Date of Award

5-2015

Level of Access

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Stephen C. Pelsue

Second Committee Member

Ah-Kau Ng

Third Committee Member

Douglas Spicer

Abstract

We’ve investigated the role of the Ttc7 protein in B cells to learn more about its function in B cell activation and differentiation. B cell numbers are significantly increased in Ttc7fsn mice, and the compartment is skewed toward subpopulations that harbor multireactive/autoreactive B cells. There is a remarkably expanded mixed population of CD93+lgM- maturing B cells in the Ttc7fsn mutant that includes a substantial proportion of CD138+ cells. Morphological comparisons confirm that Ttc7fsn samples include a sizeable population of plasmablasts/plasmacytes. Functional studies demonstrate that Ttc7fsn responses to BCR ligation are distinctly different from Ttc7WT. Calcium mobilization is severely muted and caspase-3 activation is higher. Despite this, there is no difference in viability. Instead there is a significant increase in the proportion of cycling cells. Homology modeling and in silico computations predicted clear structural differences between the two proteins. CD spectroscopy confirms that the helical structure and stability of Ttc7fsn is significantly reduced. Confocal microscopy indicated Ttc7 is localized to the cytoplasm. Anti-BCR stimulation induces Ttc7 translocation to the plasma membrane where it assumes a punctate appearance. Recent studies indicate that Ttc7 interacts with PI4Klllα and is necessary for the formation of PI4Klllα-EFR3-Ttc7 plasma membrane “patches”. We show the Ttc7-PI4Klllα interaction occurs in B cells and can be modulated by BCR ligation. In contrast, Ttc7fsn forms fewer puncta that are less distinct, and patch formation between PI4Klllα and Ttc7fsn is reduced. The PI4Klllα-EFR3-Ttc7 complex was recently shown to be necessary for PI4P production in mammalian cells. We predict Ttc7 is likewise necessary for PI4P production at the plasma membrane in B cells, via formation of the PI4Klllα-EFR3-Ttc7 complex. PI4P is a signaling partner in multiple downstream functions. We propose that failure to assemble the Ttc7-PI4Klllα-Efr3 complex in B cells reduces PI4P production, which subsequently activates ER stress/UPR signals. Normal UPR activation drives terminal B cell differentiation. We hypothesize that the altered B cell responses and development observed in Ttc7fsn B cells result from the combination of ERS/UPR activation and interaction with self-antigens, which inadvertently permit autoreactive B cells to “bypass” tolerance checkpoints and develop into terminally differentiated autoantibody producing B cells.

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