AP-2-containing clathrin coats assemble on mature lysosomes.

Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and on purified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment.

Suppression of apoptosis during cytokine deprivation of 32D cells is not sufficient to induce complete granulocytic differentiation.

The role of cytokines in the control of hematopoietic cell differentiation remains controversial. Two general models for the cytokine control of hematopoietic differentiation have been proposed. In the stochastic model, cytokines provide proliferative and survival signals to the differentiating hematopoietic cell, but they do not provide specific lineage commitment signals. In the instructive model, cytokines transmit specific signals to multipotent hematopoietic cells, thereby directing lineage commitment. To distinguish between these two models with respect to granulocyte colony-stimulating factor (G-CSF) and granulocytic differentiation, we used the 32Dcl3 cell line, which is capable of differentiating into granulocytes in response to G-CSF, 32D cells transfected with either bcl-2 or bcl-XL showed prolonged survival in medium containing no cytokine supplement. Cells surviving in these cultures developed the segmented nuclei characteristic of mature neutrophils. However, no induction of myeloperoxidase activity or increase in cathepsin G transcripts were detected. These data support a hybrid model for the role of G-CSF in granulocytic differentiation; although some features of granulocytic differentiation, namely nuclear segmentation, do not require G-CSF and appear therefore to be preprogrammed in 32D cells, the complete maturation of these cells to granulocytes appears to be dependent on G-CSF.