Yang, Xu
(2018)
UNCONVENTIONAL PROTEIN SECRETION OF KERATIN 75 BY AMELOBLASTS IN VIVO.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Keratin-75, originally identified as a hair follicle specific keratin, was recently discovered in enamel tissue. Individuals carrying a K75 A161T mutation presented with altered enamel structure and increased susceptibility to dental caries. The first objective of this dissertation is to verify the existence of K75 in enamel tissue and to study its distribution, especially the subcellular localization in ameloblasts. We confirmed the existence the K75 in ameloblasts, stratum intermediate and enamel matrix through in situ hybridization, immunofluorescence(IF), and mass spectrometry. K75 expression in forming rodent teeth was confined to the secretory stage ameloblasts in IF studies. Unlike other cytokeratins, such as K14, which forms fibrillar networks, K75 was found in large granular bodies and diffuse signal in ameloblast cell body and Tomes’ processes. Consistent with IF observations, immuno-gold transmission electron microscopy(IG-TEM) studies detected K75 in large and small membrane-delineated vesicles located in ameloblast cell body and Tomes’ processes. Moreover, the majority of K75 signal overlapped with enamel matrix proteins(EMPs) ameloblastin and amelogenin in both types of vesicles.
IF and IG-TEM data strongly indicate that K75, traditionally regarded as a component of cytoskeleton, is secreted by ameloblasts together with other EMPs. Since K75 does not have a signal peptide, how K75 is transported out of the cell is intriguing. The second objective of this dissertation is to obtain insights into the trafficking pathway of K75. To address this objective, co-localization studies of K75 with organelle markers and conventional trafficking inhibition experiments were done. Under physiological conditions, K75 showed limited overlap with ER and lysosome markers, however it was highly co-localized with ER-Golgi-Intermediate-Compartment(ERGIC) and Golgi markers. When ER-Golgi trafficking was inhibited, ameloblastin was detained in rER lumen and isolated from Golgi apparatus whereas K75 still managed to translocate into the Golgi.
In summary, we demonstrate that localization of K75 in ameloblasts is different from typical cytokeratins. It is secreted extracellularly with EMPs by secretory stage ameloblasts. Moreover, our results suggest that K75 utilizes a novel unconventional protein secretion pathway which involves ERGIC and Golgi. This is the first time a secretory pathway for a cytosolic cytokeratin, lacking signaling peptide, was revealed.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
24 April 2018 |
| Date Type: |
Publication |
| Defense Date: |
18 January 2018 |
| Approval Date: |
24 April 2018 |
| Submission Date: |
19 April 2018 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
75 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Dental Medicine > Dental Science |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Keratin-75, ameloblast, amelogenesis, unconventional protein secretion, membrane trafficking, amelogenin, ameloblastin, enamel |
| Date Deposited: |
24 Apr 2018 14:49 |
| Last Modified: |
24 Apr 2019 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/34348 |
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