Showing 1 - 8 of 8 results
Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.
Liquid-liquid phase separation plays a key role in the
assembly of diverse intracellular structures. However,
the biophysical principles by which phase separation
can be precisely localized within subregions
of the cell are still largely unclear, particularly for
low-abundance proteins. Here, we introduce an oligomerizing
biomimetic system, ‘‘Corelets,’’ and utilize
its rapid and quantitative light-controlled
tunability to map full intracellular phase diagrams,
which dictate the concentrations at which phase
separation occurs and the transition mechanism, in
a protein sequence dependent manner. Surprisingly,
both experiments and simulations show that while
intracellular concentrations may be insufficient for
global phase separation, sequestering protein ligands
to slowly diffusing nucleation centers can
move the cell into a different region of the phase diagram,
resulting in localized phase separation. This
diffusive capture mechanism liberates the cell from
the constraints of global protein abundance and is
likely exploited to pattern condensates associated
with diverse biological processes.
Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome.
Phase transitions involving biomolecular liquids are a
fundamental mechanism underlying intracellular organization.
In the cell nucleus, liquid-liquid phase
separation of intrinsically disordered proteins (IDPs)
is implicated in assembly of the nucleolus, as well
as transcriptional clusters, and other nuclear bodies.
However, it remains unclear whether and how physical
forces associated with nucleation, growth, and
wetting of liquid condensates can directly restructure
chromatin. Here, we use CasDrop, a novel
CRISPR-Cas9-based optogenetic technology, to
show that various IDPs phase separate into liquid
condensates that mechanically exclude chromatin
as they grow and preferentially form in low-density,
largely euchromatic regions. A minimal physical
model explains how this stiffness sensitivity arises
from lower mechanical energy associated with deforming
softer genomic regions. Targeted genomic
loci can nonetheless be mechanically pulled together
through surface tension-driven coalescence. Nuclear
condensates may thus function as mechanoactive
chromatin filters, physically pulling in targeted
genomic loci while pushing out non-targeted regions
of the neighboring genome.
Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9.
Anti-CRISPR proteins are powerful tools for CRISPR-Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR-Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells.
Optogenetic reversible knocksideways, laser ablation, and photoactivation on the mitotic spindle in human cells.
At the onset of mitosis, cells assemble the mitotic spindle, a dynamic micromachine made of microtubules and associated proteins. Although most of these proteins have been identified, it is still unknown how their collective behavior drives spindle formation and function. Over the last decade, RNA interference has been the main tool for revealing the role of spindle proteins. However, the effects of this method are evident only after a longer time period, leading to difficulties in the interpretation of phenotypes. Optogenetics is a novel technology that enables fast, reversible, and precise control of protein activity by utilization of light. In this chapter, we present an optogenetic knocksideways method for rapid and reversible translocation of proteins from the mitotic spindle to mitochondria using blue light. Furthermore, we discuss other optical approaches, such as laser ablation of microtubule bundles in the spindle and creation of reference marks on the bundles by photoactivation of photoactivatable GFP. Finally, we show how different optical perturbations can be combined in order to acquire deeper understanding of the mechanics of mitosis.
Near-infrared light-controlled gene expression and protein targeting in neurons and non-neuronal cells.
Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner QPAS1 is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested functionality of two BphP1-QPAS1-based optogenetic tools, such as an NIR and blue light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA), in several cell types including cortical neurons. We found that the performance of these optogenetic tools often rely on physiological properties of a specific cell type, such as nuclear transport, which may limit applicability of blue light-sensitive component of iRIS. In contrast, the NIR-light-sensing part of iRIS performed well in all tested cell types. The TA system showed the best performance in HeLa, U-2 OS and HEK-293 cells. Small size of the QPAS1 component allows designing AAV viral particles, which were applied to deliver the TA system to neurons.
Real-time observation of light-controlled transcription in living cells.
Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 min after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.
A Phytochrome-Derived Photoswitch for Intracellular Transport.
Cells depend on the proper positioning of their organelles, suggesting that active manipulation of organelle positions can be used to explore spatial cell biology and to restore cellular defects caused by organelle misplacement. Recently, blue-light dependent recruitment of specific motors to selected organelles has been shown to alter organelle motility and positioning, but these approaches lack rapid and active reversibility. The light-dependent interaction of phytochrome B with its interacting factors has been shown to function as a photoswitch, dimerizing under red light and dissociating under far-red light. Here we engineer phytochrome domains into photoswitches for intracellular transport that enable the reversible interaction between organelles and motor proteins. Using patterned illumination and live-cell imaging, we demonstrate that this system provides unprecedented spatiotemporal control. We also demonstrate that it can be used in combination with a blue-light dependent system to independently control the positioning of two different organelles. Precise optogenetic control of organelle motility and positioning will provide a better understanding of and control over the spatial biology of cells.
Ultraviolet-B-mediated induction of protein-protein interactions in mammalian cells.
Light-sensitive proteins are useful tools to control protein localization, activation and gene expression, but are currently limited to excitation with red or blue light. Here we report a novel optogenetic system based on the ultraviolet-B-dependent interaction of the Arabidopsis ultraviolet-B photoreceptor UVR8 with COP1 that can be performed in visible light background. We use this system to induce nuclear accumulation of cytoplasmic green fluorescent protein fused to UVR8 in cells expressing nuclear COP1, and to recruit a nucleoplasmic red fluorescent protein fused to COP1 to chromatin in cells expressing UVR8-H2B. We also show that ultraviolet-B-dependent interactions between DNA-binding and transcription activation domains result in a linear induction of gene expression. The UVR8-COP1 interactions in mammalian cells can be induced using subsecond pulses of ultraviolet-B light and last several hours. As UVR8 photoperception is based on intrinsic tryptophan residues, these interactions do not depend on the addition of an exogenous chromophore.