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E Wetzler

Publications and source records attributed to E Wetzler.

5 recordsLinked to original sources
PubMed1998

Intracellular storage and regulated plasma membrane expression of human complement receptor type 1 in rat basophil leukemia cell transfectants.

Polymorphonuclear neutrophils (PMN) contain multiple distinct secretory compartments that are sequentially mobilized during cell activation. Complement receptor type 1 (CR1) is a marker for a readily mobilizable secretory vesicle compartment, which can undergo exocytic fusion with the plasma membrane independently of secretion of traditional granule contents. The basis for the formation of these distinct compartments is incompletely understood. Primary and secondary granules are generated directly from the Golgi complex during different stages of development of the cell, obviating the need for sorting signals for proper packaging of their constituents. To determine whether the secretory vesicles are formed in a similar manner, we studied a stable rat basophilic leukemia cell line (RBL-CR1) transfected with a plasmid containing the cDNA of human CR1 driven by a viral promoter. The CR1 was present primarily intracellularly in small vesicles resembling the CR1 storage pools in resting PMN. Activation of RBL-CR1 resulted in translocation of intracellular CR1 to the plasma membrane, with mobilization requirements different from those of the classical RBL granules. Thus, in RBL-CR1, continuously synthesized CR1 is stored and upregulated in much the same way as in PMN. This suggests that differential timing of gene expression is not essential for proper storage of CR1 and that other sorting mechanisms are involved, which can be studied in RBL-transfectants.

Animals↗
PubMed1997

Isolation and characterization of complement receptor type 1 (CR1) storage vesicles from human neutrophils using antibodies to the cytoplasmic tail of CR1.

Neutrophil (PMN) activation is associated with increased surface expression of several membrane proteins that are translocated from intracellular pools. Indirect evidence suggests that the intracellular storage pools of complement receptor type 1 (CR1) in resting PMN are distinct from traditional granules and may be the secretory vesicles in which albumin is also stored, but it is not known if this compartment is homogeneous or heterogeneous. To isolate and characterize the CR1-containing vesicles, we used antibodies against unique sequences in the cytoplasmic tail of CR1. Affinity-purified IgG was used to adsorb CR1 storage vesicles from the light membrane fraction (gamma-band) of nitrogen cavitates of resting PMN. The immunoadsorbent could quantitatively remove the CR1-containing vesicles, whereas control adsorbents with nonimmune IgG showed no specific binding of CR1. Immunoblots of specifically isolated vesicles also showed enrichment of albumin, decay-accelerating factor, Fc gammaRIII, and CR3; whereas HLA class I was not detectable in these vesicles. Enzyme assay of specifically isolated vesicles after treatment with Triton X-100 showed that these vesicles contained most of the cells' latent alkaline phosphatase. An additional population of vesicles containing albumin, but not CR1, and that did not bind to anti-CR1 adsorbent was also identified. Immunoelectron microscopy showed that the specifically isolated vesicles had mean diameters of 0.086 to 0.1 microm and stained positive for CR1 and albumin. These results indicate that CR1 storage vesicles can be isolated with antibodies against the cytoplasmic tail of CR1 and show that these vesicles also contain albumin as well as glycosylphosphatidyl inositol-anchored proteins. These results are most compatible with the hypothesis that CR1-containing vesicles have arisen by endocytic retrieval of proteins that had been on the plasma membrane.

Adult↗
PubMed1994

Internalization of type 1 complement receptors and de novo multivesicular body formation during chemoattractant-induced endocytosis in human neutrophils.

Upon activation of human neutrophils by chemoattractants, functionally important proteins are rapidly transported from intracellular granules and storage vesicles to the plasma membrane. This is accompanied by a marked increase in the rate of endocytosis and by ligand-independent internalization of type 1 complement receptors (CR1). To define the pathway of endocytosis, we used gold-conjugated BSA in a pulse-chase protocol. This tracer was initially internalized into small endocytic vesicles which rapidly traversed the cytoplasm and coalesced to form large, conspicuous multivesicular bodies. Within 5 min after addition of the chemoattractant, multivesicular bodies contained > 60% of the cell-associated BSA-gold. CR1 colocalized with the endocytic tracer in both the early endosomes and multivesicular bodies. In unstimulated cells, there was much less uptake of BSA-gold and multivesicular bodies were rarely seen. Using the acidotropic amine, DAMP, and anti-DNP antibodies, we found that the multivesicular bodies were acidified but the early endosomes did not concentrate DAMP. Neither the early endosomes nor the multivesicular bodies initially contained the lysosomal membrane antigens hLAMP 1 or 2, but hLAMP-positive structures subsequently joined the multivesicular bodies. The rapid activation of the endocytic pathway upon stimulation of neutrophils allowed us to visualize the de novo formation and maturation of multivesicular bodies. Our observations suggest that vesicles containing ion pumps and acid hydrolases fuse with multivesicular bodies, giving them characteristics of lysosomes, and that these are the probable sites of degradation of CR1. The observations do not support models which would require transport of CR1 from multivesicular bodies to defined, pre-existing lysosomes for degradation.

Adamantane↗