Abstract. To study the construction of the ER, we used the microtubule-disrupting drug nocodazole to induce the complete breakdown of ER structure in living cells followed by recovery in drug-free medium, which regenerates the ER network within 15 rain. Using the fluorescent dye 3,3'-dihexyloxacarbocyanine iodide to visualize the ER, we have directly observed the network construction process in living cells. In these experiments, the ER network was constructed through an iterative process of extension, branching, and intersection of new ER tubules driven by the ER motility previously described as tubule branching. We have tested the cytoskeletal requirements of this process. We find that newly formed ER tubules are aligned with single microtubules but not actin fibers or vimentin intermediate filaments. Microtubule polymerization preceded the extension of ER tubules and, in experiments with a variety of different drugs, appeared to be a necessary condition for the ER network formation.Furthermore, perturbations of the pattern of microtubule polymerization with microtubule-specific drugs caused exactly correlated perturbations of the pattern of ER construction. Induction of abnormally short, nonintersecting microtubules with 20 #M taxol prevented the ER network formation; ER tubules only extended along the few microtubules contacting the aggregated ER membranes. This requirement for a continuous network of intersecting microtubules indicates that ER network formation takes place through the branching and movement of ER membranes along microtubules. Cytochalasin B had no apparent effect on the construction of the ER network during recovery, despite apparently complete disruption of actin fibers as stained by phalloidin. Blockage of protein synthesis and disorganization of intermediate filaments with cycloheximide pretreatment also failed to perturb ER construction.
The above results suggest a high degree of clustering for B-cell disorders among first-degree relatives of patients with WM, along with distinct clinical features at presentation based on familial disease cluster patterns. Genomic studies to delineate genetic predisposition to WM are underway.
When a signal sequence is attached to 0-galactosidase, the normally cytoplasmic protein is unable to fully traverse the cytoplasmic membrane. We used a genetic approach to study those features of 0-galactosidase responsible for the block in translocation. By using both in vivo and in vitro techniques, fragments of 0-galactosidase were interposed between a signal sequence and alkaline phosphatase. The alkaline phosphatase acts as a sensor for any blocking effects of ,B-galactosidase on export. From these studies, we show that multiple regions of B-galactosidase contribute to its failure to be translocated. These results are most easily interpreted if the folding of I-galactosidase or of domains of it is responsible for the block in export. In addition, in certain constructs, positively charged amino acids directly following the signal sequence interfered with export.The translocation of a protein across the cytoplasmic membrane of bacteria requires the presence of special signals within that protein. In most cases, such signals are located at the amino terminus of the protein. For instance, in Escherichia coli, periplasmic and outer membrane proteins are synthesized initially with amino-terminal hydrophobic signal sequences. These signal sequences are interchangeable; when they have been exchanged between cell envelope proteins, the signal of one protein is found to be effective in promoting the export of another (4). However, the attachment of a signal sequence to a normally cytoplasmic protein does not necessarily result in the translocation of that protein.One cytoplasmic protein that is not exportable is the E. coli 3-galactosidase. A hybrid protein which consists of the signal sequence of maltose-binding protein (MBP), half of the mature MBP sequences, and most of the P-galactosidase molecule except for approximately 25 amino acids from its amino terminus has been studied in some detail (3) (see Fig. 2). These studies have led to the following picture. The MBP portion of the hybrid, led by the signal sequence, is transferred across the membrane. Some portion of P-galactosidase is also transferred into the periplasmic space, but the process is not completed and the hybrid protein remains imbedded in the membrane. These conclusions are based on the findings that (i) despite its membrane location, the signal sequence of the MBP-p-galactosidase hybrid is processed (20) and (ii) at least some of the P-galactosidase sequences are exposed to the periplasm since they are susceptible to protease added to spheroplasts (20). Either there are sequences within 3-galactosidase which prevent its full transfer or it is missing sequences which are required for effective transfer. Preliminary results suggest that there exist, in fact, secretion-incompatible features to the sequence or structure of 3-galactosidase (3,22).
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