CMIAD Lab Focus – Ayscough Group

Group members:

Post docs:

  • Iwona Smaczynska-de Rooij - funded through BBSRC grant
  • Ellen Allwood
  • John Palmer - funded through BBSRC grant

PhD students:

  • Joe Tyler – funded through White Rose Mechanistic Biology PhD studentship
  • Harriet Knafler - funded through White Rose Mechanistic Biology PhD studentship

Apprentice:

  • Aaron Kirkby

Ayscough Group Members

ENDOCYTOSIS & CYTOSKELETONScientific Questions we are addressing:

  • How are new actin filaments initiated at cell membranes?
  • What mechanisms ensure that endocytosis is a unidirectional process?
  • How do dynamin, amphiphysin and actin function together during membrane scission?
  • What alternative mechanisms of endocytosis are present in yeast
  • What is the importance of endocytosis for virulence of the pathogenic fungus Candida albicans?

EM image of an endocytic invagination in S.cerevisiae.

Techniques we use:

  • Molecular biology: PCR, cloning, in vitro mutagenesis
  • Biochemistry: protein expression and purification, binding assays including microscale thermophoresis; immunoprecipitation and GST pull down; lipid and liposome binding, actin polymerization assays, negative staining and electron microscopy analysis of actin filaments
  • Genetics: S. cerevisiae and Candida albicans gene deletion and epitope tagging, growth, phenotypic analysis, strain crossing, tetrad dissection
  • Cell Biology and microscopy: dual colour live cell imaging including quantitative analysis and patch tracking using Deltavision microscope; yeast endocytosis assays and vital staining for mitochondria and vacuoles.
    Electron microscopy, mostly in collaboration with Martin Goldberg, Durham

Lay Summary of research within the lab

Cells are the basic unit of life and all organisms are composed of one or more cells. Central to the functioning of many cells, including human cells, is the internal skeleton, or cytoskeleton. This cytoskeleton is required for cells to have certain shapes that are often a necessary part of their functioning. However, unlike our own body skeleton that is static, the cytoskeleton is able to remodel itself to change cell shape, or allow a cell to move.

One of the most important proteins in the cytoskeleton is called actin. It is an amazing protein because it is almost the same now as hundreds of millions of years ago, long before humans, or even vertebrates existed. Staying so similar over time is called evolutionary conservation. Proteins that are very important to cell functioning are the most highly conserved. Actin is a protein that can join together with other actin proteins to form long lines or filaments. These filaments can be organised by other proteins to form large structures that are part of the cytoskeleton.

We are interested in how this protein is controlled in cells and in particular, we are trying to determine how the filaments can be started from single actin proteins in a process called nucleation. We are also aiming to understand how force can be generated by these filaments to help inward bending of the outer membrane of the cell to let parts of the membrane to be pinched off (internalised) This process, called endocytosis allows the surface composition of the cell to be effectively regulated.

Impact

Academic impact:

  1. KA pioneered use of the drug Latrunculin for disruption of the actin cytoskeleton. This allowed questions to be addressed about the importance of actin in cells, particularly in the model organism S. cerevisiae (budding yeast).
  2. Studies by PhD student Soheil Aghamohammadzadeh in the lab led to a breakthrough in our understanding of the differential requirement for actin during endocytosis in yeast and mammalian cells.
  3. KA is a member of the BBSRC Grant Panels and has sat on the committees of the British Society of Cell Biology and the Biochemical Society.

Public Understanding:

  • Kathryn, Ellen and Iwona have undertaken a number of demonstrations and workshops at schools for children aged 5-11. These have focused on the “Bacteria and fungi in our environment and the importance of handwashing”, and also on “DNA and Cells”.

Selected Recent Publications

  1. Palmer SE, Smaczynska-de Rooij II, Marklew CJ, Allwood EG, Mishra R, Johnson S, Goldberg MW, Ayscough KR. A Dynamin-Actin Interaction Is Required for Vesicle Scission during Endocytosis in Yeast. (2015) Curr Biol. 25:868-78.
  2. Aghamohammadzadeh S, Smaczynska-de Rooij II, Ayscough KR.  (2014) An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited. PLoS One. 9:e103311.
  3. B.Chapa-y-Lazo, E.G. Allwood, I.I. Smaczynska-de Rooij, M.L. Snape and K.R. Ayscough Yeast endocytic adaptor AP-2 binds the stress sensor Mid2 and functions in polarised cell responses (2014) Traffic. 15:546-557
  4. Alpadi K, Kulkarni A, Namjoshi S, Srinivasan S, Sippel KH, Ayscough K, Zieger M, Schmidt A, Mayer A, Evangelista M, Quiocho FA, Peters C. Dynamin-SNARE interactions control trans-SNARE formation in intracellular membrane fusion. (2013) Nat Commun.4:1704.
  5. Urbanek AN, Smith AP, Allwood EG, Booth WI, Ayscough KR. A novel actin binding motif in Las17/WASP nucleates actin filaments independently of Arp2/3. (2013) Curr Biol 23: 196-203
  6. I.I. Smaczynska-de Rooij, E.G. Allwood, R. Mishra, W.I. Booth, S. Aghamohammadzadeh,  M.W. Goldberg, K.R. Ayscough. (2012) Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis. Traffic. 13 (2) 317-328.
  7. Aghamohammadzadeh, S. and Ayscough K.R (2009) Differential Requirements for Actin during Yeast and Mammalian Endocytosis. Nature Cell Biol. 11. 1039-1042.
  8. Robertson, A.S., Allwood, E.G., Smith, A.P., Gardiner, F.C., Costa, R., Winder, S.J. and Ayscough, K.R. (2009) The WASP homolog Las17 activates the novel actin-regulatory activity of Ysc84 to promote endocytosis in yeast.  Mol. Biol. Cell. 20. 1618-1628

Student awards/prizes

  • 2009 – meeting Actin 2009: Poster prize to Aga Urbanek
  • 2013 – North of England Cell Biology Prize for Best Talk
  • 2014 – BMS Third year PhD symposium award Sarah Palmer
  • 2015 – BMS Second year PhD symposium award Joe Tyler

Funding:

BBSRC project grants

  • Elucidating the mechanism of endocytic invagination and scission. Supporting IS, CM, KV.
  • Defining factors that ensure unidirectionality of endocytosis. Supporting EA, AU

BBSRC White Rose Mechanistic Biology PhD studentship

  • Analysis of a Novel Actin Nucleating Machinery regulating Membrane trafficking, supports JT

Collaborators

  • Martin Goldberg (University of Durham)
  • Teresa Zoladek (Polish Academy of Science, Warsaw, Poland)
  • Robert Robinson (A*Star, Singapore)