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      In vivo Confocal Laser Scanning Microscopy and Micropuncture in Intact Rat

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          Abstract

          Background: Intravital microscopy theoretically provides the optimal conditions for studying specific organ functions. However, the application of microscopy in intact organs in vivo has been limited so far due to technical difficulties. The purpose of this study was to establish a method of in vivo confocal laser scanning microscopy (CLSM) for the study of endocytosis in proximal tubules of intact kidney. Methods: The left kidney of rats placed on a modified microscope stage was exposed and stabilized in a thermostatically controlled cup. The stage was then attached to an upright confocal microscope. Surface proximal tubules were microinfused with fluorescent albumin or transferrin. Single or time-series images of microinfused proximal tubules were recorded in reflection and/or fluorescence mode. Results: The stability of the kidney and the resolution of images were sufficient to visualize intracellular vesicles. Albumin and transferrin were initially observed at the brush border, then later internalized by proximal tubules and accumulated in lysosomes over a time period of 15 min. Furthermore, fusion of vesicles was observed in time-lapse images. Conclusion: These results show that in vivo CLSM in intact kidney may be an excellent method to evaluate proximal tubular endocytosis and ligand trafficking.

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          Most cited references 15

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          Distinct Membrane Domains on Endosomes in the Recycling Pathway Visualized by Multicolor Imaging of Rab4, Rab5, and Rab11

          Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.
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            Two-photon excitation fluorescence microscopy.

            Two-photon fluorescence microscopy is one of the most important recent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast images and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine.
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              Single-molecule imaging of EGFR signalling on the surface of living cells.

              The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.
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                Author and article information

                Journal
                NEE
                Nephron Exp Nephrol
                10.1159/issn.1660-2129
                Cardiorenal Medicine
                S. Karger AG
                1660-2129
                2005
                January 2005
                14 January 2005
                : 99
                : 1
                : e17-e25
                Affiliations
                Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark
                Article
                81794 Nephron Exp Nephrol 2005;99:e17–e25
                10.1159/000081794
                15637463
                © 2005 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 9, References: 25, Pages: 1
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/81794
                Categories
                Original Paper

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