Cytoplasmic dynein regulation by subunit heterogeneity and its role in apical transport.

Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin's cytoplasmic COOH-terminal tail, which encodes an apical targeting determinant in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that Tctex-1 and RP3 compete for binding to dynein intermediate chain and that overexpressed RP3 displaces endogenous Tctex-1 from dynein complexes in MDCK cells. Furthermore, replacement of Tctex-1 by RP3 selectively disrupts the translocation of rhodopsin to the MDCK apical surface. These results directly show that cytoplasmic dynein function can be regulated by its subunit composition and that cytoplasmic dynein is essential for at least one mode of apical transport in polarized epithelia.

Rhodopsin trafficking and its role in retinal dystrophies.

We review the sorting/targeting steps involved in the delivery of rhodopsin to the outer segment compartment of highly polarized photoreceptor cells. The transport of rhodopsin includes (1) the sorting/budding of rhodopsin-containing vesicles at the trans-Golgi network, (2) the directional translocation of rhodopsin-bearing vesicles through the inner segment, and (3) the delivery of rhodopsin across the connecting cilium to the outer segment. Several independent lines of evidence suggest that the carboxyl-terminal, cytoplasmic tail of rhodopsin is involved in the post-Golgi trafficking of rhodopsin. Inappropriate subcellular targeting of naturally occurring rhodopsin mutants in vivo leads to photoreceptor cell death. Thus, the genes encoding mutations in the cellular components involved in photoreceptor protein transport are likely candidate genes for retinal dystrophies.

Studies of N-hydroxy-N'-aminoguanidine derivatives by nitrogen-15 nuclear magnetic resonance spectroscopy and as ribonucleotide reductase inhibitors.

Hydroxyguanidine, with the imino group of guanidine and the hydroxyamino group of hydroxyurea, has functional groups believed to be important for both anticancer and antiviral activities (Adamson, R.H. Nature (London) 1972, 236, 400-401). Three new N-hydroxy-N'-aminoguanidine derivatives have been synthesized and found to be 20-30 times more active than the hydroxyguanidine itself as inhibitors of ribonucleotide reductase from rat Novikoff tumors (Tai, W.A.; Lai, M.M.; Lien, E.J. "Novel N-Hydroxyguanidine Derivatives as Antiviral Agents", North American Medicinal Chemistry Symposium, University of Toronto, Toronto, Canada, June 20-24, 1982; Abstr, p 144). The character of the tautomeric equilibria, the pKa values, and the protonation sites of these hydroxyguanidine derivatives have been determined by 15N NMR spectroscopy.

Novel N-hydroxyguanidine derivatives as anticancer and antiviral agents.

Hydroxyguanidine contains both features of guanidine and hydroxyurea and has antiviral and anticancer activities both in vitro and in vivo. In order to enhance the antiviral and anticancer activity of this compound, a new series of hydroxyguanidine derivatives with the following structures were synthesized: R = NNHC(= NH)NHOH, where R = aromatic or heterocyclic aldehyde. This series of compounds was prepared in order to alter the lipophilic/hydrophilic balance, as well as the electronic and steric properties of hydroxyguanidine. The anticancer activities of the compounds were tested against cultured L1210 cells. The ID50 values of the above compounds are in the range of 7.80-126 microM. They are about 10-fold more active than hydroxyurea and hydroxyguanidine. The antiviral activities were also tested by assaying the inhibition of transformation of chicken embryo fibroblasts infected with Rous sarcoma virus. The ID50 values of these new compounds are in the range of 2.76-195.2 microM. The most active ones are about 100-fold more active than hydroxyguanidine. At the ID50, no apparent toxicity to the cells was noticed.

An autoantibody targeting glycated IgG is associated with elevated serum immune complexes in rheumatoid arthritis (RA).

Advanced glycation end-products (AGE) play a role in diabetes complications and in RA. An autoantibody to IgG-AGE has been shown to correlate with RA disease activity. Thus we sought to analyse serum immune complexes (IC) and AGE-modified proteins in Caucasians and North American Indians to see if the presence of anti-IgG-AGE influenced their composition. Polyethylene glycol precipitation of IC from the serum of anti-IgG-AGE-positive or -negative RA patients, and healthy and diabetic controls were examined. Concentrations of circulating IC were highest in anti-IgG-AGE+ RA patients, followed by anti-IgG-AGE- RA patients, which were greater than healthy controls. IC amounts in the Ojibwe were consistently higher than in Caucasians. Affinity purification of AGE-modified proteins from IC and immunoblotting with antibodies against Ig gamma and mu heavy chains, kappa and lambda light chains, and AGE Nepsilon(carboxymethyl)lysine and imidazolone yielded similar results: anti-AGE+ RA patients had elevated levels relative to those without the autoantibody. Levels in both RA groups were higher than in controls. Glycated albumin amounts followed a similar distribution, but were not influenced by the presence of anti-AGE antibodies. A heavily glycated kappa-chain was present primarily in IC from anti-IgG-AGE+ patients. These studies indicate that anti-AGE antibodies have a direct impact on the accumulation of IgG-AGE but not glycated albumin, and may block the normal clearance of IgG-AGE through AGE receptors.

Localization of Tctex-1, a cytoplasmic dynein light chain, to the Golgi apparatus and evidence for dynein complex heterogeneity.

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.

Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1.

The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. However, the roles of other dynein subunits in cargo binding have been unknown. Here we demonstrate that dynein translocates rhodopsin-bearing vesicles along microtubules. This interaction occurs directly between the C-terminal cytoplasmic tail of rhodopsin and Tctex-1, a dynein light chain. C-terminal rhodopsin mutations responsible for retinitis pigmentosa inhibit this interaction. Our results point to an alternative docking mechanism for cytoplasmic dynein, provide novel insights into the role of motor proteins in the polarized transport of post-Golgi vesicles, and shed light on the molecular basis of retinitis pigmentosa.