31 research outputs found
Single Cell Analysis of Transcriptional Activation Dynamics
Gene activation is thought to occur through a series of temporally defined regulatory steps. However, this process has not been completely evaluated in single living mammalian cells.To investigate the timing and coordination of gene activation events, we tracked the recruitment of GCN5 (histone acetyltransferase), RNA polymerase II, Brd2 and Brd4 (acetyl-lysine binding proteins), in relation to a VP16-transcriptional activator, to a transcription site that can be visualized in single living cells. All accumulated rapidly with the VP16 activator as did the transcribed RNA. RNA was also detected at significantly more transcription sites in cells expressing the VP16-activator compared to a p53-activator. After alpha-amanitin pre-treatment, the VP16-activator, GCN5, and Brd2 are still recruited to the transcription site but the chromatin does not decondense.This study demonstrates that a strong activator can rapidly overcome the condensed chromatin structure of an inactive transcription site and supercede the expected requirement for regulatory events to proceed in a temporally defined order. Additionally, activator strength determines the number of cells in which transcription is induced as well as the extent of chromatin decondensation. As chromatin decondensation is significantly reduced after alpha-amanitin pre-treatment, despite the recruitment of transcriptional activation factors, this provides further evidence that transcription drives large-scale chromatin decondensation
Visualization and Analysis of 3D Microscopic Images
In a wide range of biological studies, it is highly desirable to visualize and analyze three-dimensional (3D) microscopic images. In this primer, we first introduce several major methods for visualizing typical 3D images and related multi-scale, multi-time-point, multi-color data sets. Then, we discuss three key categories of image analysis tasks, namely segmentation, registration, and annotation. We demonstrate how to pipeline these visualization and analysis modules using examples of profiling the single-cell gene-expression of C. elegans and constructing a map of stereotyped neurite tracts in a fruit fly brain
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Mechanodelivery of nanoparticles to the cytoplasm of living cells
Nanotechnology has opened up the opportunity to probe,
sense, and manipulate the chemical environment of biological
systems with an unprecedented level of spatiotemporal
control. A major obstacle to the full realization of these
novel technologies is the lack of a general, robust, and simple
method for the delivery of arbitrary nanostructures to
the cytoplasm of intact live cells. Here, we identify a new
delivery modality, based on mechanical disruption of the
plasma membrane, which efficiently mediates the delivery
of nanoparticles to the cytoplasm of mammalian cells. We
use two distinct execution modes, two adherent cell lines,
and three sizes of semiconducting nanocrystals, or quantum
dots, to demonstrate its applicability and effectiveness.
As the underlying mechanism is purely physical, we
anticipate that such “mechanodelivery” can be generalized
to other modes of execution as well as to the cytoplasmic
introduction of a structurally diverse array of functional
nanomaterials
Coupling between Protein Conformation and Local Unfolding Highlights the Role of Disorder in Protein Function and Suggests a New Target for Tuberculosis Treatment
The ATM Substrate KAP1 Controls DNA Repair in Heterochromatin: Regulation by HP1 Proteins and Serine 473/824 Phosphorylation
The Dynamics of HCF-1 Modulation of Herpes Simplex Virus Chromatin during Initiation of Infection
Successful infection of herpes simplex virus is dependent upon chromatin modulation by the cellular coactivator host cell factor-1 (HCF-1). This review focuses on the multiple chromatin modulation components associated with HCF-1 and the chromatin-related dynamics mediated by this coactivator that lead to the initiation of herpes simplex virus (HSV) immediate early gene expression