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Biomedical subjects

Alois Sonnleitner

Publications and source records attributed to Alois Sonnleitner.

7 recordsLinked to original sources
PubMed2005

The significance of lipid composition for membrane activity: new concepts and ways of assessing function.

In the last decade or so, it has been realised that membranes do not just have a lipid-bilayer structure in which proteins are embedded or with which they associate. Structures are dynamic and contain areas of heterogeneity which are vital for their formation. In this review, we discuss some of the ways in which these dynamic and heterogeneous structures have implications during stress and in relation to certain human diseases. A particular stress is that of temperature which may instigate adaptation in poikilotherms or appropriate defensive responses during fever in mammals. Recent data emphasise the role of membranes in sensing temperature changes and in controlling a regulatory loop with chaperone proteins. This loop seems to need the existence of specific membrane microdomains and also includes association of chaperone (heat stress) proteins with the membrane. The role of microdomains is then discussed further in relation to various human pathologies such as cardiovascular disease, cancer and neurodegenerative diseases. The concept of modifying membrane lipids (lipid therapy) as a means for treating such pathologies is then introduced. Examples are given when such methods have been shown to have benefit. In order to study membrane microheterogeneity in detail and to elucidate possible molecular mechanisms that account for alteration in membrane function, new methods are needed. In the second part of the review, we discuss ultra-sensitive and ultra-resolution imaging techniques. These include atomic force microscopy, single particle tracking, single particle tracing and various modern fluorescence methods. Finally, we deal with computing simulation of membrane systems. Such methods include coarse-grain techniques and Monte Carlo which offer further advances into molecular dynamics. As computational methods advance they will have more application by revealing the very subtle interactions that take place between the lipid and protein components of membranes - and which are so essential to their function.

Animals↗
PubMed2005

DIC image reconstruction on large cell scans.

The width of the emission spectrum of a common fluorophore allows only for a limited number of spectral distinct fluorescent markers in the visible spectrum, which is also the regime where CCD-cameras are used in microscopy. For imaging of cells or tissues, it is required to obtain an image from which the morphology of the whole cell can be extracted. This is usually achieved by differential interference contrast (DIC) microscopy. These images have a pseudo-3D appearance, easily interpreted by the human brain. In the age of high throughput and high content screening, manual image processing is not an option. Conventional algorithms for image processing often use threshold-based criteria to identify objects of interest. These algorithms fail for DIC images as they have a range from dim to bright with an intermediate intensity equal to the background, so as to produce no clear object boundary. In this article we compare different reconstruction methods for up to 100 MB-large DIC images and implement a new iterative reconstruction method based on the Hilbert Transform that enables identification of cell boundaries with standard threshold algorithms.

Algorithms↗
PubMed2004

Single-molecule reader for high-throughput bioanalysis.

We report here the development of a device for single-molecule imaging on large surface areas. A CCD camera operated in time delay and integration mode is synchronized with the movement of a sample scanning stage, enabling continuous data acquisition. Experiments on single fluorescent lipid molecules in supported lipid bilayers and on stained living cells demonstrate the capabilities of the method. Areas of up to 5 x 5 mm(2) were recorded within 11 min at a pixel size of 129 nm.

Biological Assay↗
PubMed2004

Co-localization of CD3 and prion protein in Jurkat lymphocytes after hypothermal stimulation.

While long-term effects of temperature treatment in respect of, e.g., gene-expression and cellular function have already been studied in some detail, nothing is known on the physiological responses of lymphocytes during short-term hypothermal shifts. In this report, we characterized the effects of such a stimulation using the human lymphocyte cell line Jurkat E6.1 and present evidence that warming from 4 to 37 degrees C for only 2 min is sufficient to cause co-localization of CD3, prion protein and the lipid-raft ganglioside GM1 paralleling lymphocyte activation as observed by Ca(2+) mobilization and mitogen-activated protein kinase-phosphorylation.

Actins↗
PubMed2003

A rugged energy landscape mechanism for trapping of transmembrane receptors during endocytosis.

Efficient clathrin-mediated endocytosis of transmembrane receptors requires that clathrin-coated pits retain the receptors long enough to allow vesicle formation and internalization. In many cases, however, the receptors can exhibit mean lifetimes in coated pits much shorter than the lifetime of the pit at the plasma membrane. A rugged energy landscape for binding, which produces a broad distribution of residence times, ensures a significant probability of times much greater than the mean and would allow efficient trapping of proteins in these cases. We used fluorescence correlation spectroscopy and total internal reflection microscopy to measure the kinetics of movement of a C5a receptor-yellow fluorescent protein fusion in living cells. These experiments demonstrate that clusters of trapped receptors exhibit fluctuations in fluorescence intensity that vary in time scale over 2 orders of magnitude. Most of the variation in intensity is likely due to the motion of the receptors in the plane of the plasma membrane, although it is not possible to rule out a small contribution from motion orthogonal to the plane of the membrane. The broad time scale distribution of the intensity fluctuations is consistent with a rugged energy landscape mechanism for trapping of the receptors. This mechanism, which allows efficient trapping to coexist with rapid exchange, may also be relevant to other biological processes involving binding in heterogeneous chemical environments.

Antigens, CD↗
PubMed2002

Structural rearrangements in single ion channels detected optically in living cells.

Total internal reflection fluorescence microscopy was used to detect single fluorescently labeled voltage-gated Shaker K(+) channels in the plasma membrane of living cells. Tetramethylrhodamine (TMR) attached to specific amino acid positions in the voltage-sensing S4 segment changed fluorescence intensity in response to the voltage-driven protein motions of the channel. The voltage dependence of the fluorescence of single TMRs was similar to that seen in macroscopic epi-illumination microscopy, but the exclusion of nonchannel fluorescence revealed that the dimming of TMR upon voltage sensor rearrangement was much larger than previously thought, and is due to an extreme, approximately 20-fold suppression of the elementary fluorescence. The total internal reflection voltage-clamp method reveals protein motions that do not directly open or close the ion channel and which have therefore not been detected before at the single-channel level. The method should be applicable to a wide assortment of membrane-associated proteins and should make it possible to relate the structural rearrangements of single proteins to simultaneously measured physiological cell-signaling events.

Animals↗