Abstract
To date, much attention has been focused on the heavy and intermediate chains of the multisubunit cytoplasmic dynein complex; however, little is known about the localization or function of dynein light chains. In this study, we find that Tctex-1, a light chain of cytoplasmic dynein, localizes predominantly to the Golgi apparatus in interphase fibroblasts. Immunofluorescent staining reveals striking juxtanuclear staining characteristic of the Golgi apparatus as well as nuclear envelope and punctate cytoplasmic staining that often decorates microtubules. Tctex-1 colocalization with Golgi compartment markers, its distribution upon treatment with various pharmacological agents, and the cofractionation of Tctex-1-associated membranes with Golgi membranes are all consistent with a Golgi localization. The distribution of Tctex-1 in interphase cells only partially overlaps with the dynein intermediate chain and p150(Glued) upon immunofluorescence, but most of Tctex-1 is redistributed onto mitotic spindles along with other dynein/dynactin subunits. Using sequential immunoprecipitations, we demonstrate that there is a subset of Tctex-1 not associated with the intermediate chain at steady state; the converse also appears to be true. Distinct populations of dynein complexes are likely to exist, and such diversity may occur in part at the level of their light chain compositions.
Highlights
Cytoplasmic dynein has been shown to be involved in a wide range of intracellular motile events, including microtubule (Ϫ)end-directed organelle movement [1], endosomal transport [2], centrosomal localization of the Golgi complex [3], anaphase chromosome segregation [4], mitotic spindle alignment [5, 6], and nuclear distribution [7, 8]
We examined the intracellular localization of Tctex-1 in mammalian fibroblasts and found that Tctex-1 was predominantly localized to the Golgi complex of interphase cells by both immunocytochemical and biochemical methods
Intracellular Localization of Tctex-1 and Its Proposed Functions—Several previous reports, all performed in nonpolarized cells, have indicated that cytoplasmic dynein mediates multiple functions at the Golgi apparatus
Summary
Cytoplasmic dynein has been shown to be involved in a wide range of intracellular motile events, including microtubule (Ϫ)end-directed organelle movement [1], endosomal transport [2], centrosomal localization of the Golgi complex [3], anaphase chromosome segregation [4], mitotic spindle alignment [5, 6], and nuclear distribution [7, 8]. Our anti-Tctex-1 antibodies labeled tubulo-vesicular structures in a juxtanuclear distribution suggestive of the Golgi complex (Fig. 3A). 10 g/ml BFA treatment of NRK cells rapidly dispersed Tctex-1 juxtanuclear staining within 15 min into a fine punctate pattern against diffuse cytoplasmic background staining (Fig. 6C).
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