Subpopulations of microtubules with differential sensitivity to nocodazole: role in the structural organization of the Golgi complex and the lysosomal system

Abstract

The role of cytoplasmic microtubules (MTs) in the structural organization of the Golgi complex and the lysosomal system was studied in L929 mouse fibroblasts using a combination of cytochemical and electron microscopic methods. Immunocytochemical staining demonstrated a radiating pattern of MTs, originating in the juxtanuclear region containing the centrioles. The stacks of Golgi cisternae with associated vesicles and tubules (the trans Golgi network), as well as most of the endosomes and lysosomes, were also located in and immediately around this region. Wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) bound to the plasma membrane at 4 degrees C. After warming to 37 degrees C, the conjugate was rapidly internalized by endocytosis and via small vesicles transferred to endosomes and lysosomes in the juxtanuclear region. Slightly later it was also found in the trans Golgi network. A weaker but otherwise similar staining was obtained if the conjugate was applied to fixed and permeabilized cells. Treatment with 3.0 microM nocodazole induced depolymerization of MTs and rounding up of the cells. At the same time, the Golgi complex was disorganized with dispersion of its stacks of cisternae throughout the cytoplasm and atrophy of the trans Golgi network. The lysosomes gathered around the dispersed cisternal stacks and the endosomes became fewer in number. Moreover, the uptake of WGA-HRP was markedly inhibited. Following withdrawal of the drug, MTs reformed and the structural organization of the cell was again normalized. If the recovery was allowed to take place in the presence of low concentrations of nocodazole (0.03 or 0.15 microM), MTs of short and medium length were formed, the latter showing a curly path. In parallel, a partial or nearly complete normalization of cell morphology occurred. Internalization and transport of WGA-HRP labeled membrane to the lysosomes and the trans Golgi network was also resumed. In contrast, the cells were not able to pass through mitosis in a normal manner and multimicronucleated cells accumulated. Taken together, the findings indicate that L929 cells contain a subset of nocodazole-resistant MTs which play an important role in the structural organization of the cell, especially with regard to the Golgi complex and the lysosomal system. They support recent notions that there exists subpopulations of MTs with different physicochemical properties and functions.