Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation

Abstract

In cells, proteins encoded by the same gene do not all behave uniformly but engage in functional subpopulations induced by spatial or temporal segregation. While conventional microscopy has limitations in revealing such spatial and temporal diversity, single-molecule tracking (SMT) microscopy circumvented this problem and allows for high-resolution imaging and quantification of dynamic single-molecule properties. Particularly in the nucleus, SMT has identified specific DNA residence times of transcription factors (TFs), DNA-bound TF fractions and positions of transcriptional hot-spots upon cell stimulation. By contrast to cell stimulation, SMT has not been employed to follow dynamic TF changes along stages of cell differentiation. Herein, we analysed the serum response factor (SRF), a TF involved in the differentiation of many cell types to study nuclear single-molecule dynamics in neuronal differentiation. Our data in living mouse hippocampal neurons show dynamic changes in SRF DNA residence time and SRF DNA-bound fraction between the stages of adhesion, neurite growth and neurite differentiation in axon and dendrites. Using TALM (tracking and localization microscopy), we identified nuclear positions of SRF clusters and observed changes in their numbers and size during differentiation. Furthermore, we show that the SRF cofactor MRTF-A (myocardin-related TF or MKL1) responds to cell activation by enhancing the long-bound DNA fraction. Finally, a first SMT colocalization study of two proteins was performed in living cells showing enhanced SRF/MRTF-A colocalization upon stimulation. In summary, SMT revealed modulation of dynamic TF properties during cell stimulation and differentiation.

Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function

“…While Hnf4g functions only in the gastrointestinal tract, it will be interesting to investigate whether the revealed principle of Hes1 dynamics holds true in other differentiating tissues [68][69][70] . Independently, differential TF dynamics have been shown in various embryonic and adult differentiation contexts 44,49,71 , suggesting a broader applicability for TF dynamics as a regulatory principle during differentiation. Arguing against simple gene expression regulation through binding of a trans-acting TF to cis-regulatory elements, exciting future avenues will include interrogating the molecular mechanisms underlying these differential TF dynamics, such as post-translational modifications and association with co-factors 2 , or the formation of protein microenvironments 72 , as well as to quantitatively probe the lineage TF concentration dependence 73 for cell state transitions during differentiation and malignant transformation.…”

“…However, academic-scale SMT has been limited in throughput and the information gained has typically been restricted to parameters describing TF diffusion. While first studies have compared two distinct undifferentiated and differentiated states 44,49 , they were obtained through directed differentiation protocols and TF dynamics have thus been measured at different time points outside the multicellular context of a differentiating tissue model. In contrast, livecell SMT has been performed in embryos of various species [50][51][52][53] .…”

“…Over the last decade, SMT emerged as a powerful technique to probe TF dynamics [42][43][44][45][46] .…”

MRTF may be the missing link in a multiscale mechanobiology approach toward macrophage dysfunction in space

“…To address knowledge gaps, we introduce the role of emerin—a putative gravi-sensitive nuclear envelope protein ( Aventaggiato et al, 2020 ; Vahlensieck et al, 2022 )—, novel microgravity mechanisms for arginase-1 ( ARG1 ) regulation, and, most notably, a novel scale in the multiscale space milieu via the MRTF-A/SRF (serum response factor) pathway. Compared to live-cell imaging, transcriptomic analysis has traditionally been blind to the dynamic, intracellular localization of MRTF-A ( Hipp et al, 2019 ; Kuchler et al, 2022 ). Furthermore, MRTF-A is currently not included in any KEGG database pathway, and its transcription program may be concealed by overarching pro-inflammatory signaling pathways.…”