Membrane Biophysics and Biophotonics group (MB&B)
Research at the Membrane Biophysics and Biophotonics group (MB&B) is focused in application of Biophotonic techniques to biological systems. The experimental techniques used in our laboratory are mostly based in multiphoton excitation microscopy, such as fluorescence imaging, fluorescence lifetime imaging (FLIM), fluorescence polarization imaging, fluorescence correlation spectroscopy (FCS), Raster image correlation spectroscopy (RICS) and second harmonic generation microscopy (SHG). With these techniques it is possible to reconstruct 3D images of the specimen of interest and learn about their structural and dynamical aspects. We are particularly interested in exploring the lateral structure of different model and biological membranes, both at equilibrium and not equilibrium situations. Also we are currently applying the aforementioned techniques to understand different physical aspects of skin tissue.
MB&B is associated to MEMPHYS- Center for Biomembrane Physics, a center of excellence supported by The Danish National Research Foundation. Also our group has active participation in a national bioimaging infrastructure (DaMBIC) and NanoCAN a center financed by Lundbeck Foundation devoted to application of nano-devices to fight cancer.
Head of research: Professor with special responsibilities Luis A. Bagatolli
We are performed diverse research activities mostly related with the structural and dynamical aspects of compositionally simple and complex membranes (models and biological relevant membranes) and skin tissue:
Physical (structural and dynamical) properties of ex and in vivo skin tissue.
Transdermal penetration of liposomes in skin tissue.
Mimicking bacterial membranes: interaction of lipopolysacharides and surfactant protein D.
Application of biophysical approaches to evaluate food surface cleaning procedures (SonoSteam).
The action of sphyngomyelinase D from Loxosceles spider venom on model and biological membranes.
Developing novel 3D imaging analysis of giant vesicles displaying phase coexistence.
Lung surfactant membranes. Chronic Obstructive Pulmonary Disease and early biomarkers.
A description of three representative research projects from our lab follows:
1) Ascertaining skin physical properties in vivo by means of Biophotonics techniques
In vivo studies of human skin (including, for example, UV irradiation response, carcinogenesis, drug testing and wound healing) have been limited on ethical and practical grounds. While a number of in vivo skin-related studies have been conducted using animal models (mouse for example), it is still under debate if the experimental observations obtained from these models faithfully capture the inherent characteristics of the human counterpart. The majority of the studies focus on structural and dynamical aspects of human skin use samples from cadaver. Since conditions and time of storage largely influence physical and biochemical aspects of the specimens, it is still debated how accurate it is to establish direct connections between the ex vivo and in vivo situations. Alternatively, skin organotypic cultures represent a valid alternative to “native” skin in vivo studies. However this model is restricted by, among other constraints, their short culture life span, precluding long term studies. A very attractive alternative has been recently proposed to facilitate in vivo studies on human skin. This strategy is based on stable regeneration of normal or diseased human skin in appropriate hosts by means of tissue engineering. Recently, bioengineered human-skin-engrafted immunodeficient mice have been established as an in vivo human skin model system. This model, named “skin humanized mice”, is based on the optimized grafting of a new bioengineered human skin and allows performing a large variety of in vivo skin studies (such as regenerative medicine, gene therapy, genomics and pathology in a human context of homogeneous samples). To our knowledge, however, systematic studies addressing physicochemical aspects of the tissue using skin humanized mice have not been reported so far. For these types of studies, the use of non-invasive biophotonics related approaches are an excellent alternative. Biophotonics related techniques have been described as very promising tool to perform functional imaging of human skin in vivo. Among others, multiphoton excitation fluorescence microscopy (MPEFM and its related techniques) provides an excellent choice for both ex vivo and in vivo imaging. In particular MPEFM has the intrinsic ability to provide inherent spatial resolution and optical sectioning at greater depths (up to 900 micrometers) with minimal photobleaching and photodamage. These features make MPEFM ideal to study thick specimens such as tissues. Combinations of multiphoton excitation based techniques, such as fluorescence lifetime imaging (FLIM), fluorescence correlation spectroscopy (FCS), Raster image correlation spectroscopy (RICS) and second harmonic generation microscopy (SHG), offer a very powerful experimental tool to explore particular structural and dynamical aspects of biological specimens. Examples of measurable physicochemical parameters using these experimental techniques are: i) diffusion coefficients of model substances (as well as local number of particles), ii) local proton activity and iii) local polarity. In this particular project we intend to apply these techniques to systematically study in vivo 3D maps (constructed with information obtained at the different tissue strata) of the abovementioned parameters using the skin-humanized model. These in vivo studies will focus first in characterizing the architecture and properties of skin under normal conditions and, subsequently, performing comparative studies under controlled diseased conditions. Another related project in our lab is dedicated to study transdermal delivery of liposomes as drug carriers. Last but not least, a related project in collaboration with Unilever is currently performed in our laboratory.
2) Effect of “membrane active” spider venom’s components on biological membranes
The phenomenon of envenomation has been known and studied since antiquity. Venom biochemistry has had a principal role in the identification of the molecular species involved in toxicity and some bulk aspects thereof, such as associated enzymatic activities and the structure of the toxins. These classical approaches have been extended to “venomics” projects which will continue to contribute toxins but will scarcely clarify mechanistic principles. Biophysical approaches on the other hand, refine and complement the biochemical aspects with essential information about structure and dynamics at different levels of organization, which may be affected by particular agents. These perspectives are generally seldom combined in experimental systems representative of particular toxin related phenomenon. Loxosceles spiders have a worldwide distribution and are considered one of the most medically important groups of spiders. Envenomation (loxoscelism) can result in dermonecrosis and, less commonly, a systemic illness that can be fatal. Even though the characteristic dermonecrotic lesion results from the direct effects of the venom on the cellular and basal membrane components, still the mechanism of venom action is multifactorial and incompletely understood. Loxosceles spiders have venoms with a toxic component for mammals with a rare enzymatic activity, termed sphingomyelinase D (SMD). SMD is a relatively abundant protein component of pure Loxosceles venom, comprising a significant portion of venom protein along with components of lower molecular weight. Sphingomyelinases have a molecular mass of approximately 33 kDa and catalyze the cleavage of sphingomyelin (SM), releasing ceramide-1-phosphate (Cer-1-P) and choline. Sphingomyelins, in turn, are integral components of many cell membranes (such as erythrocytes and cells of the vascular epithelium), and although the natural targets of SMD action are not known, there is convincing evidence which points to the role of the enzymatic activity in the initiation of events which may lead to extensive necrosis and, on occasion, severe systemic poisoning. Cer-1-P is one of the simplest sphingophospholipids found in biological membranes and contrary to SM, its average concentration is usually very small. Recent evidence suggests an important role of this lipid as a novel second messenger with important roles in (intra)cellular processes as diverse as potassium channel function, phagocytosis, inflammatory responses, and cell survival and tumorigenesis. The mechanism of action is unknown, however it is speculated that Cer-1-P function through the activation/recruitment of effector proteins by direct interaction, such as PLA2, or by a modulation of membrane properties such as curvature and electrostatics. Very scarce number of studies exploring the physical chemistry, and particularly the phase behavior and electrostatics of this bioactive lipid (i.e. Cer-1-P) has been reported. In addition and to our knowledge, studies of membrane physical aspects during SMD action are not reported in the literature. The main aim of this project is to evaluate the effect of the interaction of SMD, obtained from Loxosceles spiders, on membrane systems at different compositional complexity levels (i.e. from simple lipid model mixtures to native biological membranes). The experimental approach is based in a multidisciplinary strategy that use biophysical, biochemical and cell biology related methods. For instance, biochemical aspects (enzyme kinetics) are planned to be characterized and correlated with structural and dynamical aspects of different model membranes upon SMD interactions. The project also aims to establish a correlation between membrane structure and cellular response after cell/SMD interaction. Since systemic loxoscelism produce massive hemolysis (1), red blood cells (that in turn contains in the membrane a high proportion of the natural substrate of SMD, i.e. sphyngomyelin)- SMD interaction will be explored. As other factors have been noted to contribute to the severity of tissue damage in loxoscelims, such as local abundance of adipose tissue (1), adypocites-SMD interactions will be also explored. The types of studies that will be performed in cells include mostly membrane biophysics-based approaches. In the vast literature on venoms there are scarce reports combining biochemistry and biophysics (both in model membranes and cells), using the same experimental approaches. In our opinion this particular multidisciplinary strategy will reveal clues about the unexplored mechanisms of SMD at different molecular and supramolecular levels.
3) Identification and characterization of early chronic obstructive pulmonary disease (COPD) biomarkers from respiratory airways: impact in early COPD diagnosis.
Nearly 15% of Danes older than 45 years can be diagnosed with symptoms of chronic obstructive pulmonary disease (COPD), and by the year 2020 it is estimated that COPD will be fifth among burdens to societies on a worldwide scale. It is already a large socioeconomic burden, and in Denmark alone, it is yearly associated with 23.000 hospitalizations and 10 deaths every day. Its prevalence, manifestation and progression are clearly associated with smoking; and the most important single step COPD patients can do to slow down disease progression is to quit smoking. Unfortunately, COPD is most often diagnosed when the disease state is too progressive to allow for remission. Therefore we are in need of biomarkers that allow for early diagnosis of COPD or biomarkers that defines certain smokers as people with a high risk of developing COPD.
Pulmonary surfactant is the surface active proteo-lipid material that is produced in large amounts by type II pneumocyte cells in the lower respiratory airways. It creates a unique interface that a) helps to regulates O2/CO2 gas exchange at the alveolar cell surface, b) reduces surface tension and maintains lung volumes at the end of the expiration and c) acts as an anti-microbial layer. Surfactant exerts it effects not only in the lung, but also in the upper airways. Pulmonary Surfactant is composed mainly of phospholipids (PL’s) (representing 80-90% of the surfactant molecular mass) including surfactant proteins (SP), -A, -B, -C, and -D. These proteins play important roles in surfactant homeostasis and host defence. SP-A and -D are relatively abundant, hydrophilic proteins (with relatively weak tension-active qualities) that bind complex carbohydrates, including those on the surface of bacteria, viruses, and other lung pathogens. They act as opsonins by increasing the microbial uptake by alveolar macrophages and by modulating the subsequent activation of alveolar macrophages. In contrast, SP-B and -C are small, hydrophobic proteins that play a critical role by enhancing the rate of spreading and stability of phospholipids. The last is crucial to regulate changes in surface tension within the lungs during respiration. Despite the very obvious and straightforward relationship between normal lung function and surfactant integrity, there is a dearth of information about abnormalities of lung surfactant and COPD. Most studies have focused on inflammatory mediators, infiltrating cells and enzymes associated with remodeling of the lung tissue. An alternative approach is to think of surfactant inactivation, due to type II pneumocyte malfunctioning and stress, as a direct cause of COPD and the subsequent progressive inflammation and remodeling. Our working hypothesis is that smoking induced COPD may, at a very early point before any symptoms appear, affect the composition and function of both surfactant and mucus in the lung and upper airways. By studying the aforementioned patterns (abnormal concentration of the surfactant components, surfactant supramolecular structure and dynamics, stress signalling molecules) we want to identify and correlate potential COPD biomarkers among nasal lavages, broncho-alveolar lavages and condensed exhaled air samples from smokers/non-smokers patients (males and females).
Selected publications from Luis Bagatolli:
1) M. Fidorra, A. Garcia, J. Ipsen, S. Hartel and L.A. Bagatolli “Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach”, 2009 Biochim. Biophys. Acta 1788(10):2142-2149 view►
2) U. Bernchou, J. R. Brewer, H. S. Midtiby, J. H. Ipsen, L. A. Bagatolli and A. C. Simonsen, “Texture of lipid bilayer domains” 2009, Journal of the American Chemical Society 131(40):14130-1431. view►
3) D.C. Carrer, C.Vermehren and L.A Bagatolli. 2008. “Pig skin structure and transdermal delivery of liposomes: A two photon microscopy study ”. J. Controlled Release, 132(1):12-20. view►
4) L-R Montes, A. Alonso, F. Goñi and L.A Bagatolli. 2007. “Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions ”. Biophys. J 93(10):3548-3554. view►
5) Plasencia-Gil, L. Norlen,and L.A: Bagatolli. 2007 “Direct visualization of lipid domains in human skin stratum corneum’s lipid membranes: effect of pH and temperature” Biophys. J 93(9):3142-3155 view►
6) Bagatolli L.A. 2006. “To see or not to see: lateral organization of biological membranes and fluorescence microscopy” Review article. Biochim Biophys Acta 1758:1541-1556.view►
7) Bernardino de la Serna J., J. Perez-Gil, A. C. Simonsen and L.A. Bagatolli. 2004. “Cholesterol rules: direct observation of the coexistence of two fluid phases in native pulmonary surfactant membranes at physiological temperatures” J. Biol. Chem. 279:40715-40722.view►
8) Dietrich C., L. A. Bagatolli, Z. Volovyk, N. L. Thompson, M. Levi, K. Jacobson and E. Gratton. 2001. “Lipid Rafts Reconstituted in Model Membranes”. Biophys. J. 80: 1417-1428.view►