| ObjectiveLysosomes generally were described as spherical organelles which included more than 70 hydrolytic enzymes. Lysosomes were mobile and dynamic organelles. They move actively through the cytoplasm to carry out their functions in intracellular digestion, i. e. in heterophagy and autophagy. It was observed for the first time that lysosomes of cultured chick embryo cells contained long, tubular extensions, which were acid phosphatase ( ACPase)-positive in 1973. Afterwards, these tubular extensions were observed one by one in more than 20 types, i. e. in macrophages, hepatocytes, exocrine acinar cells, smooth muscle cells, neurons of central nervous system, and were named as " nematolyso-somes". Recently, nematolysosomes are found in spinal neurons, but little is known about the physiological characteristics. On the basis of the findings of nematolysosomes, a new concept was proposed that lysosomes were not discrete spherical structures, but could be thread-like structures, branched and became three-dimensional network system. The function of nematolysosomes is not clear enough. Nematolysosomes are associated with endocytotic transport and degradation in some cell types. It is suggested that nematolysosomes have close relationship between cellular regulation of distribution and transport.According to some reports, nematolysosomes were largely associated with cytoskeleton. It has been reported that nocodazole leads to disassembly of micro-tubules and fragmentation of the nematolysosomes, or even became spherical ly-sosomes. Meanwhile, the regulation of array was also disrupted. Comparatively, PMA leads to assembly of microtubules and increases the number of nematolysosomes. It has been identified in our previous studies that morphology and distribution of nematolysosome in spinal neurons depended on the integrity of microtubules. Treated with colchicine, nematolysosomes seemed to have shrunk and changed their shape from thread-like to spherical and these changes were reversible with the removal of colchicines and repolymerization of microtubules. Therefore , in the present study, the main problem is which proteins of cytoskeletonal proteins are associated with nematolysosomes. This is the key to reveal the transport mechanism of nematolysosomes. How nematolysosomes mobile along with the pathway of cytoskeletonal proteins from cytoplasm to ending? To solve this problem, it is necessary to illustrate the relationship among the cytoskeletonal proteins, such as tublin, MAP, F-actin, NFP, a-spectrin and vinculin. In this paper, our aim is to investigate the relationship between microtubule associated protein 5 and nematolysosome.In our experiments, the distribution and effects of nocodazole and PMA, and relation between nematolysosome and MAP5 were determined by means of CLSM, fluorescence microscopy, phase contrast microscopy, light microscopy, and electron microscopy. When relationship and effects between nematolysosomes and MAP5 were found by using immunocytochemistry, enzyme cytochemistry and fluorescence-labled chemistry, we could certainly give some important evidents for the transport pathway between nematolysosome and MAPS in neurons.Methods1. Primary spinal neurons were cultured using newborn Wistar rats. Growth characterization of neurons was observed by phase contrast microscopy. In order to make sure which day was the best day for immunochemistry and enzyme chemistry technique, neurons were observed day by day.2. The distribution of ACPase, a marker enzyme for lysosomes, in spinal neurons was observed using enzyme cytochemistry technique by light microsco-py-3. The distribution of nematolysosomes was observed using electron enzyme cytochemistry technique by electron microscopy.4. Cytoskeletonal protein was tested using immunocytochemistry technique. When primary antibody and second antibody were specificly combined, VEC-TASTAIN ABC reagents were employed for immunoperoxidase staining. The distribution of MAP5 was observed by light microscopy and electron micros... |
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