In polarized MDCK cells basolateral vesicles arise from clathrin-gamma-adaptin-coated domains on endosomal tubules.

Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices. These buds generate vesicles that carry TR to the basolateral border. The lattices contain gamma-adaptin and are dispersed by treatment with brefeldin A (BFA). Since BFA treatment abrogates the vectorial trafficking of TR in polarized MDCK cells, we propose that the clathrin-coated domains of the endosome tubules contain the polarized sorting mechanism responsible for their preferential basolateral distribution.

Intrinsic signals in the unique domain target p56(lck) to the plasma membrane independently of CD4.

In T lymphocytes, the Src-family protein tyrosine kinase p56(lck) (Lck) is mostly associated with the cytoplasmic face of the plasma membrane. To determine how this distribution is achieved, we analyzed the location of Lck in lymphoid and in transfected nonlymphoid cells by immunofluorescence. We found that in T cells Lck was targeted correctly, independently of the cell surface proteins CD4 and CD8 with which it interacts. Similarly, in transfected NIH-3T3 fibroblasts, Lck was localized at the plasma membrane, indicating that T cell-specific proteins are not required for targeting. Some variation in subcellular distribution was observed when Lck was expressed in HeLa and MDCK cells. In these cells, Lck associated with both the plasma membrane and the Golgi apparatus, while subsequent expression of CD4 resulted in the loss of Golgi-associated staining. Together, these data indicate that Lck contains intrinsic signals for targeting to the plasma membrane. Furthermore, delivery to this site may be achieved via association with exocytic transport vesicles. A mutant Lck molecule in which the palmitoylation site at cysteine 5 was changed to lysine (LC2) localized to the plasma membrane and the Golgi region in NIH3T3 cells. However, the localization of a mutant in which the palmitoylation site at cysteine 3 was changed to serine (LC1) was indistinguishable from wild-type Lck. Chimeras composed of only the unique domain of Lck linked to either c-Src or the green fluorescent protein similarly localized to the plasma membrane of NIH-3T3 cells. Thus, the targeting of Lck appears to be determined primarily by its unique domain and may be influenced by the use of different palmitoylation sites.

Detection of non-palpable prostate cancer. A mathematical and laboratory model.

Prostate cancer is currently the most commonly diagnosed cancer among males in the United States. As technology improves and the search for this enigmatic condition intensifies, we are detecting greater numbers of non-palpable tumours. These tumours are generally treated aggressively, given the uncertainty of their behaviour, but this approach may be over-zealous for small volume disease. The likelihood of detecting any cancer volume can be derived from Bayes' theorem of conditional probability. A laboratory model using coloured clay was created to contrast tumour volumes of 2.5, 5 and 20% (n = 75). Six random systematic biopsies were taken from each model in a blind fashion; 36% of the 2.5%, 44% of the 5% and all of the 20% models had at least 1 positive biopsy. Twenty-two of the 25 models representing 20% tumour had 3 or more biopsy cores positive. These data suggest that low volume disease with low biological potential will be found by random biopsy as the mathematical probability predicts. The high incidence of occult prostate cancer in the older population makes this a worrying observation. Also, and perhaps more important, there is a direct correlation between the volume of disease and the number of positive biopsies. This correlation is easily seen in both models and may allow for an estimation of tumour volume. This ability to estimate tumour volume may be a useful clinical tool that helps to guide therapy and assess prognosis.