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1.  Alpha-adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development. 
Molecular Biology of the Cell  1997;8(8):1391-1403.
A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to support high levels of endocytosis; thus, this coated vesicle protein seems to be an excellent marker for endocytic activity. The expression patterns of dynamin, detected in the embryo by in situ hybridization methods, are very similar to those reported here for alpha-adaptin reflecting the likely coordinated expression of endocytic components. Taken together with previous evidence, our results suggest that endosomal vesicle trafficking, membrane recycling, and the regulation of endocytosis play critical roles in the wide range of developmental processes.
PMCID: PMC276164  PMID: 9285813
2.  Alteration of clathrin light chain expression by transfection and gene disruption. 
Molecular Biology of the Cell  1993;4(6):647-660.
The light chain subunits of clathrin, LCa and LCb, have been implicated in the regulation of coated vesicle disassembly and other aspects of clathrin cycling within the cell. The potential for functional specialization of each light chain is suggested by tissue-specific variation in the relative amounts of the two light chains and by conservation of differences between LCa and LCb sequences during evolution. To investigate whether there might be exclusive roles for LCa and LCb in clathrin function, the expression of LCa was manipulated in C1R lymphoid cells and PC12 pheochromocytoma cells by transfection with light chain cDNA. These two cell lines differ in their ratios of LCa to LCb, expressing 86 and 25% LCa, respectively. After transfection with exogenous human LCa cDNA, a PC12 cell derivative was produced that completely lost the ability to manufacture LCa. Loss of LCa expression was found to be because of gene disruption and consequent lack of mRNA transcription. In C1R cells, the normally high level of LCa expression was reduced to 25% by overexpression of transfected LCb cDNA under the control of an inducible promoter. The C1R transfectants with reduced levels of LCa and the LCa-negative PC12 transfectant grow normally and show no change in clathrin distribution, clathrin assembly level, or impairment of endocytosis or secretion compared with wild-type cells and cells transfected with vectors lacking light chain cDNA. However, subtle alterations in the hsc70-mediated clathrin uncoating process were observed for vesicles derived from the LCa-negative cells, reflecting the preferential activity of LCa in stimulating the in vitro uncoating reaction.
PMCID: PMC300971  PMID: 8374173

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