Monthly Lab Focus – Ciani Group

SIGNALING IN MEMBRANE TRAFFICKINGPrincipal Investigator:

Post docs: Recruiting now!

PhD students:

  • Jessica Willan
  • Liam Marshall

Technician:

  • Jennifer Louth

Membrane Remodelling

Scientific Questions we are addressing:

  • How does protein structure translate into function?
  • How do protein assemblies remodel membranes?
  • Can we design a functional protocell from the bottom-up using natural and designed biological components? 

Techniques we use:

  • Molecular biology: PCR, cloning, in vitro mutagenesis.
  • Biochemistry: protein expression and purification, binding assays including fluorescence anisotropy and NMR; immunoprecipitation and GST pull down; protein assembly polymerization assays, negative staining and electron microscopy analysis of protein filaments.
  • Protein structure determination via Multidimensional NMR spectroscopy.
  • Cell Biology and microscopy: dual colour live cell imaging including deconvolution using Deltavision microscope (in collaboration with Prof Philip Woodman in Manchester).
  • Giant Unilamellar Vesicles preparation for studying membrane remodelling proteins (in collaboration with Dr Paul Beales in Leeds). 

Lay Summary of research within the lab

We take inspiration from Nature to manufacture mimics of biological structures from proteins to the compartmentalised structure of biological cells. For this purpose, we study the biological mechanisms of the ESCRT membrane remodelling complexes, a coiled-coil based protein assembly. We employ ESCRT to dynamically trigger changes in lipid architectures in a highly responsive manner. These membrane architectures will be used for carrying out multiplexed and parallelised chemical reactions enabling specific functions. A long-term aim is the bottom-up design and mimicry of protocellular systems starting from simple biological components. We are also very much interested in how the molecular mechanisms behind ESCRT membrane remodelling functions also support non-canonical functions of these complexes, and specifically their roles in regulation of mitosis.

Impact

Academic impact:

  • We have further advanced the understanding of the molecular basis of protein oligomerisation via assembly of natural coiled-coil sequences. Coiled coils are a protein motif widely employed by Nature to control protein-protein interactions, but they are also used extensively by chemists and bioengineers as building block for templating functional biomaterials and a large variety of applications in biotechnology;
  • We have elucidated the mechanisms that govern protein structure-function relationships through the rational design of coiled coil-based secondary structure switches, beta-sheet assemblies and enzyme structure. Each of these structures and protein assemblies are the causative link in major diseases, such as neurodegeneration and cancer;
  • Our active collaborations with groups within the medical school have contributed to the understanding of the molecular mechanisms underlying pathologies such as rheumatoid arthritis and cardiac dysfunction.

Selected Recent Publications:

  • Muthana M, Hawtree S, Wilshaw A, Linehan E, Roberts H, Khetan S, Adeleke G, Wright F, Akil M, Fearon U, Veale D, Ciani B, Wilson AG. C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis. Proc Natl Acad Sci U S A. 2015.
  • Beales PA, Ciani B & Cleasby AJ (2015) Nature's lessons in design: nanomachines to scaffold, remodel and shape membrane compartments. Phys Chem Chem Phys, 17(24), 15489-15507.
  • Patel N, Exell JC, Jardine E, Ombler B, Finger LD, Ciani B & Grasby JA (2013) Proline Scanning Mutagenesis Reveals a Role for the Flap Endonuclease-1 Helical Cap in Substrate Unpairing. J. Biol. Chem., 288. pp. 34239-34248.
  • Hameed A, Bennett E, Ciani B, Hoebers LP, Milner R, Lawrie A, Francis SE, Grierson AJ (2013) No evidence for cardiac dysfunction in Kif6 mutant mice. PLoS One. 2013;8(1):e54636.
  • Ciani B, Bjelic S, Honnappa S, Jawhari H, Jaussi R, Payapilly A, Jowitt T, Steinmetz MO & Kammerer RA (2010) Molecular basis of coiled-coil oligomerization-state specificity. Proc Natl Acad Sci U S A, 107(46), 19850-19855.
  • Macdonald PR, Progias P, Ciani B, Patel S, Mayer U, Steinmetz MO & Kammerer RA (2006) Structure of the extracellular domain of Tie receptor tyrosine kinases and localization of the angiopoietin-binding epitope. J Biol Chem, 281(38), 28408-28414.
  • Layfield R, Ciani B, Ralston SH, Hocking LJ, Sheppard PW, Searle MS & Cavey JR (2004) Structural and functional studies of mutations affecting the UBA domain of SQSTM1 (p62) which cause Paget's disease of bone. Biochem Soc Trans, 32(Pt 5), 728-730.
  • Searle MS & Ciani B (2004) Design of beta-sheet systems for understanding the thermodynamics and kinetics of protein folding. Curr Opin Struct Biol, 14(4), 458-464.

Student awards/prizes:

  • Past PhD student Alexa Cleasby received the 3rd prize Biochemical Society 2012 Science Communication Competition.
  • FEBS travel award to Jessica Willan for the DNA Repair meeting in Smolenice 2013.

Funding:

EPSRC project grant

  • Harnessing Nature's ability to create membrane compartmentalisation through redesign of a protein machinery. Recruiting now – get in touch!

Leverhulme Trust research project

  • Designing recyclable self-assembled fibrous biomaterials. Recruiting now – get in touch! 

Collaborators

  • Dr Helen Bryant (University of Sheffield, Oncology)
  • Dr Munitta Muthana (University of Sheffield, Oncology)
  • Professor Philip Woodman (University of Manchester)
  • Dr Paul Beales (University of Leeds)
  • Dr Frederik Claeyssens (University of Sheffield, Materials Engineering)