Grassart and Cheng et al. use genome editing and quantitative microscopy to examine the dynamics of actin and dynamin2 during clathrin-mediated endocytosis.

Clathrin coat proteins form invaginated pits at the plasma membrane, which are subsequently released by the GTPase dynamin into the cytoplasm as coated vesicles. The dynamics of clathrin and dynamin assembly are incompletely understood, however, in part because overexpressing fluorescently tagged versions of these proteins might interfere with the endocytic process. Grassart and Cheng et al. therefore used genome-edited cell lines that express fluorescent versions of dynamin2 and clathrin light chain at wild-type levels uniformly across the cell population, allowing the researchers to quantitatively analyze their dynamics in an unbiased manner without perturbing endocytosis.

In the initial phase of endocytosis, which was highly variable in duration, clathrin accumulated in membrane punctae that transiently recruited small numbers of dynamin2 molecules. In a final, more regular phase lasting around 20 seconds, approximately 26 molecules of dynamin2—enough to form a single loop around the neck of the invaginated pit—stably associated with each clathrin puncta before it disappeared from the plasma membrane and internalized into the cell.

The researchers then examined genome-edited cells expressing fluorescently tagged actin. Whether actin is an integral component of the clathrin-mediated endocytosis machinery has been uncertain, but the researchers found that actin accumulated at almost every endocytic site, typically before the appearance of dynamin2. Treating cells with actin inhibitors such as jasplakinolide or cytochalasin D indicated that actin polymerization promotes dynamin2 recruitment and aids vesicle scission.

Senior author David Drubin now wants to analyze the many accessory factors that assist dynamin and clathrin in order to build a quantitative model of clathrin-mediated endocytosis.

References

References
Grassart
A.
,
Cheng
A.T.
et al
.
2014
.
J. Cell Biol.
.

Author notes

Text by Ben Short