Variations of endocytic and of lysosomal functions during the cell cycle have been investigated in synchronized hepatoma cells (derived from Morris hepatoma 7288c) by following the cellular uptake of horseradish peroxidase, dextran (mol wt, 70,000), and chloroquine .Cell fractionation and cytochemistry show that in asynchronously growing cells exposed for 1 h to 5 mg/ml peroxidase, the bulk of the enzyme taken up by the cells is found in phagosomes . By using the same experimental system with synchronized HTC cells, large variations of endocytosis are observed during the cell cycle . Peroxidase uptake is lowest during mitosis, increases 5-10 times during G 1 phase, reaches a plateau, and finally decreases at the end of S phase and during G2 phase . A similar evolution is observed for the uptake of dextran (0 .5 or 1 mg/ml), but it is likely that a significant part of the polysaccharide is still associated with the pericellular surface after 1 h . Moreover, dextran is transferred more slowly than peroxidase to lysosomes . Cellular accumulation of chloroquine is related to intralysosomal pH or to the buffering capacity of lysosomes . Our results show that this drug is taken up more rapidly during G 1 and S phases while the rate of accumulation is lowest in mitotic cells .
A method for the detection and characterization of clusters of particles observed in section with the electron microscope is presented. Cluster analysis is performed by the division method described by Berthet et al. (1976). Starting from a single cluster, profiles from each electron micrograph are successively classified in sets containing an increasing number of clusters. The decrease in the mean free distance, lambda, between profiles in the clusters, is used for terminating the subdivision procedure. The function relating the mean free distance with the number of clusters is evaluated in each subdivision set. The actual number of clusters is selected on the basis of the slope of that function, at a point where lambda has a value close to the average profile diameter. The method assumes a convex shape for the clusters; the salient feature is that it provides a physical delineation of clusters in the section. Hence, an evaluation of some characteristics of clusters in the three-dimensional sample may be obtained by using standard stereological procedures. Characterization of the volume to which the individual particles of a population are eventually restricted can as a result be performed. Practical problems in the acquisition of the data needed for cluster analysis are discussed and a system using for that purpose a Quantimet 720 image analyser in a basic configuration, connected on line with a PDP 11/10 minicomputer, is presented. Application of the method is illustrated by the analysis of lysosomes in cultured hepatoma (HTC) cells, at the end of mitosis and during the S phase. Cluster analysis shows that in cells actively synthesizing DNA they are grouped in clusters representing 5.7% of the cellular volume. Moreover, the average number of particles per cluster falls from a minimum of thirteen at mitosis to only six at the S phase.
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