Current Projects

 Anomalous diffusion of proteins due to molecular crowding (Daniel Banks)

 Ran-GTP/Ran-GDP gradient across the nuclear membrane (Asmahan Abu-Arish)

 Membrane insertion of the pro-apoptotic protein Bax (Dmitri Satsoura)

 Interaction of focused laser beam and erythrocyte membrane (Felix Wong)

 Molecular Beacons (James Jonkman)


Study of anomalous diffusion of proteins due to molecular crowding


Daniel Banks

Daniel is investigating the diffusion of proteins in crowded environments using a model system constituted of polymers and proteins.

  • Why study anomalous diffusion in crowded environments?

    • A cell depends on the diffusion of many of its proteins for its proper functioning. Much study is being done on the function of individual proteins, and how they are transported in the cell. Cells are full of obstacles. Membranes, organelles, and other proteins obstruct diffusion. To properly understand what is happening inside a cell, the possibility of anomalous diffusion must be taken into account.


  • What is anomalous diffusion?

    • An uninhibited particle in solution diffuses according to Brownian motion, but in complicated environments the diffusion may become anomalous. While the mean square displacement (MSD) of a particle following normal diffusion is proportional to time: <r2(t)> ~ t, anomalous diffusion is defined by: <r2(t)> ~ ta, with a<1. As a result, the apparent diffusion coefficient (D ~ <r2(t)>/t) is a function of time.

    The diffusion rate of a particle motion obstructed by obstacles depends on the size, number, and random positioning of the obstacles. This makes an analytical determination of a general solution to this problem enormously difficult, if not impossible. Instead, Monte Carlo simulations and other numerical methods are used. The result is of the form expected for anomalous diffusion: <r2(t)> ~ ta, with the anomalous coefficient a depending on the concentration, size, mobility and reactivity of the obstacles.

    In case of anomalous diffusion, autocorrelation curves recorded with FCS will have the form:




    as opposed to:

    ,




    for normal 3-D diffusion.


  • What is the model system in this study?

    • Our simple model uses a fluorescently labeled protein in solution with non-fluorescent obstacles (dextrans of different sizes). We can vary the concentration and size of the obstacles. We can measure the diffusion coefficient and the anomalous exponent characterizing the obstructed diffusion. We want to understand the diffusion process in our model system, and to determine how the size and concentration of obstacles influence this process. Our results will include a range of data that should be useful for comparison with in vivo studies of anomalous protein diffusion.


References:

[1] Bunde, A.; Havlin, S., Eds. (1995) Fractals and Disordered Systems. 2nd edit., pp. 115- 175, Springer-Verlag, Berlin.
[2] Saxton, Micheal J. (1994) Anomalous Diffusion Due to Obstacles: A Monte Carlo Study. Biophysical Journal, 66:394-401.
[3] Wachsmuth, M., Waldeck, W. & Langowski, J. (2000) Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy. J. Mol. Biol. 298:677-689.

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Ran-GTP/Ran-GDP Gradient across the Nuclear Membrane

Asmahan Abu-Arish

To help improve our understanding of the mechanism of nuclear transport Asmahan is characterizing the distribution of Ran inside cells.

  • Why study the protein Ran?

    • The GTP-binding protein Ran is the major regulator of nucleo-cytoplasmic transport across the nuclear pore complex (NPC). It has been suspected for a long time that there is a gradient from Ran-GDP to Ran-GTP when going from the cytoplasm to the nucleus. This gradient is proposed to drive both nuclear import and export [1]. It has been recently visualized using a fluorescent protein construct [2].

  • What is the Ran construct used in this study?

    • The group of Dr. Karsten Weis designed a biological construct (called YRC) formed of a Ran-binding domain (RBD) associated with two fluorophores (YFP & CFP).YRC has low affinity for Ran-GDP but binds Ran-GTP in an extended conformation. This construct produces fluorescence resonance energy transfer (FRET) in closed conformation, that is, only when it is free in solution. YRC-FRET can then be used as a probe to detect the Ran-GTP/ Ran-GDP gradient.

  • How is the gradient of Ran-GTP/ Ran-GDP characterized?

    • We are using Fluorescence Correlation Spectroscopy (FCS) to measure the local concentration and detect the dynamics of the conformational changes of the probe in the presence/absence of Ran-GTP/Ran-GDP.

    With the help of Vera Matulovic, Asmahan is using FCS to investigate the photophysical behavior of individual fluorescent particles with respect to both temperature and pH.

References:

[1] T. D. Allen, J. M. Cronshaw, S. Bagley, E. Kiseleva & M. W. Goldberg. (2000) J. Cell Sci. 113, 1651-1659.
[2] P. Kalab, K. Weis, R. Heald. (2002) Science. 295, 2452-2456.

Asmahan has also worked on finding the three-dimensional structure of Agrobacterium T-complex using transmission electron microscopy:

A. Abu-Arish, D. Frenkiel-Krispin, T. Fricket, Tavi Tzfira, V.Citovsky, S.G. Wolf, and M. Elbaum, 2004, Three-dimensional reconstruction of Agrobacterium VirE2 Protein with Single-stranded DNA, JBC. 279, 25359-63.

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Membrane insertion of the pro-apoptotic protein Bax


Dmitri Satsoura

Dmitri studies the interaction between the protein Bax and the mitochondrial membrane. The aim of the study is the characterization of the pore-forming behaviour of Bax during apoptosis.

  • Why study the insertion of Bax in the mitochondrial membrane?

    • The Bax protein is a pro-apoptotic member of the Bcl-2 family of proteins that regulate the mitochondrial apoptosis pathway. Bax plays a pivotal role in the regulation of release of cell death-activating factors from mitochondria. The exact details of the mechanism of escape of these factors across the outer mitochondrial membrane remain unclear.

  • What is the system used in this study to investigate the interaction between Bax and membranes?

    • Traditionally, small or large unilamellar vesicles have been used in this context as in vitro systems to study such interactions. In this work, we use giant unilamellar vesicles (GUVs), which due to their large radii have minimum membrane curvature and thus are better models for cell membranes than conventional vesicles. Moreover, GUVs are suitable for optical microscopy thus permitting the study of protein-membrane interactions by single-molecule spectroscopy.

  • How are the giant unilamellar vesicles (GUVs) prepared?

    • GUVs are prepared by electroformation. In this method, lipids are spin-coated on an ItO coated glass slide, and an alternative electric field is applied, causing the formation of GUVs. The figure below shows a typical GUV obtained by electroformation.


    Figure: the lipid used was 1-stearoyl-2-linoleoyl-sn-glycero-3-phospholcholine or SLPC, and the GUV was prepared in pure water at 10 Hz and 1V.

This work is done in collaboration with Dr. David Andrews and Dr. Brian Leber.

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Interaction Between Focused Laser Beam and Red Blood Cell Membrane


Felix Wong
Felix is studying the interaction of laser beams with biological membranes, and in particular with the membrane of red blood cells.

  • Why study the interaction between light and biological systems?

    • Due to various optical properties of lasers, interactions between laser beams and cells can be used in medicine and biological research.
    1. Optical Tweezers are a good example of such an application. The forces of radiation pressure created by the laser can help trap and manipulate cells and biological molecules in piconewton range [2].
    2. Another important application is photodynamic therapy (PDT), a photosensitizer is used in conjunction with laser light to kill tumor cells by creating singlet oxygens or free radicals [2].

  • Why focus on RBCs?

    • When using lasers to study cells (for example during optical tweezers, FRAP, and FCS experiments), it is important to damage cells as little as possible. Understanding the photo-damage done by focused laser beams to RBC membranes in the presence of a fluorophore is a first step towards the study of membrane elasticity using FCS, for which we need to obtain a correlation function by exciting fluorescent particles diffusion close to the membrane with a focused laser [3].

  • So does the laser damage the membrane?

    • Yes, it clearly does. After a few minutes of irradiation, the cell bursts through a well-known process called hemolysis (or, since in this case it is caused by light, hemolysis. Also, very quickly after irradiation started, a small imprint is observed on the membrane at the laser focus [4]. We try to understand the reasons for the creation of this imprint.

    Fig. 1: Photoinduced damage on RBC. A) Before illumination. B) Imprint creation after laser illumination started. C) RBC turned to spherical in shape. D) Cell bursts.

References:

[1] Ashkin, A., Optical trapping and manipulation of neutral particles using lasers. Proceedings of the National Academy of Sciences of the United States of America, 1997. 94(10): p. 4853-4860.
[2] Hopper, C., Photodynamic therapy: a clinical reality in the treatment of cancer. Lancet Oncol, 2000. 1: p. 212-9.
[3] Fradin, C., et al., Fluorescence Correlation Spectroscopy Close to a Fluctuating Membrane. Biophys. J., 2003. 84(3): 2005-2020.
[4] Bloom, J.A. and W.W. Webb, "Photodamage to Intact Erythrocyte-Membranes At High Laser Intensities - Methods of Assay and Suppression," Journal of Histochemistry & Cytochemistry 32(6), 608-616, 1984.

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Molecular Beacons


James Jonkman
James collaborates with the group of Dr. Ying-fu Li on this project. He is helped in his investigation of the dynamics of molecular beacons by Aleksandra Dabkowska.

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