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Biophysical studies of pigment transport in frog melanophores: impedance measurements and advanced microscopy analyses
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Small proteins, other molecules and large organelles are frequently transported from one location to another within cells. These transports employ cytoskeletal networks and enable cells to maintain regions with different functions and attain an asymmetrical shape.

The aim of this work was to explore biophysical methods for monitoring intracellular transport processes and associated structural changes. For these studies we have used pigment cells, melanophores, from the African clawed frog Xenopus laevis. In response to external stimuli, these cells can change colour by redistributing pigment granules in the cytoplasm.

Transparent "cell clinics" equipped with gold electrodes were developed for impedance studies. The results show that impedance measurements at different frequencies not only can be used to monitor cell attachment and spreading, but also events like pigment aggregation. Significant F-actin breakdown and a cell area decrease may explain the impedance decrease seen during latrunculin-induced aggregation. In aggregation induced by melatonin there was, however, a small increase of the cell area, no F-actin breakdown and still lowered impedance, indicating that some other, likely intracellular mechanism is involved. In addition, confocal laser scanning microscopy (CLSM) studies showed that aggregation was associated with an increase in the cell height, more prominent for latrunculin than for melatonin. This height increase did not seem to involve influx of water through aquaporin channels at the cell membrane, or newly formed or remodelled microtubules in the cells.

Besides impedance measurements, Image Correlation Spectroscopy (ICS) was applied to analyse pigment aggregation. The study shows for the first time that ICS can be used to analyse aggregation of non-fluorescent particles and suggests that the method may provide new information on the state of aggregation of granules in pigment cells.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 803
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26657Local ID: 11222ISBN: 91-7373-491-8 (print)OAI: oai:DiVA.org:liu-26657DiVA: diva2:247206
Public defence
2003-09-26, Victoriasalen, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-17Bibliographically approved
List of papers
1. The cell clinic: closable microvials for single cell studies
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2002 (English)In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 4, no 3, 177-187 p.Article in journal (Refereed) Published
Abstract [en]

We present the development of a cell clinic. This is a micromachined cavity, or microvial, that can be closed with a lid. The lid is activated by two polypyrrole/Au microactuators. Inside the microvials two Au electrodes have been placed in order to perform impedance studies on single or a small number of cells. We report on impedance measurements on Xenopus leavis melanophores. We could measure a change in the impedance upon cell spreading and identify intracellular events such as the aggregation of pigment granules. The electrical data is correlated to optical microscopy.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26486 (URN)10.1023/A:1016092228965 (DOI)11042 (Local ID)11042 (Archive number)11042 (OAI)
Note

The original publication is available at www.springerlink.com: Edwin WH Jager, Charlotte Immerstrand, Kajsa Holmgren Peterson, Karl-Eric Magnusson, Ingemar Lundström and Olle Inganäs, The cell clinic: Closable microvials for single cell studies, 2002, Biomedical microdevices (Print), (4), 3, 177-187. http://dx.doi.org/10.1023/A:1016092228965 Copyright: Springer Verlag (Germany) http://www.springerlink.com/

Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13
2. Altered impedance during pigment aggregation in Xenopus laevis melanophores
Open this publication in new window or tab >>Altered impedance during pigment aggregation in Xenopus laevis melanophores
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2003 (English)In: Medical and Biological Engineering and Computing, ISSN 0140-0118, E-ISSN 1741-0444, Vol. 41, no 3, 357-364 p.Article in journal (Refereed) Published
Abstract [en]

Melanophores are dark-brown pigment cells located in the skin of amphibia, fish and many invertebrates. The skin colour of these organisms is regulated by the translocation of pigment organelles, and the pigment distribution can be altered by external stimuli. The ability to change colour in response to stimuli makes these cells of interest for biosensing applications. It was investigated whether pigment aggregation in Xenopus laevis melanophores can be detected by impedance measurements performed in transparent microvials. The results show that cell attachment, cell spreading and pigment aggregation all resulted in impedance changes, seen particularly at the highest frequency tested (10 kHz). The mechanisms behind the impedance changes were investigated by the addition of latrunculin or melatonin, both of which cause pigment aggregation. The latrunculin-induced aggregation was associated with cell area decrease and filamentous actin (F-actin) breakdown, processes that can influence the impedance. Lack of F-actin breakdown and an increase in cell area during melatonin-induced aggregation suggest that some other intracellular process also contributes to the impedance decrease seen for melatonin. It was shown that impedance measurements reflect not only cell attachment and cell spreading, but also intracellular events.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26494 (URN)10.1007/BF02348443 (DOI)11050 (Local ID)11050 (Archive number)11050 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. Height changes associated with pigment aggregation in Xenopus laevis melanophores
Open this publication in new window or tab >>Height changes associated with pigment aggregation in Xenopus laevis melanophores
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2004 (English)In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 24, no 3, 203-214 p.Article in journal (Refereed) Published
Abstract [en]

Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgC12-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-31125 (URN)10.1007/s10540-005-2581-6 (DOI)16209129 (PubMedID)16859 (Local ID)16859 (Archive number)16859 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically approved
4. Image correlation spectroscopy for quantification of pigment aggregation
Open this publication in new window or tab >>Image correlation spectroscopy for quantification of pigment aggregation
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In the skin of amphibia, fish, and other organisms, the distribution of the pigment granules within melanophores regulates skin colour. In this study we have applied Image Correlation Spectroscopy (ICS; Petersen et al. 1998; Petersen et al. 1993) to monitor the aggregation of pigment granules. Normally in ICS, images from confocallaser scanning rnicroscopy are used to calculate autocorrelation functions from which the density of fluorescent particles in the image, like membrane receptors, can be obtained. The present study differs from traditional ICS in that the images are obtained by light microscopy and then Gaussian filtered to give the particles the appropriate intensity profile required for ICS analysis. ICS appears to be more sensitive than particle counting and image mean intensity for quantification of the degree of pigment aggregation. This study demonstrates for the first time that ICS can be used to analyse the aggregation of non-fluorescent particles, such as pigment granules in Xenopus laevis melanophores.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84684 (URN)
Available from: 2012-10-17 Created: 2012-10-17 Last updated: 2012-10-17Bibliographically approved

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