Abstract
Clathrin-mediated endocytosis (CME) is essential for maintaining many basic cellular processes. We monitored the dynamics of clathrin in live Drosophila melanogaster hemocytes overexpressing clathrin light chain fused to enhanced green fluorescent protein (EGFP) using evanescent wave microscopy. Membrane-associated clathrin-coated structures (CCS) constitutively appeared at the peripheral filopodial membrane, moved centripetally while growing in intensity, before being eventually endocytosed within a few tens of seconds. This directed CCS traffic was independent of microtubules but could be blocked by latrunculin A. Taking advantage of available mutants of Drosophila, we expressed clathrin-EGFP in wasp and shibire mutant backgrounds to study the role of actin and dynamin in CCS dynamics and CME in hemocytes. We show that actin plays an essential role in CME in these cells, and that actin and dynamin act at the same stage, but independent of each other. Drosophila melanogaster hemocytes proved to be a promising model system to uncover the molecular events during CME in combining live-cell imaging and genetic analysis.
