Biology Reference
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nucleation in cells. Therefore, to test whether cortactin could associate with
CCPs and function during endocytosis, we performed both co-localization
and endocytic assay experiments with and without perturbation of cortactin
function. Immunocytochemistry experiments using either anti-cortactin
antibodies or expressed red fluorescent protein-tagged cortactin revealed a
dramatic co-localization of cortactin with clathrin and Dyn2 puncta at the
plasma membrane (Cao et al., 2003). At the ultrastructural level, immunogold
labelling of plasma membrane replicas showed that cortactin localized to both
flat (early) and budded (mature) CCPs. Highly curved pits showed an
enrichment of cortactin labelling at the vesicle neck, not unlike what has been
shown for dynamin.
To test for cortactin's role in endocytosis, cells were microinjected with
a
nity-purified cortactin antibodies and subsequently challenged to inter-
nalize various endocytic ligands. Cortactin antibody-injected cells showed a
significant decrease in the uptake of labelled transferrin and LDL, while fluid
internalization (examined using dextran) was unchanged. Interestingly, cells
expressing the cortactin SH3 domain also exhibited markedly reduced
endocytosis of transferrin and LDL. These data demonstrate that cortactin
is an important component of the receptor-mediated endocytic (RME)
machinery where it regulates the scission of CCPs from the plasma membrane.
Thus, cortactin provides a direct link between the dynamic actin cytoskeleton
and the membrane 'pinchase' dynamin to support vesicle formation during
RME. How the cell coordinates the efforts of cortactin, dynamin, actin and
their associated proteins, including syndapin, N-WASp and endocytic coat
proteins, to support vesicle formation is currently unknown.
Dynamin and actin-based vesicle trafficking
Actin-based 'comet formation' has emerged as a novel, motor-independent
mechanism to move vesicles within cells. These actin-associated vesicles, or
comets, are known to form from lipid microdomains in the plasma membrane
and from the Golgi complex and consist of a membranous vesicle-head and an
actin tail (Figure 12.1B) (Rozelle et al., 2000). While a portion of these actin
vesicles represent fluid uptake (macropinosomes), specific protein cargoes
remain unknown. Because of their transient nature and rapid movement
(0.15 mm/s) they are seldom observed in cells. Stimulation of tyrosine kinase
signalling results in a quantifiable difference in comet formation, suggesting
that tyrosine phosphorylation is important for this process (Rozelle et al.,
2000). A significant step forward was made when it was observed that
transient expression of type I phosphatidylinositol phosphate 5-kinases
(PIP5KIa) and accumulation of phosphatidylinositol 4,5-bisphosphate
(PIP
2
) resulted in enhanced comet formation, allowing for more reliable
