Photoactivatable Green Fluorescent Protein Research Articles

Selective activation of photoactivatable fluorescent protein based on binary holography

The ability to deliver laser doses to different target locations with high spatial and temporal resolution has been a long-sought goal in photo-stimulation and optogenetics research via, for example, photoactivatable proteins. These light-sensitive proteins undergo conformational changes upon photoactivation, serving functions such as triggering fluorescence, modulating ion channel activities, or initiating biochemical reactions within cells. Conventionally, photo-stimulation on light-sensitive proteins is performed by serially scanning a laser focus or via 2D projection, which is limited by relatively low spatiotemporal resolution. In this work, we present a programmable two-photon stimulation method based on a digital micromirror device (DMD) and binary holography to perform the activation of photoactivatable green fluorescent protein (PAGFP) in live cells. This method achieved grayscale and 3D selective PAGFP activation with subcellular resolution. In the experiments, we demonstrated the 3D activation capability and investigated the diffusion dynamics of activated PAGFP on the cell membrane. A regional difference in cell membrane diffusivity was observed, indicating the great potential of our approach in interrogating the spatiotemporal dynamics of cellular processes inside living cells.

Development of a mouse embryonic stem cell model for investigating the functions of the linker histone H1-4.

The linker histone H1 C-terminal domain (CTD) plays a pivotal role in chromatin condensation. De novo frameshift mutations within the CTD coding region of H1.4 have recently been reported to be associated with Rahman syndrome, a neurological disease that causes intellectual disability and overgrowth. To investigate the mechanisms and pathogenesis of Rahman syndrome, we developed a cellular model using murine embryonic stem cells (mESCs) and CRISPR/Cas9 genome engineering. Our engineered mES cells facilitate detailed investigations, such as H1-4 dynamics, immunoprecipitation, and nuclear localization; in addition, we tagged the mutant H1-4 with a photoactivatable GFP (PA-GFP) and an HA tag to facilitate pulldown assays. We anticipate that these engineered cells could also be used for the development of a mouse model to study the in vivo role of the H1-4 protein.

The Mobility of Neurofilaments in Mature Myelinated Axons of Adult Mice.

Studies in cultured neurons have shown that neurofilaments are cargoes of axonal transport that move rapidly but intermittently along microtubule tracks. However, the extent to which axonal neurofilaments move in vivo has been controversial. Some researchers have proposed that most axonally transported neurofilaments are deposited into a persistently stationary network and that only a small proportion of axonal neurofilaments are transported in mature axons. Here we use the fluorescence photoactivation pulse-escape technique to test this hypothesis in intact peripheral nerves of adult male hThy1-paGFP-NFM mice, which express low levels of mouse neurofilament protein M tagged with photoactivatable GFP. Neurofilaments were photoactivated in short segments of large, myelinated axons, and the mobility of these fluorescently tagged polymers was determined by analyzing the kinetics of their departure. Our results show that >80% of the fluorescence departed the window within 3 h after activation, indicating a highly mobile neurofilament population. The movement was blocked by glycolytic inhibitors, confirming that it was an active transport process. Thus, we find no evidence for a substantial stationary neurofilament population. By extrapolation of the decay kinetics, we predict that 99% of the neurofilaments would have exited the activation window after 10 h. These data support a dynamic view of the neuronal cytoskeleton in which neurofilaments cycle repeatedly between moving and pausing states throughout their journey along the axon, even in mature myelinated axons. The filaments spend a large proportion of their time pausing, but on a timescale of hours, most of them move.

Reconstitution of Golgi Biogenesis in Permeabilized Trypanosoma brucei Cells.

The protozoan parasite, Trypanosoma brucei, offers a simple system to study the growth and duplication of the Golgi. Cell lines stably expressing a photoactivatable GFP attached to an endogenous Golgi protein are permeabilized using digitonin. Photoactivation followed by imaging can then be used to follow the formation of the new Golgi.

Recurrent circadian circuitry regulates central brain activity to maintain sleep

Animal brains have discrete circadian neurons, but little is known about how they are coordinated to influence and maintain sleep. Here, through a systematic optogenetic screening, we identified a subtype of uncharacterized circadian DN3 neurons that is strongly sleep promoting in Drosophila. These anterior-projecting DN3s (APDN3s) receive signals from DN1 circadian neurons and then output to newly identified noncircadian "claw" neurons (CLs). CLs have a daily Ca2+ cycle, which peaks at night and correlates with DN1 and DN3 Ca2+ cycles. The CLs feedback onto a subset of DN1s to form a positive recurrent loop that maintains sleep. Using trans-synaptic photoactivatablegreen fluorescent protein (PA-GFP) tracing and functional invivo imaging, we demonstrated that the CLs drive sleep by interacting with and releasing acetylcholine onto the mushroom body γ lobe. Taken together, the data identify a novel self-reinforcing loop within the circadian network and a new sleep-promoting neuropile that are both essential for maintaining normal sleep.

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutesand microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

Engineering Photoactivatability in Genetically Encoded Voltage and pH Indicators.

Genetically-encoded indicators of neuronal activity enable the labeling of a genetically defined population of neurons to optically monitor their activities. However, researchers often find difficulties in identifying relevant signals from excessive background fluorescence. A photoactivatable version of a genetically encoded calcium indicator, sPA-GCaMP6f is a good example of circumventing such an obstacle by limiting the fluorescence to a region of interest defined by the user. Here, we apply this strategy to genetically encoded voltage (GEVI) and pH (GEPI) indicators. Three photoactivatable GEVI candidates were considered. The first one used a circularly-permuted fluorescent protein, the second design involved a Förster resonance energy transfer (FRET) pair, and the third approach employed a pH-sensitive variant of GFP, ecliptic pHluorin. The candidate with a variant of ecliptic pHluorin exhibited photoactivation and a voltage-dependent fluorescence change. This effort also yielded a pH-sensitive photoactivatable GFP that varies its brightness in response to intracellular pH changes.

Miro2 Regulates Inter-Mitochondrial Communication in the Heart and Protects Against TAC-Induced Cardiac Dysfunction.

Rationale: The constrained mitochondria in cardiomyocytes communicate with each other, through mitochondrial kissing or nanotunneling, forming a dynamically continuous network to share content and transfer signals. However, the molecular mechanism of cardiac inter-mitochondrial communication is unclear. Objective: To determine the molecular mechanism underlying the robust inter-mitochondrial communication and its pathophysiological relevance in the heart. Methods and Results: By mitochondria-targeted expressing the photoactivatable green fluorescent protein, we revealed that most mitochondrial nanotubes bridge communicating mitochondrial pairs were associated with microtubules. Miro2 (mitochondrial Rho GTPase), the outer mitochondrial membrane protein which usually mediates mitochondrial transport within cells, accompanied with mitochondrial nanotubes along microtubules in adult cardiomyocytes. Adenovirus mediated expression of Miro2 in cardiomyocytes accelerated inter-mitochondrial communication through increasing mitochondrial nanotunneling and mitochondrial kissing between adjacent mitochondrial pairs. In transverse aortic constriction-induced hypertrophic mouse hearts Miro2 protein was declined, accompanied with decreased inter-mitochondrial communication. Miro2 transgenic mice showed ameliorated cardiac function, increased mitochondrial nanotube formation and inter-mitochondrial communication, and improved mitochondrial function after transverse aortic constriction. E3 ubiquitin ligase Parkin was increased in transverse aortic constriction mouse hearts and phenylephrine stimulation-induced hypertrophic cardiomyocytes. Inhibition of proteasome blocked phenylephrine-induced decrease of Miro2, and Parkin overexpression led to the decrease of Miro2. Conclusions: Mitochondrial Miro2 expression levels regulate inter-mitochondrial communication along microtubules in adult cardiomyocytes, and degradation of Miro2 through Parkin-mediated ubiquitination contributes to impaired inter-mitochondrial communication and cardiac dysfunction during hypertrophic heart diseases.Visual Overview: An online visual overview is available for this article.

Characterization of the C584R variant in the mtDNA depletion syndrome gene FBXL4, reveals a novel role for FBXL4 as a regulator of mitochondrial fusion

Mutations in FBXL4 (F-Box and Leucine rich repeat protein 4), a nuclear-encoded mitochondrial protein with an unknown function, cause mitochondrial DNA depletion syndrome. We report two siblings, from consanguineous parents, harbouring a previously uncharacterized homozygous variant in FBXL4 (c.1750 T > C; p.Cys584Arg). Both patients presented with encephalomyopathy, lactic acidosis and cardiac hypertrophy, which are reported features of FBXL4 impairment. Remarkably, dichloroacetate (DCA) administration to the younger sibling improved metabolic acidosis and reversed cardiac hypertrophy. Characterization of FBXL4 patient fibroblasts revealed severe bioenergetic defects, mtDNA depletion, fragmentation of mitochondrial networks, and abnormalities in mtDNA nucleoids. These phenotypes, observed with other pathogenic FBXL4 variants, confirm the pathogenicity of the p.Cys584Arg variant. Although treating FBXL4 fibroblasts with DCA improved extracellular acidification, in line with reduced lactate levels in patients, DCA treatment did not improve any of the other mitochondrial functions. Nonetheless, we highlight DCA as a potentially effective drug for the management of elevated lactate and cardiomyopathy in patients with pathogenic FBXL4 variants. Finally, as the exact mechanism through which FBXL4 mutations lead to mtDNA depletion was unknown, we tested the hypothesis that FBXL4 promotes mitochondrial fusion. Using a photo-activatable GFP fusion assay, we found reduced mitochondrial fusion rates in cells harbouring a pathogenic FBXL4 variant. Meanwhile, overexpression of wildtype FBXL4, but not the p.Cys584Arg variant, promoted mitochondrial hyperfusion. Thus, we have uncovered a novel function for FBXL4 in promoting mitochondrial fusion, providing important mechanistic insights into the pathogenic mechanism underlying FBXL4 dysfunction.

CXCL9/10 producing cell clusters optimize Th1 cell positioning for interaction with antigen presenting cells

Abstract Intracellular pathogen clearance is dependent on successful T cell mediated immunity. Th1 cells must localize to the site of infection to be activated into IFN-γ producing effector T cells. How Th1 cells find and interact with antigen presenting cells (APC) in infected tissues is poorly understood. Preliminary data using intravital imaging and the REX3 mouse, in which CXCL9 and CXCL10 producing cells express red and blue fluorescent proteins (RFP/BFP) respectively, suggest that CXCL9/10-producing cells form dense perivascular clusters that attract and arrest CXCR3+ Th1 cells. We hypothesize that these CXCL10+ cell clusters are enriched for APC that optimize Th1 cell activation and IFN-γ production. To determine the phenotype of the immune cells within the chemokine-producing clusters we crossed the REX3 mouse with a photoactivatable green fluorescent protein (PA-GFP) mouse. This will enable selective labeling of cells within chemokine-rich clusters prior to flow cytometry. Using precise multiphoton delivery of 830 nm light, we have successfully photoactivated the CXCL10+ clustered regions in the inflamed ear and have developed a flow cytometry panel to distinguish between macrophages, cDCs, and monocyte-derived cells that may be positioned within or outside these chemokine-clusters. Additionally, we will determine if these cell clusters enhance Th1 function by sorting photoactivated (in clusters) and non-photoactivated (outside clusters) Th1 cells from the inflamed ear and analyzing IFN-γ expression. Understanding how Th1 cells are positioned and activated at sites of inflammation will allow the field to boost responses to infectious agents and therapeutically attenuate Th1 responses in autoimmunity.

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity.

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

Crystal structure of a domain-swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway.

Photoactivatable fluorescent proteins (PA-FPs) are a powerful non-invasive tool in high-resolution live-cell imaging. They can be converted from an inactive to an active form by light, enabling the spatial and temporal trafficking of proteins and cell dynamics. PA-FPs have been previously generated by mutating selected residues in the chromophore or in its close proximity. A new strategy to generate PA-FPs is the genetic incorporation of unnatural amino acids (UAAs) containing photocaged groups using unique suppressor tRNA/aminoacyl-tRNA synthetase pairs. We set out to develop a photoactivatable GFP variant suitable for time-resolved structural studies. Here, we report the crystal structure of superfolder GFP (sfGFP) containing the UAA ortho-nitrobenzyl-tyrosine (ONBY) at position 66 and its spectroscopic characterization. Surprisingly, the crystal structure (to 2.7Å resolution) reveals a dimeric domain-swapped arrangement of sfGFP66ONBY with residues 1-142 of one molecule associating with residues 148-234 from another molecule. This unusual domain-swapped structure supports a previously postulated GFP folding pathway that proceeds via an equilibrium intermediate.

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in invitro and invivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

Local Oxidative Damage in the Soma and Dendrites Quarantines Neuronal Mitochondria at the Site of Insult

SummaryNeurons are highly dependent on mitochondria, but little is known about how they react to a local mitochondrial oxidative insult. We therefore developed a protocol in primary hippocampal cultures that combines the photosensitizer mito-KillerRed with fluorescent biosensors and photoactivatable GFP. We found in both the soma and dendrites that neurons restrict the local increase in mitochondria-derived reactive oxygen species and the decrease in ATP production to the damaged compartment, by quarantining mitochondria. Although the cytosol of both the soma and dendrites became oxidized after mito-KillerRed activation, dendrites were more sensitive to the oxidative insult. Importantly, the impaired mitochondria exhibited decreased motility and fusion, thereby avoiding the spread of oxidation throughout the neuron. These results establish how neurons manage oxidative damage and increase our understanding about the somatodendritic regulation of mitochondrial functions after a local oxidative insult.

Mitoflash lights single mitochondrial dynamics events in mature cardiomyocytes

Mitochondria, the powerhouse of eukaryotic cells, are highly dynamic organelle. Mitochondrial fission, fusion, kissing and contraction have been reported over and over again in non-static cells, such as fibroblast, with tubular mitochondrial networks. Even though the fluorescence propagation among mitochondria of mature cardiomyocytes had been captured using mitochondrial matrix targeted photoactivatable GFP (PAGFP) or MitoDendra proteins, there are no direct evidence that single real time mitochondrial dynamics events exist in mature cardiomyocytes with ball-like mitochondria. Here we first time revealed the visualizable single mitochondrial dynamics events in adult mature cardiomyocytes by the mitochondrial flash (mitoflash). We found fission, fusion, contraction and kissing were accompanied by a mitoflash event. Metabolism could increase mitochondrial contraction. Fusion and Kissing mediated inter-mitochondrial communication with higher frequency than fission. These results demonstrate that mitochondria of static mature cardiomyocytes are undergoing the rare, but real dynamics change.

Highly standardized multicolor femtosecond fiber system for selective microphotomanipulation of deoxyribonucleic acid and chromatin.

We present a three-color femtosecond Er/Yb:fiber laser enabling highly specific and standardized nonlinear optical manipulation of live cells. The system simultaneously provides bandwidth-limited 80-fs pulses with identical intensity envelope centered at wavelengths of 515, 775, and 1035nm in the focus of a confocal microscope. We achieve this goal by combining high-order dispersion control via, for example, chirped fiber Bragg gratings with proper bandwidth management in each nonlinear conversion step. Wavelength-selective and noninterfering induction of deoxyribonucleic acid (DNA) photoproducts and DNA strand breaks, as well as fluorescence photoactivation of a photoactivatable green fluorescent protein (PA-GFP)-histone fusion protein, are demonstrated. The capability to introduce different types of DNA lesions and perform photoswitching experiments in a selective manner is essential for quantitative studies on DNA repair and chromatin dynamics.

What We're Reading

Lack of mismatch repair (from Fayeena via Pinterest)Mismatch repair–deficient solid tumors developed more mutations and neoantigens. Cells from such tumor proliferated normally, but in immunocompetent mice, tumor growth was impaired by a dynamic T-cell response. Mismatch repair–capable tumors could be converted to mismatch repair–defective tumors, which provided the immune system with a continuously renewed supply of neoantigens, a principle that could potentially be harnessed therapeutically with drugs targeting DNA repair pathways.Germano G, …, Bardelli A. Nature 2017 Dec 7;552:116–120.Best of the bunch (by PhotoBobil via WikiMedia)Can the effectiveness of Ab-dependent killing of tumor cells be improved? Anti-CD20 binds to B-cell lymphomas and was systematically paired with mAbs to GITR, PD-L1, PD-1, CLTA-1, 4-1BB, OX40, TIGIT, or CD27 in B lymphoma-bearing mice. Anti-CD27 plus anti-CD20 had a dramatically greater effect on survival by an indirect effect: stimulated T and NK cells produced IFNγ and myeloid attractants. This led to myeloid infiltration, which enhanced both anti-CD20–dependent phagocytosis by macrophages and tumor destruction.Turaj AH, …, Lim SH. Cancer Cell 2017 Dec 11;32:777–791.e6.Lymph node niches (from Sangaletti et al., Cancer Discovery 2014;4:110)Single-cell RNA-seq can provide signatures of cells present in a tissue, and histochemistry provides localization. However, knowing both has been a Schrödinger's cat dilemma. NICHE-seq addresses this by using two-photon laser-scanning microscopy, and transgenic mice that ubiquitously express photoactivatable GFP. Precise activation of a niche in situ before tissue dissociation allows scRNA-seq of the labeled cells. Infection and tumor development induced dynamic changes that could provide insights into differences in the tissue structures of responsive and resistant tumors.Medaglia C, …, Amit I. Science 2017 Dec 22;358:1622–6.Chromosome 6p21 encodes HLA (by Mysid, based on NIH's Genetics Home Reference via Wikimedia Commons)Immunotherapies often depend on efficient binding of neoepitopes to HLA class I and presentation to antitumor CTLs. Comparison of more than 1,500 genotypes in two independent treatment cohorts revealed that heterozygosity at all HLA loci and expression of certain HLA supertypes correlated with increased survival, whereas patients with some homozygous loci or HLA molecules containing elements that interfere with neoantigen recognition had poorer outcomes.Chowell D, …, Chan TA. Science 2017 Dec 7. DOI: 10.1126/science.aao4572.Long-lived memory mammal (from Miquitos via Flickr)Memory T cells are quiescent yet primed to react upon antigenic re-exposure. Yellow fever virus induces long-lived human memory T cells, and Akondy et al. found that these cells proliferate during priming and retain chromatin structures resembling effector T cells, suggesting they derive from effector cells. Youngblood et al. used LCMV-specific murine T cells to study epigenetic changes to chromatin of long-term memory CD8+ T cells and found a similar differentiation history. Some effector methylation patterns had reversed to naïve-associated patterns, but the demethylated state of some key effector genes were retained, allowing a swift reactivation upon rechallenge.Akondy RS, …, Ahmed R. Nature 2017 Dec 21;552:362–7.Youngblood B, …, Ahmed R. Nature 2017 Dec 21;552:404–9.Intratumor T cells (from Lo et al., CCR 2017;23:925)Although identifying T cells that are specific for tumors has become easier, it is still difficult to determine a particular TCR's specificity. With the use of a yeast display library of random peptides that can bind to a particular HLA-A, peptides recognized by four intratumoral T cells were isolated, and their parent proteins identified using prediction algorithms and validated with synthetic peptides. Such tools could enable identification of immunogenic epitopes recognized by antitumor T cells.Gee MH, …, Garcia KC. Cell 2018 Jan 25;172:1–15.

Crystal Structure of Green Fluorescent Protein Clover and Design of Clover-Based Redox Sensors

We have determined the crystal structure of Clover, one of the brightest fluorescent proteins, and found that its T203H/S65G mutations relative to wild-type GFP lock the critical E222 side chain in a fixed configuration that mimics the major conformer of that in EGFP. The resulting equilibrium shift to the predominantly deprotonated chromophore increases the extinction coefficient (EC), opposes photoactivation, and is responsible for the bathochromic shift. Clover's brightness can further be attributed to a π-π stacking interaction between H203 and the chromophore. Consistent with these observations, the Clover G65S mutant reversed the equilibrium shift, dramatically decreased the EC, and made Clover photoactivatable under conditions that activated photoactivatable GFP. Using the Clover structure, we rationally engineered a non-photoactivatable redox sensor, roClover1, and determined its structure as well as that of its parental template, roClover0.1. These high-resolution structures provide deeper insights into structure-function relationships in GFPs and may aid the development of excitation-improved ratiometric biosensors.

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.

Spatial information from NICHE-seq

Single-Cell Genomics Immune functions depend on the interactions of heterogeneous cells in a range of microenvironments in the body. Although information regarding immune cell function has been collected using single-cell RNA-sequencing methods, these techniques have traditionally lacked spatial information. Medaglia et al. describe NICHE-seq, a technique that allows the sorting and analysis of cells from within visually selected territories in transgenic mice that express photoactivatable green fluorescent protein. The method successfully identified T and B cell-specific niches in mouse lymph nodes and spleens after virus infection. The approach will allow us to bridge the gap between cellular and spatial information in studies of organs. Science , this issue p. [1622][1] [1]: /lookup/doi/10.1126/science.aao4277