The efficient sorting and targeting of endocytosed macromolecules is critical for epithelial function. However, the distribution of endosomal compartments in these cells remains controversial. In this study, we show that polarized Madin-Darby canine kidney (MDCK) cells target the apical endosomal protein endotubin into an apical early endosomal compartment that is distinct from the apical recycling endosomes.
Cyclic stretch has been shown to alter cell physiology, cytoskeletal structure, signal transduction, and gene expression in a variety of cell types. To determine the effects of stretch on the gene transfer process, we compared the transfection efficiencies of human A549 cells grown either statically or exposed to 10% cyclic stretch (Delta surface area) at 60 cycles/min (1 Hz) for 24 hours prior to, and/or after transfection with pEGFP-N1 and pCMV-lux-DTS using lipoplex or electroporation. Stretching the cells prior to transfection had no effect on gene transfer. By contrast, cyclic, but not continuous, stretch applied immediately after transfection for as little as 30 minutes resulted in a 10-fold increase in gene transfer and expression by either transfection technique. These stretch conditions did not result in rupture of the plasma membrane based on the fact that DNA was unable to enter stretched cells unless either an electric field was applied or the DNA was complexed with liposomes. Taken together with the timing of the stretch response and the known effects of stretch on transcription, these findings suggest that cyclic stretch may be altering the intracellular transport of plasmids to increase gene expression.
Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N-glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid-rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac-antigen chimeras and expressed them in Madin-Darby canine kidney cells. We show that both the apically targeting wildtype endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX-100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.
Endosomes are the site of sorting of internalized receptors and ligands in all cell types and, in polarized cells, the apical endosomal compartment is involved in the selective transepithelial transport of immunoglobulins and growth factors. The biochemical composition of this specialized compartment remains largely unresolved. We have characterized a glycoprotein, called endotubin, that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. A monoclonal antibody against endotubin recognizes a broad band of 55-60kDa, which upon isoelectric focusing can be resolved into two bands, and a faint band of 140kDa. Metabolic labelling followed by immunoprecipitation indicates that endotubin is synthesized as a 140kDa precursor that is cleaved to the 55-60kDa forms. High pH washing of endosomal membranes removes the 55-60kDa forms from the membrane, whereas the high-molecular-mass form remains membrane associated and appears to be an integral membrane protein. Immunoblotting with a polyclonal antibody against the putative cytoplasmic tail of the protein identifies a 140kDa band and a band of 74kDa, presumably the cleavage product. Immunoprecipitation with antibodies against the 55-60kDa form results in coprecipitation of a 74kDa protein, and immunoprecipitation with antibody against the 74kDa protein results in coprecipitation of the 55-60kDa form. Epitope mapping of the monoclonal antibody binding site supports a proposed type I membrane protein orientation. We propose that endotubin is proteolytically processed into a heterodimer with the 55-60kDa fragment remaining membrane-associated through a non-covalent association with the membrane-bound 74kDa portion of the molecule.
uclear trafficking of macromolecules usually brings to mind the nuclear import of NLS-containing proteins and certain RNAs and the export of NES-containing proteins and mRNAs. One macromolecule whose nuclear import is often overlooked and under-appreciated is exogenously administered DNA. While extrachromosomal DNA may not be a "normal" species in the cell, its nuclear localization is integral to the life cycles of many pathogens and necessary for the success of transfections in the laboratory and gene therapy in the clinic. Moreover, the movement of DNA from the cytoplasm to the nucleus remains one of the major barriers to efficient gene transfer and expression (Fig. 1). Without localization of DNA to the nucleus, no transcription, replication, integration, maintenance, or "gene therapy" can take place. Surprisingly, there has been relatively litde attention directed toward either discovering or exploiting the mechanisms used by the cell to direct DNA to the nucleus, despite its importance in gene therapy. The discussion that follows will highlight our working knowledge of the mechanisms of DNA nuclear import in both non-viral and viral systems. The Nuclear Envelope Is a Barrier to Gene DeliveryThat the nuclear envelope presents a major barrier to gene transfer and viral infections was realized over 20 years ago in seminal experiments by Capecchi and others in which plasmids that had been microinjected into the cytoplasm were found to be virtually incapable of directing gene expression while those injected into the nucleus were highly proficient for gene expression. Using similar microinjection strategies, Graessman demonstrated that when 1000 to 2000 copies of a plasmid were injected into the cytoplasm, less than 3% of the expression was seen as compared to cells injected in the nucleus with the same number of plasmids.^^ Other experiments in a variety of mammalian cell types,^^'^^ as well as in Xenopus oocytes has confirmed that plasmids injected into the cytoplasm are much less capable of directing gene expression than those injected into the nucleus. The same is true for many viral genomes: direct nuclear injection, instead of cytoplasmic injection, of the DNA genome from SV40 or a reverse-transcribed retroviral genome residted in 10 to 100-fold more infectious virus particles in a given time.^^' ^'During mitosis, the nuclear envelope breaks down, eliminating a major barrier to gene transfer. If plasmids or viral DNA genomes are present in the cytoplasm, they have unencumbered access to the nuclear compartment during this stage of the cell cycle. By contrast, in non-dividing cells, the nuclear envelope provides a substantial barrier to the DNA (see above). Indeed, it has been demonstrated that retroviruses cannot productively infect non-dividing cells due to the fact that their reverse-transcribed viral genomes (rtDNA) cannot traverse the nuclear envelope to gain access to the nucleus. However, when the cells undergo mitosis, rtDNA can localize to the nucleus, integrate, and lead to new rounds of replication...
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