Abstract
The actin-based molecular motor myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the myosin VI-binding partner Dab2 and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI.
Highlights
Myosin VI turnover during endocytosis is underexplored
This table displays the average percent recovery of each construct, a value representing the average percentage of initial fluorescence returning to a given bleached region with time
The average percent recovery of all constructs analyzed was comparable, with an overall average of 78.74 Ϯ 0.02%. This recovery level falls within the range seen in other fluorescence recovery after photobleaching (FRAP) studies of proteins involved in the endocytic pathway [37, 38]
Summary
Myosin VI turnover during endocytosis is underexplored. Results: Wild type myosin VI exchanges at the same rate as its binding partner and faster than artificially dimerized myosin VI. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic halflife common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes This half-life is shared by the myosin VI-binding partner Dab and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/ dimer equilibrium Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI
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