The DNM1 Knockout K-562 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population harboring targeted disruptions in the DNM1 gene within the human K-562 suspension cell line. This heterogeneous knockout pool provides a robust loss-of-function model for studying dynamin-1-mediated processes without the need for clonal selection, enabling bulk biochemical and cell-based assays that capture population-level phenotypes.
The K-562 host cell line, derived from a 53-year-old female with BCR-ABL1-positive chronic myeloid leukemia in blast crisis, is a highly undifferentiated suspension line that co-expresses erythroid and myeloid markers. Its well-characterized signaling landscape and capacity for multidifferentiation make it a versatile model for hematopoiesis and leukemogenesis, while its suspension growth supports large-scale culture, transfection, and high-throughput screening applications.
Dynamin-1 is a mechanochemical GTPase integral to clathrin-mediated endocytosis and synaptic vesicle recycling. Its activity is regulated by phosphorylation at Ser-774 and Ser-778 by cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase-3?? (GSK3??), and by calcium/calcineurin-dependent dephosphorylation following neuronal depolarization. Upon recruitment to clathrin-coated pits, dynamin-1 assembles into helical polymers around the vesicle neck, and GTP hydrolysis drives a conformational change that severs the membrane, releasing the nascent vesicle. This process is orchestrated through direct interactions with a network of SH3-domain-containing proteins, including amphiphysin, endophilin, syndapin, and cortactin, which connect dynamin-1 to the clathrin coat, AP-2 adaptor complex, and the actin cytoskeleton. Beyond its canonical role in endocytosis, dynamin-1 participates in EGFR internalization, mTOR signaling, and calcium signaling, highlighting its broader regulatory functions.
In the K-562 background, ectopic expression of dynamin-1 provides a simplified platform to dissect core endocytic machinery without the complexities of neuronal differentiation. The knockout polyclonal cells enable direct measurement of clathrin-mediated uptake via transferrin internalization assays, visualization of clathrin-coated pits by immunofluorescence, and analysis of dynamin-1 protein interactions through co-immunoprecipitation with adaptor proteins. Additionally, phospho-specific western blotting allows investigation of upstream regulatory pathways, including CDK5- and GSK3??-mediated phosphorylation events.
This polyclonal knockout model supports a wide spectrum of research applications, from mechanistic studies of vesicle scission and clathrin coat disassembly to screening of small-molecule dynamin GTPase inhibitors. The cells can be employed in GTPase activity assays, electron microscopic examination of endocytic intermediates, and functional rescue experiments. Furthermore, they offer a relevant system for exploring the molecular pathology of DNM1-linked neurological disorders, such as early infantile epileptic encephalopathy 31 (EIEE31). For additional technical information or support, please contact Ascent Research.
