Lab Overview
We are taking a biochemical approach to elucidate the underlying molecular
mechanisms driving endocytic clathrin coated vesicle (CCV) formation. A major
emphasis of our work is understanding the in vivo function and mechanism of
action of the GTPase dynamin a key regulator or clathirin mediated endocytosis.
We use cell-free assays that reconstitute CCV formation, enzymological assays
of wild-type and mutant dynamins and in vivo analysis using adenoviral-mediated
expression of mutant proteins to elucidate function.
Highlight
Marcel Mettlen
Clathrin-mediated endocytosis (CME) depends on the coordinated assembly of
multiple molecular players and recent progress in total internal reflection
fluorescence microscopy (TIR-FM) has revealed a striking heterogeneity in
the dynamic behaviors of clathrin coated structures (CCSs). Here, we address
the role of different cargo receptors in influencing clathrin-coated pit
(CCP) dynamics by a combination of biochemistry and TIR-FM imaging, using
a newly developed automated tracking software that is able to process accurately
large datasets and produce unbiased estimates of CCS lifetimes
Using BSC1 cells - transformed or not with either GFP-labeled clathrin light
chain (CLC) or GFP-s2 subunit of the adaptor protein AP2 - we show that overexpression
of transferrin receptor (TfnR) or a CD8-low-density lipoprotein receptor chimera
(CD8/LDLR) under a tetracycline (Tet)-regulatable promoter can be reproducibly
titrated over a 40-fold range by varying Tet concentrations. Biochemical measurements
of endocytosis establish a corresponding increase in surface receptors attributable
to a rate-limiting step in endocytosis, rather than accelerated recycling,
with a direct, inverse correlation between efficiency of receptor uptake and
level of overexpression. TIR-FM analyses under these conditions demonstrate
an unaltered CCS density at the cell surface. Furthermore, we evidence with
our new tracking software the existence of 3 different sub-populations of CCSs
at the cell surface. Interestingly, overexpression of cargo receptors leads
to a partial depletion of the intermediate-lifetime population, while increasing
the relative contribution of short-lifetime (possibly fast cycling) CCSs and
of (possibly non-terminal) long-lived structures.
Taken together, these results suggest that cargo receptors stabilize formerly
called 'abortive' CCSs, leading to an increase of a fast-maturating population
of CCP. Furthermore, under saturated endocytosis, the relative concentration
of cargo and its clathrin/adaptor-mediated clustering is not rate-limiting
for vesicle detachment, but fission mechanism could become saturated.
2006 Publications
Miwako, I., and S.L. Schmid. 2006. A cell-free biochemical complementation
assay reveals complex and redundant cytosolic requirements for LRP endocytosis.
Exp Cell Res. 312:1335-44.
Soulet, F., S.L. Schmid, and H. Damke. 2006. Domain requirements for an endocytosis-independent,
isoform-specific function of dynamin-2. Exp Cell Res. 312:3539-3545
