A number of years ago a small group of investigators was torn apart by a debate over the existence or fate of somewhat obscure endosomal vesicles not even represented in text books. During the last decade, however, interest has shifted with the fireworks of newly discovered molecules involved in the regulation of protein transport. We are now entering the post-genomic era, where individual components can be re-assembled into functional, multiprotein cellular machines. Borders between disciplines in Life Sciences are fading away as our molecular understanding approaches atomic resolution. It is these exciting developments that were captured by the latest edition of the ESF conference series on endocytosis.

Animals↗

PubMed2000

Both calmodulin and the unconventional myosin Myr4 regulate membrane trafficking along the recycling pathway of MDCK cells.

In epithelial cells, endocytosed transferrin and its receptor, which cycle basolaterally, have been shown to transit through recycling endosomes which can also be accessed by markers internalized from the apical surface. In this work, we have used an in vitro assay to follow transfer of an endocytosed marker from apical or basolateral early endosomes to recycling endosomes labeled with transferrin. We show that calmodulin (CaM) function is necessary for transfer and identified myr4, a member of the unconventional myosin superfamily known to use CaM as a light chain, as a possible target protein for CaM. Since myr4 is believed to act as an actin-based mechanoenzyme, we tested the role of polymerized actin in the assay. Our data show that conditions which either prevent actin polymerization or induce the breakdown of existing filaments strongly inhibit interactions between recycling endosomes and either set of early endosomes. Altogether, our data indicate that trafficking at early steps of the endocytic pathway in Madin-Darby Canine Kidney cells depends on the actin-based mechanoenzyme myr4, its light chain CaM, and polymerized actin.

Actins↗

PubMed2000

SNARE hypothesis 2000.

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Exocytosis↗

PubMed2000

Another star on drugs.

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Kinesins↗

PubMed2000

The recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components.

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.

Animals↗

PubMed1996

Endosomal fractions from viral K-ras-transformed MDCK cells reveal transformation specific changes on two-dimensional gel maps.

We have investigated the effects of viral Kirsten ras oncogene expression in Madin-Darby canine kidney (MDCK) II epithelial cell on the differential protein expression of organelle proteins. MDCK cells, stably transformed via infection with a helper-independent retroviral vector construct, were grown on permeable filter supports. Whereas normal cells form highly polarized monolayers, ras-transformed cells display an unpolarized phenotype, detaching from the substratum and developing multilayers (Schoenenberger, C.-A. et al., J. Cell Biol. 1991, 112, 873-889). We postulate that this breakdown of epithelial polarity reflects disturbed intracellular protein transport and sorting, namely, proteins will no longer be sorted correctly in intracellular organelles and will therefore not reach their appropriate target membrane. Here we emphasize the role of endosomes as sorting platform in epithelial cells. We found significant differences in the molecular composition of endosomes from normal vs. oncogenic transformed epithelial cells, strengthening previous evidence indicating that oncogenic transformation results in abnormal expression of normal genes (Celis, J. E., Olsen, E., Electrophoresis 1994, 15, 309-344) as well as the expression of new ones (Huber, L. A. et al., Electrophoresis 1994, 15, 468-473).

Animals↗

PubMed1994

IFAP 300 is common to desmosomes and hemidesmosomes and is a possible linker of intermediate filaments to these junctions.

The distribution of IFAP 300, a protein previously characterized as cross-linking vimentin intermediate filaments (IF), has been investigated in epithelial cells. In frozen sections of bovine tongue epithelium the staining obtained with IFAP 300 antibodies is concentrated in the peripheral cytoplasm of keratinocytes, including the entire peripheral region of basal cells. Further immunofluorescence studies reveal that in primary cultures of mouse keratinocytes the distribution of IFAP 300 is similar to that of the desmosomal protein desmoplakin. In rat bladder carcinoma 804G cells the staining pattern of IFAP 300 antibodies coincides with that obtained with antibodies against the hemidesmosomal protein BP 230. By immunogold electron microscopy IFAP 300 is mainly located at sites where IF appear to attach to desmosomes and hemidesmosomes. Morphometric analyses of the distribution of the gold particles show that IFAP 300 overlaps with desmoplakin and BP 230, but also that it extends deeper into the cytoplasm than these latter two proteins. The staining reaction seen in epithelial cells by immunofluorescence and immunogold is specific for IFAP 300 as shown by immunoblotting. Immunoblotting also reveals that IFAP 300 is present in both cell-free preparations of desmosomes and hemidesmosomes. These morphological and biochemical results are intriguing since, in recent years, the proteins appearing in these two types of junctions have been found to be different. One possible exception is plectin, a protein that has been suggested to be very similar to IFAP 300. However, we show here that IFAP 300 differs from plectin in several respects, including differences at the primary sequence level. We also show that purified IFAP 300 pellets with in vitro polymerized IF prepared from desmosome-associated keratins under conditions in which IFAP 300 alone is not sedimentable. This indicates that IFAP 300 can associate with keratin IF. These data, taken together with the immunogold results, suggest that IFAP 300 functions in epithelial cells as a linker protein connecting IF to desmosomes as well as to hemidesmosomes, possibly through structurally related proteins such as desmoplakin and BP 230, respectively.

Amino Acid Sequence↗