carl Zeiss Award from the German Society for Cell Biology this month.
During the Fall Term of 2015 members of the lab took new independent positions. Naomi Courtemanche is Assistant Professor of Genetics, Cell Biology and Development at the University of Minnesota and Qian Chen is Assistant Professor of Biology at the University of Toledo. Three new postdoctoral associates joined the laboratory: Moon Chatterjee from Inari Kursula’s lab at the University of Hamburg in Germany, Wasim Sayyad from Vincent Torre’s lab at the International School for Advanced Studies in Trieste, Italy, and Sumit Kumar Dey from Siddhartha S Jana’s lab at the Indian Association for the Cultivation of Science in Kolkata India.
Chris Jurgenson (now an assistant professor at Delta State University) has a paper accepted in Acta Crystallogr F Struct Biol Commun entitled “Crystals of Arp2/3 complex in two new space groups with structural information about actin related protein 2 and potential WASP binding sites.”
Rajesh Arasada has a paper in press in Journal of Cell Science showing that the F-BAR protein Rga7p is required to deliver ß-glucan synthetase Bgs4p from the late Golgi compartments to the plasma membrane adjacent to the contractile ring.
Tom Pollard will receive the 2015 National Academy of Sciences Award for Scientific Reviewing – presented in the field of biochemistry. “He is honored for his reviews tracing the history of cell motility from its beginnings, critically analyzing the biochemical reactions responsible for cellular movements, critiquing the methods and assumptions used in the field, and synthesizing the information available into creative models that have guided the development of the field.”
Naomi Courtemanche published a paper in Structure (23:68-79) with a refined model for how formin FH2 domains interact with the barbed end of the actin filament. Naomi contributed biochemical experiments on mutant proteins to test ideas that emerged from molecular dynamics simulations by collaborators in the laboratory of Greg Voth at the University of Chicago.