Stamou Lab

Single Transporter Activity Recordings (STARs).

In a breakthrough paper in Science [1] we developed a method that resolved ionic currents with a million-fold higher sensitivity than the Nobel prize awarded method of patch clamp (atto-amperes). This allowed us to observe for the first time the function of single transporters, revealing the existence of functional heterogeneity based on hitherto unknown off-cycle states (i.e. ultra-stable inactive and leaky states with lifetimes 105-fold longer than those of states in the classical transport cycle). The dramatic consequence of this unforeseen complexity is that many fundamental mechanistic tenets that were based on macroscopic experiments of transport will have to be revised. This truly transformative innovation is the crowing achievement of a decade of research in our lab[2,3] and we believe is likely to spark off a technological and conceptual paradigm shift in the transport field.

Membrane curvature

Eukaryotic life is defined by the existence of intracellular membrane-bound organelles. Endomembranes are overall more curved than the plasma membrane their heterogeneous geometrical shapes however are not arbitrary, on the contrary they are so characteristic that they have become a hallmark of the different eukaryotic organelles.[4] To elucidate why this phenotype is so remarkably conserved we have pioneered several high-throughput nanoscopic methods to study how membrane curvature is affecting the function(s) of membranes and membrane proteins, with an emphasis on Ras and G protein coupled receptors.[5-7] Our contributions have overall helped establish the notion that the properties of biological membranes are defined equally by their lipid composition and their geometrical shape.[4]

Nanoscopic heterogeneity of biological membranes

Spatiotemporal compositional and functional heterogeneities are a hallmark of biological membranes. We are investigating the implications of these heterogeneities for biological function and also exploit them for technological applications. For example, with high-throughput single proteoliposome measurements we quantified the composition of single proteoliposomes revealing dramatic heterogeneities.[8] As we showed, compositional heterogeneities can severely skew ensemble-average proteoliposome measurements however, if averaging is avoided such heterogeneities can be exploited to enable high-content screens that enable a dramatic reduction in protein consumption (~billion-fold) as compared to conventional assays.[8] We are currently extending this project to investigate nanoscopic compositional and functional heterogeneities of G protein coupled receptors in live cells.

References

1. Science, 2016. 351 (6280): p. 1469-1473        
   Direct observation of proton pumping by a eukaryotic P-type ATPase 
   Salome Veshaguri, Sune M. Christensen, Gerdi C. Kemmer, Mads P. Møller, Garima Ghale, Christina Lohr, Andreas L.Christensen, Bo H. Justesen, Ida L. Jørgensen, Jürgen Schiller, Nikos S. Hatzakis, Michael Grabe, Thomas Günther Pomorski, Dimitrios Stamou                  

2. Proceedings of the National Academy of Sciences. 2009. 106 (30): p. 12341
   Quantification of nano-scale intermembrane contact areas using fluorescence resonance energy transfer.                 
   Poul Martin Bendix, M. S. Pedersen and Dimitrios Stamou.

3. Nature Nanotechnology, 2012. 7 (1): p. 51–55                   
   Mixing sub-attolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics.    S. M. Christensen; P.Y. Bolinger; N.S. Hatzakis; M.W. Mortensen and Dimitrios Stamou

4. Nature Chemical Biology, 2015. 11 (11): p. 822-825          
   Membrane curvature bends the laws of physics and chemistry
   Lars Iversen, Signe Mathiasen, Jannik Bruun Larsen, Dimitrios Stamou

5. Nature Chemical Biology, 2009. 5 (11): p. 835                   
   How Curved Membranes Recognize Amphipathic Helices and Protein Anchoring Motifs.                   
   N. S. Hatzakis, V. K. Bhatia, J. Larsen, K. L. Madsen, P. Y. Bolinger, A. H. Kunding, J. Castillo, U. Gether, P. Hedegård and Dimitrios Stamou.

6. Nature Chemical Biology, 2015. 11 (3): p. 192-194            
   Front Cover Page 
   Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases
   Jannik Bruun Larsen, Martin Borch Jensen, Vikram K. Bhatia, Søren L. Pedersen, Thomas Bjørnholm, Lars Iversen, Mark Uline, Igal Szleifer, Knud J. Jensen, Nikos S. Hatzakis and Dimitrios Stamou

7. Nature Chemical Biology, 2017. 13: p. 724-729.
   Front Cover Page
   Membrane curvature regulates sorting of GPCRs within the plasma membrane of living cells in a ligand-specific manner
   Kadla R. Rosholm, Natascha Leijnse, Anna Mantsiou, Vadym Tkach, Søren L. Pedersen, Volker F. Wirth, Lene B. Oddershede, Knud J. Jensen, Karen L. Martinez, Nikos S. Hatzakis, Poul Martin Bendix, Andrew Callan-Jones and Dimitrios Stamou

8. Nature Methods, 2014. 11 (9): p. 931-934     
   Nanoscale high content analysis using compositional heterogeneities of single proteoliposomes
   Signe Mathiasen, Sune M. Christensen,, Juan Jose Fung, Soren G. F. Rasmussen, Jonathan F. Fay, Sune K. Joergensen, Salome Veshaguri, David L. Farrens, Maria Byrne, Brian Kobilka, Dimitrios Stamou

Head of group:

Dimitrios Stamou
Professor
stamou@nano.ku.dk 

Postdocs:

Aleksander Cvjetkovic
acv@chem.ku.dk 

Christopher Shuttle
c.shuttle@chem.ku.dk 

Salome Veshaguri
s.veshaguri@gmail.com 

Theresa L. Boye
tlb@chem.ku.dk 

Ph.D students:

Eleftherios Kosmidis
eleftherios.kosmidis@nbi.ku.dk 

Eleftheria Kazepidou
elka@chem.ku.dk 

Gabriele Kockelckoren
Gabriele@chem.ku.dk 

Line Lauritsen
line.lauritsen@nbi.ku.dk 

Mads Møller
mads.moeller@nano.ku.dk 

Jesper Holmkvist
jflh@chem.ku.dk 

M.Sc. students:

Michelle Berling
hfk576@alumni.ku.dk

Postdoctoral Fellows
Brian Lohse, Ph.D.
Faculty, University of Copenhagen

Poul Martin Bendix, Ph.D.
Faculty, University of Copenhagen

Nikos Hatzakis, Ph.D.
Faculty, University of Copenhagen

Lars Iversen, Ph.D.
Senior Research Scientist, Novozymes, Copenhagen

Elena Bertseva
Data engineer, Ørsted, Copenhagen

Garima Ghale
Venture Manager, Health Stream, Toronto

Ph.D. students
Vikram Bhatia
Senior Research Scientist, Symphogen, Copenhagen

Andreas Kunding
CEO and founder, SELMA Diagnostics, Copenhagen

Sune Christensen
Senior Research Scientist, Novozymes, Copenhagen

Li Wei
Scientist, Novozymes, Copenhagen

Christina Lohr
Product Manager Research Metrics, Elsevier, Amsterdam

Andreas Lauge
Patent Attorney Associate, Plougmann Vingtoft, Copenhagen

Jannik Larsen
Faculty, Technical University of Denmark

Asger Tønnesen
Research Assistant, Technical University of Denmark

Kadla Rosholm
Scientist, Sophion, Copenhagen

Signe Mathiasen
Postdoctoral Fellow, Columbia University Medical Center

Samuel Walsh
Adjunkt at Diplomingeniøruddannelse Bioteknologi, Center for Engineering Absalon, Copenhagen

Artu Breuer
Just graduated

Science, 2016. 351 (6280): p. 1469-1473 Full text
Direct observation of proton pumping by a eukaryotic P-type ATPase. S. Veshaguri, S. M. Christensen, G. C. Kemmer, M. P. Møller, G. Ghale, C. Lohr, A. L.Christensen, B. H. Justesen, I. L. Jørgensen, J. Schiller, N. S. Hatzakis, M. Grabe, T. G. Pomorski and D. Stamou

Nature Chemical Biology, 2015. 11 (11): p. 822-825 Full text
Invited Review Commentary for celebrating the 10th anniversary of Nature Chemical Biology Membrane curvature bends the laws of physics and chemistry. L. Iversen, S. Mathiasen, J. B. Larsen and D. Stamou

Nature Chemical Biology, 2015. 11 (3): p. 192-194 (Front cover page) Full text
Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases. J.B. Larsen, M.B. Jensen, V.K. Bhatia, S.L. Pedersen, T. Bjørnholm, L. Iversen, M. Uline, I. Szleifer, K.J. Jensen, N.S. Hatzakis and D. Stamou

Nature Methods, 2014. 11 (9): p. 931-934 Full text
Nanoscale high content analysis using compositional heterogeneities of single proteoliposomes. S. Mathiasen, S.M. Christensen,, J.J. Fung, S.G.F. Rasmussen, J.F. Fay, S.K. Joergensen, S. Veshaguri, D.L. Farrens, M. Byrne, B. Kobilka and D. Stamou

Science, 2014. 345 (6192): p. 50-54 Full text
Single molecule analysis of Ras activation by SOS reveals allosteric regulation via altered fluctuation dynamics. L. Iversen, H.-L. Tu, W.-C. Lin, S. M. Christensen, S. M. Abel, J. Iwig, H.-J. Wu, J. Gureasko, C. Rhodes, R. S. Petit, S. D. Hansen, P. Thill, C.-H. Yu, D. Stamou, A. K. Chakraborty, J. Kuriyan and J. T. Groves.

Nature Nanotechnology, 2012, 7 (1): p. 51–55 Full text
Mixing sub-attolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics. S.M. Christensen, P.Y. Bolinger, N.S. Hatzakis, M.W. Mortensen and D. Stamou

Nature Chemical Biology, 2009. 5 (11): p. 835 Full text
How Curved Membranes Recognize Amphipathic Helices and Protein Anchoring Motifs. N.S. Hatzakis, V.K. Bhatia, J. Larsen, K.L. Madsen, P.Y. Bolinger, A.H. Kunding, J. Castillo, U. Gether, P. Hedegård and D. Stamou

Proceedings of the National Academy of Sciences, 2009. 106 (30): p. 12341 Full text
Quantification of nano-scale intermembrane contact areas using fluorescence resonance energy transfer. P.M. Bendix, M.S. Pedersen and D. Stamou