- Title
- Elucidating the role of myosin phosphatase in the contractility of myometrial smooth muscle
- Creator
- Butler, Trent Andrew John
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2017
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Myometrium is the smooth muscle layer of the uterus, and proper regulation of myometrial contractions is key to a successful birth. The myometrium is relatively dormant during most of gestation. A phenotypic transformation occurs at the onset of labour, which results in the generation of forceful, phasic myometrial contractions approximately every 3-5 minutes to expel the foetus. Contractions are generated through interactions of active myosin with actin filaments. Calcium-dependent kinase pathways activate myosin through MLC20 phosphorylation. In vascular and gastrointestinal smooth muscles, kinase pathways tissue-specifically regulate the activity of MP-MYPT1, which is the main phosphatase that dephosphorylates MLC20 in these muscles, and this regulation of MP-MYPT1 activity is equally important in the control of MLC20 phosphorylation as calcium-dependent mechanisms. It is yet to be confirmed whether MP-MYPT1 is responsible for dephosphorylating MLC20 in myometrium. This thesis aimed to investigate whether MP-MYPT1 may dephosphorylate MLC20 in human myometrium, and further explored the kinase pathways that may regulate myometrial MP-MYPT1. This study was performed when medical research was going through a reproducibility crisis. It is discovered that Western blotting, a key technique used in the field to analyse protein abundance and modifications, has limitations that contribute to poor reproducibility and can cause incorrect results when applied to analysis of human myometrial tissue. A novel approach based on commercially developed improvements to this technology was used to tackle these issues. Using this approach as well as confocal immunofluorescence microscopy, novel findings on MYPT1 in human myometrium were made including: 1) MYPT1 co-localises with myosin filaments in human myometrial-derived cells. 2) MPRIP, a MYPT1 scaffolding protein, is expressed in myometrium and co-localises with myosin filaments in human myometrial-derived cells. 3) Myometrial levels of MYPT1 and MPRIP do not change with labour in humans. 4) MYPT1 is phosphorylated at Ser-668 via the cAMP-PKA pathway. This study confirmed that MYPT1 co-localises with ROCK2 in human myometrial-derived cells. These data therefore suggest that in human myometrium, MP-MYPT1 has a role in dephosphorylating MLC20, and that pro-contractile Rho-ROCK1/2 and pro-relaxatory cAMP-PKA kinase pathways converge on MYPT1 to oppositely regulate MP-MYPT1 activity. It is concluded that regulation of MP-MYPT1 activity may be important in the control of myometrial force production, but MYPT1 or MPRIP expression is not targeted at the protein level to initiate labour.
- Subject
- myometrium; uterus; smooth muscle; contraction; MYPT1
- Identifier
- http://hdl.handle.net/1959.13/1335911
- Identifier
- uon:27510
- Rights
- Copyright 2017 Trent Andrew John Butler
- Language
- eng
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 27 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 137 KB | Adobe Acrobat PDF | View Details Download |