title: Investigating SMAD Signaling Dynamics and Function in Single Cells
Creators: name: Huang, Zixin
Publisher: name: UNSPECIFIED
Year: 2024

description: SMAD signaling is crucial for regulating key cellular processes such as differentiation, proliferation, 
apoptosis, migration, and extracellular matrix production. The pathway gets activated when ligands of 
the TGF-beta superfamily bind to specific serine/threonine kinase receptors on the cell surface and 
induce the formation of receptor complexes that phosphorylate SMAD proteins at their C-terminal 
domains. Phosphorylated SMADs then form oligomes that translocate to the nucleus to regulate gene 
expression. Despite extensive knowledge of TGF-beta pathway components, understanding how 
pathway activation translates into distinct cellular responses, especially at the single-cell level and 
across various ligands, remains limited. To address this, I explored SMAD signaling dynamics using 
quantitative, time-resolved measurements of fluorescent reporters and computer-aided data analysis. 
Through live-cell imaging and fluorescent reporter systems, I investigated the signaling dynamics of 
SMAD2/3 and SMAD1/5/9 in response to different ligands and cellular conditions, focusing on their 
roles in cells of the mammary gland epithelium and endothelium. This research aims to deepen our 
understanding of the mechanisms underlying SMAD dynamics in regulating cell fate decisions.
Our previous studies of TGF-beta superfamily ligands revealed varied SMAD2 responses depending 
on cellular states. TGF-beta triggered a strong SMAD2 response in proliferating cells, which was 
diminished in quiescence. In contrast, Activin A induced consistent SMAD2 responses across different 
cellular states and GDF11 led to opposing effects— enhanced SMAD2 activity in quiescent cells and 
attenuated responses in proliferating cells. Interestingly, there was evidence that the resulting cell fate 
decisions at the single-cell level are more influenced by SMAD signaling intensity than by ligand 
specificity. To build on these findings, the first part of my research addressed two key questions: Is the 
intensity of the SMAD response indeed correlated with cell fate decisions across different TGF-beta 
superfamily ligands? And what mechanisms modulate the SMAD response across cell states in the 
presence of different ligands? Using Activin A treatment, I could show that SMAD2 nuclear 
accumulation in MCF10A cells correlates strongly with cellular phenotype, with cell state being a more 
significant factor than ligand type. EGF was identified as a crucial modulator of the SMAD2 response 
network, capable of rescuing apoptosis in quiescent cells regardless of the ligand. Furthermore, I 
dissected the role of phosphorylation in the SMAD2 linker region for stimulus- and state-specific 
signaling by generating reporter cell lines with mutations of the corresponding phosphorylation sites. 
When examining the role of SMAD3, I observed that its knockout led to uniform SMAD2 nuclear 
translocation in both proliferating and quiescent cells upon GDF11 stimulation, but did not change the 
response pattern to TGF-beta. Reintroducing SMAD3 attenuated SMAD2 accumulation in response to 
GDF11, with attenuation levels correlating with SMAD3 expression. To gain deeper insights into the 
factors influencing state- and ligand-specific SMAD signaling, I utilized RNA sequencing to identify 
proteins that modulate the SMAD3-mediated SMAD2 response induced by GDF11. This analysis highlighted several genes regulated by the SMAD3 pathway. From these observations, I propose that 
an extracellular feedback mechanism driven by SMAD3 may play a critical role in modulating the 
SMAD2 response, offering new perspectives on how SMAD signaling is dynamically regulated in 
different cellular contexts. 
Building on insights from SMAD2/3 signaling in epithelial cells, I investigated the SMAD1/5/9 
pathway in vascular endothelial cells. My primary goal was to compare how SMAD1, SMAD5, and 
SMAD9 respond to various TGF-beta superfamily ligands, revealing both unique and overlapping 
activation effects of these signaling components. Furthermore, I aimed to explore how the balance 
between the SMAD1/5/9 and SMAD2/3 pathways shifts with different ligands and impacts cellular 
responses. To achieve this, I developed fluorescent reporter cell lines for SMAD1, SMAD5, and 
SMAD9 in EA.hy926 vascular endothelial cells and characterized the dynamics and sensitivities of the 
signaling mediators. To understand how SMAD1/5/9 respond to various TGF-beta superfamily ligands, 
I focused on their distinct and overlapping effects. My findings revealed that only BMP9 and BMP10 
robustly induced SMAD nuclear accumulation. I identified FKBP12 as a key inhibitor of ALK2-
mediated SMAD1/5/9 activation which restricts their activation by ligands such as BMP7 and Activin 
A. Notably, these ligands activate both the SMAD2/3 and SMAD1/5/9 pathways in the absence of 
FKBP12. Using a dual SMAD1-SMAD2 reporter system, I investigated the balance between SMAD1 
and SMAD2 and I identified distinct receptor complexes involved in SMAD2/3 activation: BMP7-
induced activation relies on ALK4/ALK2 heterodimers, while Activin A-induced activation involves 
both ALK4/ALK2 heterodimers and ALK4 homodimers. Both ligands use ALK2 homodimers for 
SMAD1/5/9 activation. Finally, I investigated the cellular responses initiated through SMAD1/5/9 
and/or SMAD2/3 activation and confirmed a cooperative interaction between the SMAD1/5/9 and 
SMAD2/3 pathways, highlighting FKBP12's role as a key regulator of SMAD1/5/9 activity.
Collectively, these findings offer new insights into how SMAD dynamics influence single-cell outputs 
in both epithelial and endothelial cells. Specifically, SMAD3 mediates the GDF11-induced SMAD2 
response in MCF10A cells in a dose-dependent manner, while FKBP12 plays a crucial role in regulating 
SMAD1/5/9 responses to BMP7 and Activin A in EA.hy926 cells. This research elucidates key factors 
involved in the regulation of SMAD signaling in response to various ligand stimuli, deepening our 
understanding of their roles in diverse cellular processes.