Abstract. Antisera raised to a detergent-and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial-and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER-Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.

Exocytic transport from the endoplasmic reticulum (ER) to the Golgi complex has been visualized in living cells using a chimera of the temperature-sensitive glycoprotein of vesicular stomatitis virus and green fluorescent protein (ts-G-GFP[ct]). Upon shifting to permissive temperature, ts-G-GFP(ct) concentrates into COPII-positive structures close to the ER, which then build up to form an intermediate compartment or transport complex, containing ERGIC-53 and the KDEL receptor, where COPII is replaced by COPI. These structures appear heterogenous and move in a microtubule-dependent manner toward the Golgi complex. Our results suggest a sequential mode of COPII and COPI action and indicate that the transport complexes are ER-to-Golgi transport intermediates from which COPI may be involved in recycling material to the ER.

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A chimera with the green fluorescent protein (GFP) has been constructed to visualize the dynamic properties of the endosome-microtubule linker protein CLIP170 (GFP-CLIP170). GFP-CLIP170 binds in stretches along a subset of microtubule ends. These fluorescent stretches appear to move with the growing tips of microtubules at 0.15-0.4 microm/s, comparable to microtubule elongation in vivo. Analysis of speckles along dynamic GFP-CLIP170 stretches suggests that CLIP170 treadmills on growing microtubule ends, rather than being continuously transported toward these ends. Drugs affecting microtubule dynamics rapidly inhibit movement of GFP-CLIP170 dashes. We propose that GFP-CLIP170 highlights growing microtubule ends by specifically recognizing the structure of a segment of newly polymerized tubulin.

Indirect immunofluorescence labeling of normal rat kidney (NRK) cells with antibodies recognizing a lysosomal glycoprotein (LGP 120; Lewis, V., S.A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100:1839-1847) reveals that lysosomes accumulate in the region around the microtubule-organizing center (MTOC). This clustering of lysosomes depends on microtubules. When the interphase microtubules are depolymerized by treatment of the cells with nocodazole or during mitosis, the lysosomes disperse throughout the cytoplasm. Lysosomes recluster rapidly (within 30-60 min) in the region of the centrosomes either upon removal of the drug, or, in telophase, when repolymerization of interphase microtubules has occurred. During this translocation process the lysosomes can be found aligned along centrosomal microtubules. Endosomes and lysosomes can be visualized by incubating living cells with acridine orange. We have analyzed the movement of these labeled endocytic organelles in vivo by video-enhanced fluorescence microscopy. Translocation of endosomes and lysosomes occurs along linear tracks (up to 10 microns long) by discontinuous saltations (with velocities of up to 2.5 microns/s). Organelles move bidirectionally with respect to the MTOC. This movement ceases when microtubules are depolymerized by treatment of the cells with nocodazole. After nocodazole washout and microtubule repolymerization, the translocation and reclustering of fluorescent organelles predominantly occurs in a unidirectional manner towards the area of the MTOC. Organelle movement remains unaffected when cells are treated with cytochalasin D, or when the collapse of intermediate filaments is induced by microinjected monoclonal antivimentin antibodies. It can be concluded that translocation of endosomes and lysosomes occurs along microtubules and is independent of the intermediate filament and microfilament networks.