Biology subjects

Garcia, H. G.

Publications and source records attributed to Garcia, H. G..

26 records · Page 2Linked to original sources

bioRxiv2020

Live imaging and biophysical modeling support a button-based mechanism of somatic homolog pairing in Drosophila

The spatial configuration of the eukaryotic genome is organized and dynamic, providing the structural basis for regulated gene expression in living cells. In Drosophila melanogaster, 3D genome organization is characterized by somatic homolog pairing, where homologous chromosomes are intimately paired from end to end; however, the process by which homologs identify one another and pair has remained mysterious. A recent model proposed that specifically interacting "buttons" encoded along the lengths of homologous chromosomes drive somatic homolog pairing. Here, we turned this hypothesis into a precise biophysical model to demonstrate that a button-based mechanism can lead to chromosome-wide pairing. We tested our model and constrained its free parameters using live-imaging measurements of chromosomal loci tagged with the MS2 and PP7 nascent RNA labeling systems. Our analysis showed strong agreement between model predictions and experiments in the separation dynamics of tagged homologous loci as they transition from unpaired to paired states, and in the percentage of nuclei that become paired as embryonic development proceeds. In sum, as a result of this dialogue between theory and experiment, our data strongly support a button-based mechanism of somatic homolog pairing in Drosophila and provide a theoretical framework for revealing the molecular identity and regulation of buttons.

developmental biology↗

bioRxiv2020

Quantitative imaging of RNA polymerase II activity in plants reveals the single-cell basis of tissue-wide transcriptional dynamics

The responses of plants to their environment often hinge on the spatiotemporal dynamics of transcriptional regulation. While live-imaging tools have been used extensively to quantitatively capture rapid transcriptional dynamics in living animal cells, lack of implementation of these technologies in plants has limited concomitant quantitative studies. Here, we applied the PP7 and MS2 RNA-labeling technologies for the quantitative imaging of RNA polymerase II activity dynamics in single cells of living plants as they respond to experimental treatments. Using this technology, we count nascent RNA transcripts in real-time in Nicotiana benthamiana (tobacco) and Arabidopsis thaliana (Arabidopsis). Examination of heat shock reporters revealed that plant tissues respond to external signals by modulating the number of cells engaged in transcription rather than the transcription rate of active cells. This switch-like behavior, combined with cell-to-cell variability in transcription rate, results in mRNA production variability spanning three orders of magnitude. We determined that cellular heterogeneity stems mainly from the stochasticity intrinsic to individual alleles. Taken together, our results demonstrate that it is now possible to quantitatively study the dynamics of transcriptional programs in single cells of living plants.

plant biology↗

bioRxiv2020

Genetically-encoded fluorescent biosensor for rapid detection of protein expression

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min that causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in live cells. This sensor for transiently-expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold in vivo following the specific and rapid binding of a protein tag (Kd 120 nM, kon 1.7 x 105 M-1s-1). In live E. coli cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short-timescale processes in research model animals with high spatiotemporal resolution.

synthetic biology↗

bioRxiv2018

Enhancer priming enables fast and sustained transcriptional responses to Notch signaling

Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2/MCP system to visualize nascent transcripts in single cells in Drosophila embryos we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.

developmental biology↗

bioRxiv2018

The Drosophila pioneer factor Zelda modulates the nuclear microenvironment of a Dorsal target enhancer to potentiate transcriptional output

Connecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long term goal of developmental biology. Many key elements have been identified in the establishment of spatial-temporal control of transcription in the early Drosophila embryo, a model system for transcriptional regulation. The dorsal/ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (DL), a homologue of the NF-{kappa}B family of transcriptional activators found in humans [1,2]. A second maternally deposited factor, Zelda (ZLD), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors such as DL [3-9]. Here we utilized the MS2 live imaging system to evaluate the expression of the DL target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. Our experiments indicate that ZLD modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. Our results further show that this effective rate increase is due to an increased accumulation of DL at the site of transcription, suggesting that transcription factor \"hubs\" induced by ZLD [10] functionally regulate transcription.

developmental biology↗

bioRxiv2018

Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene

We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high temporal resolution as its seven stripe expression pattern forms, and developed tools to characterize and visualize how transcriptional bursting varies over time and space. We find that despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, we show that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. Our work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shape complex developmental patterns.

developmental biology↗

bioRxiv2018

Binary transcriptional control of pattern formation in development

Predicting developmental outcomes from regulatory DNA sequence and transcription factor patterns remains an open challenge in physical biology. Using stripe 2 of the even-skipped gene in Drosophila embryos as a case study, we dissect the regulatory forces underpinning a key step along the developmental decision-making cascade: the generation of cytoplasmic mRNA patterns via the control of transcription in individual cells. Using live imaging and computational approaches, we found that the transcriptional burst frequency is modulated across the stripe to control the mRNA production rate. However, we discovered that bursting alone cannot quantitatively recapitulate the formation of the stripe, and that control of the window of time over which each nucleus transcribes even-skipped plays a critical role in stripe formation. Theoretical modeling revealed that these regulatory strategies--bursting and the time window--obey different kinds of regulatory logic, suggesting that the stripe is shaped by the interplay of two distinct underlying molecular processes.

developmental biology↗

bioRxiv2018

Nucleo-cytoplasmic trafficking regulates nuclear surface area during nuclear organogenesis

Throughout development, nuclei must be assembled following every cell division to establish a functional organelle from compact, mitotic chromatin. During nuclear organogenesis, chromatin expands to establish a nucleus of a given size seperate from the cytoplasm. Determining how nuclear organogenesis is regulated is particularly significant in the context of certain cancers in which scaling relationships between cell and nuclear sizes are not maintained. Controlling cell size in vitro using a microfluidics approach, we determined that neither nuclear volume nor surface area scale directly with cell size. Looking to explain differential nuclear scaling relationships, we developed a simple mechano-chemical mathematical model. In simulating biological perturbations in silico, our model predicted crucial roles for nucleo-cytoplasmic trafficking in regulating nuclear expansion and in restricting the recruitment of a potential nuclear surface area factor. In mammalian tissue culture, inhibiting nuclear export increased nuclear expansion rates and reduced the amount of nuclear lamin, a candidate surface area factor, being recruited to assembling nuclei, supporting our models predictions. Targeting the principal nuclear export component in the Drosophila syncytial embryo, Embargoed, we show that nuclear expansion rates are also increased in this developmental context, consistent with our model. Using the MS2-reporter system in fly embryos, we demonstrate a role for nuclear export in regulating transcription activation timing and dynamics, suggesting that regulating nuclear assembly is crucial for downstream nuclear function. Taken together, we propose a simple model through which nuclear organogenesis is achieved and demonstrate a role for nuclear export in regulating nuclear assembly.

cell biology↗