Date of Award

Spring 5-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. Specifically, exocrine cells like pancreatic acinar and zymogenic chief cells have a highly developed secretory architecture that is rapidly established upon differentiation. What is known in these cells is that the evolutionarily conserved transcription factor MIST1 is required for final maturation. We hypothesized that MIST1 directly regulates specific subcellular components that facilitate functional maturation of secretory cells.

We show that MIST1 binds to conserved CATATG E-boxes to activate transcription of 6 genes, including the small GTPases RAB26 and RAB3D. We next demonstrate that RAB26 and RAB3D expression is significantly downregulated in Mist1-/- ZCs and upregulated in gastric cancer cell lines stably expressing MIST1 and induced to form secretory granules. Moreover, granule formation in these MIST1-expressing cells requires Rab activity because treatment with a Rab prenylation inhibitor or transfection of dominant negative RAB26 abrogates granule formation.

We next sought to elucidate how a scaling factor like MIST1 rearranges cellular architecture through transcriptional targets, specifically RAB26. We confirm that RAB26 expression was tissue specific and confined to acinar secretory cells. Functional studies in gastric cell lines showed RAB26 association with lysosomes but not with secretory granules. In addition, increasing RAB26 expression caused lysosomes to coalesce in a central, perinuclear region, causing redistribution of other organelles including mitochondria into distinct subcellular neighborhoods.

In mouse exocrine cells that are null for Mist1 and lack RAB26, we found lysosomes to be similarly abnormally distributed. Normally, lysosomes cluster centrally and basally away from secretory granules, whereas in Mist1-/- cells, they rapidly accumulate apically and degrade secretory vesicles. We confirmed active granule degradation when much of the vesicle phenotype in Mist1-/- mice was rescued upon crossing with mice deficient in the lysosome acid hydrolase processing enzyme, GlcNAc-1-phosphotransferase, alpha and beta subunits (Gntpab-/-).

Taken together, we propose that MIST1 promotes normal maturation of secretory granules through blocking their targeting by the cellular degradation/recycling machinery. These results illustrate how a transcription factor can regulate cell architecture and have implications for disease processes like acute pancreatitis where MIST1 is lost, and secretory vesicles are targeted to lysosomes.

Language

English (en)

Chair and Committee

Jason C Mills

Committee Members

Paul H Taghert, Stuart A Kornfeld, Indira U Mysorekar, Babak Razani, Joel D Schilling

Comments

Permanent URL: https://doi.org/10.7936/K7CZ35B0

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