The association of microtubules with an acid phosphatase-containing tubular compartment in macrophages.

Abstract

Resident peritoneal murine macrophages typically have acid phosphatase (AcPase) activity in lysosomes which are spherical in shape and have a perinuclear distribution.Upon stimulation with certain phorbol esters there is a rapid and dramatic modification of the AcPase-containing compartment into tubular structures which are often interconnected. The formation and maintenance of these tubular structures are dependent upon the presence of an intact microtubule system. Treatment of cells with the microtubule-depolymerizing agent nocodazole blocks the formation of the tubular structures and causes these tubular compartments to collapse toward the nucleus when nocodazole is administered after they are formed. We have employed whole mount electron microscopy and enzyme cytochemistry to document the association of the phorbol ester-induced AcPase-containing compartment and cytoplasmic microtubules. This methodological approach has the advantage of increased resolution when compared to optical microscopy and of an improved vantage point for analysis when compared to conventional thin section electron microscopy.

A method for co-localization of tubular lysosomes and microtubules in macrophages: fluorescence microscopy of individual cells.

“…However, this approach has been largely restricted to morphological analysis at the ultrastructural level. There are relatively few reports in which these methods have been employed for obtaining chemical information through enzyme cytochemical or immunocytochemical studies of tubular compartments (e.g., Pettitt et al, 1995;Samuels et al, 1995;Robinson and Karnovsky, 1991). Moreover, the rapid-freezing and freeze-substitution methodology has been largely applied to the electron microscope level analysis, with few cases of its use at the light microscopic level (e.g., Terasaki and Reese, 1992;Dailey and Bridgman, 1989).…”

“…These advantages include the ability to (a) monitor living cells under various experimental conditions, (b) localize tubular compartments labeled with exogenous fluorescent probes, (c) localize one or more antigens by immunofluorescence, and (d) increase sampling efficiency for statistical analysis. However, in conventional light microscopy preparations, the preservation of various tubular compartments can range from severe disruption to structures having a "beads on a string'' appearance or a mixture of straight segments interspersed with beaded structures, depending on the fixatives employed (e.g., Swanson et al, 1992;Wood and Brown, 1992; Lippincott-Schwartz, 1991; Robinson and Karnovsky, 1991). The reason for the extreme lability of some types of tubular structures, such as macrophage tubular lysosomes, remains unclear.…”

“…An elaborate tubular lysosomal compartment forms in murine macrophages after stimulation with certain agents (e.g., phorbol esters) (Luo and Robinson, 1992;Swanson et al, 1987) and after manipulation of the cytoplasmic pH (Heuser, 1989a). Endocytosis of peroxidase-anti-peroxidase immune complexes by rat alveolar macrophages also leads to the formation of tubular structures (Araki and Ogawa, 1987).…”

“…The tubular lysosomal compartment (as well as tubular endosomal structures) has been difficult to preserve, for morphological analysis, in a form reflective of its native structure owing to sensitivity to routine chemical f i t i o n protocols (e.g., Robinson and Luo, 1992;Robinson and Kamovsky, 1991;Hopkins et al, 1990;Heuser, 1989b;Robinson et al, 1986). Tubular lysosomes have been preserved for ultrastructural analysis by rapidfreezing and freeze-substitution techniques (Robinson and Karnovsky, 1991;Robinson et al, 1986 fixation have been stabilized by rapid-freezing and freezesubstitution in both plant (Samuels et al, 1995) and animal cells (Pettitt et al, 1995). Application of this methodology has lead to new interpretations of structure in these cells.…”

HVEM Observation of Phagosome-Lysosome Fusion in the Pigment Epithelium.

“…The inner balloon may result from fusion of lysosomes with phagosomes, and the outer one from autophagy of the lysosomal wrapping. Microtubules are estimated to function in wrapping mechanism as nematolysosome, because an intimate relationship between the lysosome and the microtubules has been observed [1, 5,22]. The reason why the second lysosome does not fuse laterally like the first phagolysosome remains to be answered.…”