JOURNAL OF STRUCTURAL BIOLOGY 105. 14&153 (19%)) Secondary Lysosomes as an Integral Part of the Cytoskeleton: A Morphological Study in Rat Kupffer Cells RICHARD A. MARUGG,~ PETER GEHR, AND MARGREET DE LEEUW* Department of Anatomy, University of Berne, CH-3000 Berne 9, Switzerland; and *TN0 Institute for Experimental Gerontology, Rijswijk, The Netherlands Received May 14, 1990, and in revised form August 11, 1990 Rat Kupffer cells contain the three major cytoskeletal components: microfilaments (MF), microtubules (MT), and intermediate filaments (IF) of the vimentin type. Previous cytomagnetometric data obtained from alveolar macrophages and rat Kupffer cells in culture provided evidence that actin filaments contribute to the move- ments of lysosomes. The lysosomal transport in living cells was affected, when the MFs were selectively dis- turbed, whereas the depolymerization of the MTs had no effect on the lysosomal movement measured by cytomag- netometric means. Immunofluorescence and ultrastruc- tural studies of isolated and cultured rat Kupffer cells, presented in this paper, will investigate the relationship between lysosomes and the cytoskeleton. The principal filamentous structure in the peripheral cytoplasm of Kupffer cells in a dense meshwork of actin filaments. The dimension of the meshes combined with the dimensions of lysosomes implies the necessity of either (i) disintegra- tion of the actin filament cross-links, (ii) depolymeriza- tion and redistribution of MF’s, or (iii) a displacement of actin filaments by the lysosomes during the organelle transport. The presence of microtubules in cytoplasmic protrusions and their track from the periphery to the perinuclear region during interphase might play a role in the transport mechanism of lysosomes, the more so be- cause microtubules could often be demonstrated in clos- est association with lysosomes even in the first phase of endocytosis. The distribution pattern of vimentin, found as a dense interconnected framework surrounding the ly- sosomes like a basket, could play a role in positioning the organelles. The dynamic functions of MF’s and MT’s and their multifunctionality led to an adaptive and flexible organization of these filaments which may both be in- volved in lysosomal motion. Our morphological findings, although static in nature, led to the conclusion that all three major cytoskeletal components, microtubules, mi- crofilaments, and the intermediate filament vimentin could participate in the complex interaction between ly- sosomes and the cytoskeleton, including the mechanism of lysosomal transcellular movements and the position- ing of these organelles. The exact force-generating mech- anism and the molecular basis involved in the organelle movements, however, remain obscure. 0 1990 Academic Press, Inc. INTRODUCTION Kupffer cells of the liver represent the largest pop- ulation of stationary mononuclear phagocytes in the body. They are located along the endothelial layer of the liver sinusoids and are mainly involved in the clearance of substances from the blood, including bacteria, endotoxins, viruses, tumor cells, cellular debris, and various specific macromolecules (Jones, 1982). Their function is reflected by the prominent presence of morphological characteristics such as pseudopodia, endocytotic vesicles, and lysosomes both in uiuo and under culture conditions. The move- ments which lead to the fusion of the primary lyso- some with the endocytotic vesicle do not appear to be a random process; they are rather the result of or- chestrated actions. The digested material will be ex- creted or stored within the cell as residual bodies. The most interesting sequences as far as the inter- action between lysosomes and the cytoskeleton is concerned are the internalization and the oriented intracellular movement described as saltatory movement (Rebhuhn, 1971). The oriented move- ments of the phagosomes and primary and secondary lysosomes are a combination of translation and ro- tation of these organelles within the cytosol. The rotational movements can be estimated in a popula- tion of phagocytes in culture by cytomagnetometric means (Gehr et al., 1985; Valberg, 1984; Valberg and Albertini, 1985; Valberg and Butler, 1987). There are several hypotheses about cytoskeleton- based organelle motion: tubulin-dynein, tubulin- kinesin, and actin-myosin interactions have been 1 Present address: Department of Anatomy, University of Zur- ich-Irchel, CH-8057 Zurich, Switzerland. 146 1047-8477/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.