Type II 5'-deiodinase (D2) catalyzes the conversion of T4 to the transcriptionally active T3. When T4 levels are high, D2 activity levels are low. Conversely when T4 levels are low, D2 catalytic activity is high. Immunocytochemistry and biochemical data from cultured rat astrocytes revealed that physiological concentration of T4 and the non-transcriptionally active metabolite rT3, but not T3, initiates the budding of D2 containing endosomes and their subsequent translocation to the perinuclear space. Further analysis showed that this process required a polymerized actin cytoskeleton but not cellular transcription or translation; however the precise mechanism remained unknown. In this present investigation, we characterized the requirement of an unconventional myosin motor protein, myosin 5a, in the actin-based endocytosis of D2 containing vesicles.
We developed an in vitro actin binding assay that exploited the T4 dependent binding of D2 containing vesicles to F-actin, and showed that D2p29:F-actin interactions are calcium, magnesium and ATP-dependent suggesting that a calmodulin (CaM) regulated myosin ATPase is required. Introduction of in vitro transcribed and translated vesicle-binding tail, which lacked the actin binding head, of myosin 5a to the in vitro actin binding assay created a dominant negative inhibitor of D2 binding to the actin cytoskeleton by competing with the native myosin 5a. A replication deficient adenoviral vector expressing the fusion protein of the 29 kDa substrate binding subunit of D2 with a green fluorescent protein reporter molecule enabled us to directly examine T4 dependent regulation of D2 in vitro as well as in living cells. Using immunoprecipitation we showed a T4 dependent association between the vesicle binding tail of myosin 5a and D2 containing vesicles. Biochemical analysis of the interaction of the myosin 5a tail with D2 containing vesicles revealed that the last 21 amino acids of myosin 5a were both necessary and sufficient for the attachment of D2 containing vesicles to the F-actin cytoskeleton.
Using rapid acquisition time-lapse digital microscopy in p29GFP expressing rat astrocytes, we showed directed T4 dependent p29GFP movement from the plasma membrane to the perinuclear region. This hormone dependent vesicle movement was not observed in cells treated with T3 or no hormone. Time lapse motion studies allowed for the calculation of the velocity and of the distance traveled for individual fusion protein containing vesicles. The velocity for cells treated with T4 or rT3 was identical to that reported for vesicle-laden myosin 5a in mouse melanophores. In contrast cells treated with T3 or those receiving no hormone treatment had velocities similar to diffusion of proteins within the plasma membrane. Astrocytes constitutively expressing both p29GFP and dominant negative myosin 5a inhibitors failed to show hormone induced centripetal movement. These data demonstrate that myosin 5a is the molecular motor responsible for thyroid hormone dependent actin based endocytosis in astrocytes.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1307 |
| Date | 27 March 2001 |
| Creators | Stachelek, Stanley J. |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved. |
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