Shedding light on the spatiotemporal regulation of RGS14 Open Access

Branch, Mary Rose (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/np193980p?locale=en
Published

Abstract

RGS14 is a multifunctional scaffold protein that integrates various cellular pathways and interacts with active (GTP-bound) and inactive (GDP-bound) Gα subunits to negatively regulate G protein signaling. RGS14 is enriched in area CA2 of the hippocampus where it acts as a natural suppressor of long-term potentiation. The functional role of RGS14 in CA2 neurons and the mechanisms by which it limits synaptic plasticity, however, are unknown. The subcellular localization of proteins in their native cellular environment can provide critical insight into their in vivo functions. All previous information about the subcellular localization of RGS14 relied largely on exogenous expression of recombinant protein in non-host cells. Since mislocalization of exogenously expressed proteins is a common problem that can give a false impression of the endogenous protein's distribution and in vivo functionalities, the primary aim of this study was to investigate the subcellular localization of endogenous RGS14 in its native cellular environment under basal conditions and following G protein activation. Because CA2 neurons cannot be isolated for study, we studied RGS14 localization in B35 neuroblastoma cells, the only cell line known to natively express RGS14. Using immunocytochemistry and confocal imaging, we found that endogenous RGS14 localizes to multiple cellular compartments in host B35 cells and that both inactive and activated G proteins can regulate the spatiotemporal dynamics of RGS14. Our results suggest that RGS14 may have multiple and complex functions in CA2 neurons, highlighting the need for future studies to determine which of these functions is critical for RGS14-mediated suppression of synaptic plasticity.

Table of Contents

Table of Contents

Background and Significance..1

Rationale...2

Hypotheses and Aims...4

Material and Methods...5

Cell Culture and Transfection...5

G protein Activation with AlF4-...5 Immunofluorescence...6 Microscopy...7 Immunoblotting...7 Analysis of Translocation...8 Statistical Analysis...9

Results...12

Subcellular localization of endogenous RGS14 in B35 cells...17

The cellular distribution of RGS14 changes throughout the cell cycle...20 AlF4- -induced G protein activation affects the subcellular localization of endogenous RGS14 in B35 cells...23

Discussion...30

RGS14 in cell division...31

RGS14 and the cytoskeleton...32

RGS14 at the nuclear membrane...33

RGS14 in the nucleus...34

RGS14 in Mitochondria...38

Conclusion...43

Illustrations

Figures

Figure 1: Immunofluorescence protocols flowchart...11

Figure 2: Natively expressed RGS14 in mouse brain and B35 cells...13

Figure 3: Immunostaining w/ anti-RGS14 antibodies detects endogenous RGS14 in B35 cells...15

Figure 4: Localization of endogenous and exogenous RGS14 in B35 cells...16

Figure 5: Subcellular distribution of endogenous RGS14 in B35 cells.. 19

Figure 6: Distribution of endogenous RGS14 is cell cycle-dependent in B35 cells...22

Figure 7: G protein activation with AlF4- induces translocation of endogenous RGS14...26

Figure 8: Co-expression of FLAG-RGS14 and Gαi/o-EE after G protein activation w/AlF4...29

Figure 9: Amino acid sequence of RGS14 predicts conserved NLS, NES, and SUMOylation motifs...37

Figure 10: Proposed model for the spatiotemporal regulation of endogenous RGS14 in B35 cells after G protein activation...41

Figure 11: Schematic of possible mechanisms by which RGS14 limits synaptic plasticity...42

Supplemental Figure 1: Quantification of relative enrichment of RGS14 at the nuclear membrane...52

Supplemental Figure 2: Effect of paraformaldehyde and methanol fixation on RGS14...53

Supplemental Figure 3: Native RGS14 immunofluorescence intensity levels...54

Supplemental Figure 4: FLAG-RGS14 localized in in various cell lines...55

Tables

Table 1: List of antibodies used in this study...10

Table 2: Subcellular localization of endogenous RGS14 in B35 cells...31

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