Search

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.

We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.

Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.

The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.

We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Far-red and near-infrared (NIR) genetically encoded calcium ion (Ca2+ ) indicators (GECIs) are powerful tools for in vivo and multiplexed imaging of neural activity and cell signaling. Inspired by a previous report to engineer a far-red fluorescent protein (FP) from a biliverdin (BV)-binding NIR FP, we have developed a far-red fluorescent GECI, designated iBB-GECO1, from a previously reported NIR GECI. iBB-GECO1 exhibits a relatively high molecular brightness, an inverse response to Ca2+ with ΔF/Fmin  = -13, and a near-optimal dissociation constant (Kd ) for Ca2+ of 105 nM. We demonstrate the utility of iBB-GECO1 for four-color multiplexed imaging in MIN6 cells and five-color imaging in HEK293T cells. Like other BV-binding GECIs, iBB-GECO1 did not give robust signals during in vivo imaging of neural activity in mice, but did provide promising results that will guide future engineering efforts. SIGNIFICANCE: Genetically encoded calcium ion (Ca2+ ) indicators (GECIs) compatible with common far-red laser lines (~630-640 nm) on commercial microscopes are of critical importance for their widespread application to deep-tissue multiplexed imaging of neural activity. In this study, we engineered a far-red excitable fluorescent GECI, designated iBB-GECO1, that exhibits a range of preferable specifications such as high brightness, large fluorescence response to Ca2+ , and compatibility with multiplexed imaging in mammalian cells.

m6A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m6A-modified base. The recruitment of the mRNA m6A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits βarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.

BackgroundThe ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging.MethodsWe recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1-GFP line (Cx3cr1+/GFP), naïve microglia in Cx3cr1-Dendra2 mice (Cx3cr1+/D2) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo.ResultsDendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration.ConclusionsThe ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1-Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation.

Synaptic refinement is a critical physiological process that removes excess synapses to establish and maintain functional neuronal circuits. Recent studies have shown that focal exposure of phosphatidylserine (PS) on synapses acts as an "eat me" signal to mediate synaptic pruning. However, the molecular mechanism underlying PS externalization at synapses remains elusive. Here, we find that murine CDC50A, a chaperone of phospholipid flippases, localizes to synapses, and that its expression depends on neuronal activity. Cdc50a knockdown leads to phosphatidylserine exposure at synapses and subsequent erroneous synapse removal by microglia partly via the GPR56 pathway. Taken together, our data support that CDC50A safeguards synapse maintenance by regulating focal phosphatidylserine exposure at synapses.

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and superresolved imaging, we identify a module that we term DISCO (distal centriole complex). The DISCO components CEP90, MNR, and OFD1 underlie human ciliopathies. This complex localizes to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR do not generate cilia, fail to assemble distal appendages, and do not transduce Hedgehog signals. Disrupting the satellite pools does not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruits the most proximal known distal appendage component, CEP83, to root distal appendage formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricts centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies.

Genetically encoded Ca2+ indicators (GECIs) enable long-term monitoring of cellular and subcellular dynamics of this second messenger in response to environmental and developmental cues without relying on exogenous dyes. Continued development and optimization in GECIs, combined with advances in gene manipulation, offer new opportunities for investigating the mechanism of Ca2+ signaling in fungi, ranging from documenting Ca2+ signatures under diverse conditions and genetic backgrounds to evaluating how changes in Ca2+ signature impact calcium-binding proteins and subsequent cellular changes. Here, we attempted to express multi-color (green, yellow, blue, cyan, and red) circularly permuted fluorescent protein (FP)-based Ca2+ indicators driven by multiple fungal promoters in Fusarium oxysporum, F. graminearum, and Neurospora crassa. Several variants were successfully expressed, with GCaMP5G driven by the Magnaporthe oryzae ribosomal protein 27 and F. verticillioides elongation factor-1α gene promoters being optimal for F. graminearum and F. oxysporum, respectively. Transformants expressing GCaMP5G were compared with those expressing YC3.60, a ratiometric Cameleon Ca2+ indicator. Wild-type and three Ca2+ signaling mutants of F. graminearum expressing GCaMP5G exhibited improved signal-to-noise and increased temporal and spatial resolution and are also more amenable to studies involving multiple FPs compared to strains expressing YC3.60.

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

Although fluorescent proteins have been utilized for a variety of biological applications, they have several optical limitations, namely weak red and near-infrared emission and exceptionally broad (>200 nm) emission profiles. The photophysical properties of fluorescent proteins can be enhanced through the incorporation of novel cofactors with the desired properties into a stable protein scaffold. To this end, a fluorescent phosphorus corrole that is structurally similar to the native heme cofactor is incorporated into two exceptionally stable heme proteins: H-NOX from Caldanaerobacter subterraneus and heme acquisition system protein A (HasA) from Pseudomonas aeruginosa. These yellow-orange emitting protein conjugates are examined by steady-state and time-resolved optical spectroscopy. The HasA conjugate exhibits enhanced fluorescence, whereas emission from the H-NOX conjugate is quenched relative to the free corrole. Despite the low fluorescence quantum yields, these corrole-substituted proteins exhibit more intense fluorescence in a narrower spectral profile than traditional fluorescent proteins that emit in the same spectral window. This study demonstrates that fluorescent corrole complexes are readily incorporated into heme proteins and provides an inroad for the development of novel fluorescent proteins.

High vascularization is a biological characteristic of glioblastoma (GBM); however, an in-vitro experimental model to verify the mechanism and physiological role of vasculogenesis in GBM is not well-established. Recently, we established a self-organizing vasculogenic model using human umbilical vein endothelial cells (HUVECs) co-cultivated with human lung fibroblasts (hLFs). Here, we exploited this system to establish a realistic model of vasculogenesis in GBM. We developed two polydimethylsiloxane (PDMS) devices, a doughnut-hole dish and a 5-lane microfluidic device to observe the contact-independent effects of glioblastoma cells on HUVECs. We tested five patient-derived and five widely used GBM cell lines. Confocal fluorescence microscopy was used to observe the morphological changes in Red Fluorescent Protein (RFP)-HUVECs and fluorescein isothiocyanate (FITC)-dextran perfusion. The genetic and expression properties of GBM cell lines were analyzed. The doughnut-hole dish assay revealed KNS1451 as the only cells to induce HUVEC transformation to vessel-like structures, similar to hLFs. The 5-lane device assay demonstrated that KNS1451 promoted the formation of a vascular network that was fully perfused, revealing the functioning luminal construction. Microarray analysis revealed that KNS1451 is a mesenchymal subtype of GBM. Using a patient-derived mesenchymal GBM cell line, mature de-novo vessel formation could be induced in HUVECs by contact-independent co-culture with GBM in a microfluidic device. These results support the development of a novel in vitro research model and provide novel insights in the neovasculogenic mechanism of GBM and may potentially facilitate the future detection of unknown molecular targets.

Signal transduction processes are a necessary component of multicellular life, and their dysregulation is the basis for a host of syndromes and diseases. Thus, it is imperative that we discover the complex details of how signal transduction processes result in specific cellular outcomes. One of the primary mechanisms of regulation over signaling pathways is through spatiotemporal control. However, traditional methods are limited in their ability to reveal such details. To overcome these limitations, researchers have developed a variety of genetically encodable, fluorescent protein-based biosensors to study these dynamic processes in real time in living cells. Due to the complexities and interconnectedness of signaling pathways, it is thus desirable to use multiple biosensors in individual cells to better elucidate the relationships between signaling pathways. However, multiplexed imaging with such biosensors has been historically difficult. Nevertheless, recent developments in designs and multiplexing strategies have led to vast improvements in our capabilities. In this review, we provide perspectives on the recently developed biosensor designs and multiplexing strategies that are available for multiplexed imaging of signal transduction pathways.

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein-protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.

BackgroundWithin the last decade, genetic engineering and synthetic biology have revolutionized society´s ability to mass-produce complex biological products within genetically-modified microorganisms containing elegantly designed genetic circuitry. However, many challenges still exist in developing bioproduction processes involving genetically modified microorganisms with complex or multiple gene circuits. These challenges include the development of external gene expression regulation methods with the following characteristics: spatial-temporal control and scalability, while inducing minimal permanent or irreversible system-wide conditions. Different stimuli have been used to control gene expression and mitigate these challenges, and they can be characterized by the effect they produce in the culture media conditions. Invasive stimuli that cause permanent, irreversible changes (pH and chemical inducers), non-invasive stimuli that cause partially reversible changes (temperature), and non-invasive stimuli that cause reversible changes in the media conditions (ultrasound, magnetic fields, and light).MethodsOpto-control of gene expression is a non-invasive external trigger that complies with most of the desired characteristics of an external control system. However, the disadvantage relies on the design of the biological photoreceptors and the necessity to design them to respond to a different wavelength for every bioprocess needed to be controlled or regulated in the microorganism. Therefore, this work proposes using biocompatible metallic nanoparticles as external controllers of gene expression, based on their ability to convert light into heat and the capacity of nanotechnology to easily design a wide array of nanostructures capable of absorbing light at different wavelengths and inducing plasmonic photothermal heating.ResultsHere, we designed a nanobiosystem that can be opto-thermally triggered using LED light. The nanobiosystem is composed of biocompatible gold nanoparticles and a genetically modified E. coli with a plasmid that allows mCherry fluorescent protein production at 37 °C in response to an RNA thermometer.ConclusionsThe LED-triggered photothermal protein production system here designed offers a new, cheaper, scalable switchable method, non-destructive for living organisms, and contribute toward the evolution of bioprocess production systems.

Upon infection of Pseudomonas cells, jumbo phages 201Φ2-1, ΦPA3, and ΦKZ assemble a phage nucleus. Viral DNA is enclosed within the phage-encoded proteinaceous shell along with proteins associated with DNA replication, recombination and transcription. Ribosomes and proteins involved in metabolic processes are excluded from the nucleus. RNA synthesis occurs inside the phage nucleus and messenger RNA is presumably transported into the cytoplasm to be translated. Newly synthesized proteins either remain in the cytoplasm or specifically translocate into the nucleus. The molecular mechanisms governing selective protein sorting and nuclear import in these phage infection systems are currently unclear. To gain insight into this process, we studied the localization of five reporter fluorescent proteins (GFP+, sfGFP, GFPmut1, mCherry, CFP). During infection with ΦPA3 or 201Φ2-1, all five fluorescent proteins were excluded from the nucleus as expected; however, we have discovered an anomaly with the ΦKZ nuclear transport system. The fluorescent protein GFPmut1, expressed by itself, was transported into the ΦKZ phage nucleus. We identified the amino acid residues on the surface of GFPmut1 required for nuclear targeting. Fusing GFPmut1 to any protein, including proteins that normally reside in the cytoplasm, resulted in transport of the fusion into the nucleus. Although the mechanism of transport is still unknown, we demonstrate that GFPmut1 is a useful tool that can be used for fluorescent labelling and targeting of proteins into the ΦKZ phage nucleus.

This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate a neural stem cell culture from Gt(ROSA)26Sortm4(ACTB-tdTomato, EGFP)Luo/J mice, and validate the MADR plasmids in vitro and in vivo by neonatal mouse brain electroporation. This protocol can be generalized to analyze any transgenic element using MADR technology. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019).

Developing sustainable agricultural practices will require increasing our understanding of plant-microbe interactions. To study these interactions, new genetic tools for manipulating nonmodel microbes will be needed. To help meet this need, we recently reported development of chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE relies on cassette exchange between two pairs of mutually exclusive lox sites and allows direct, single-step chromosomal integration of large, complex gene constructs into diverse bacterial species. We then extended CRAGE by introducing a third mutually exclusive lox site, creating CRAGE-Duet, which allows modular integration of two constructs. CRAGE-Duet offers advantages over CRAGE, especially when a cumbersome recloning step is required to build single-integration constructs. To demonstrate the utility of CRAGE-Duet, we created a set of strains from the plant-growth-promoting rhizobacterium Pseudomonas simiae WCS417r that expressed various fluorescence marker genes. We visualized these strains simultaneously under a confocal microscope, demonstrating the usefulness of CRAGE-Duet for creating biological systems to study plant-microbe interactions.

Cnidarian fluorescent protein (FP) derivatives such as GFP, mCherry, and mEOS2 have been widely used to monitor gene expression and protein localization through biological imaging because they are considered functionally inert. We demonstrate that FPs specifically bind amyloid fibrils formed from many natural peptides and proteins. FPs do not bind other nonamyloid fibrillar structures such as microtubules or actin filaments and do not bind to amorphous aggregates. FPs can also bind small aggregates formed during the lag phase and early elongation phase of fibril formation and can inhibit amyloid fibril formation in a dose-dependent manner. These findings suggest caution should be taken in interpreting FP-fusion protein localization data when amyloid structures may be present. Given the pathological significance of amyloid-related species in some diseases, detection and inhibition of amyloid fibril formation using FPs can provide insights on developing diagnostic tools.

Neurotropic strains of mouse hepatitis virus (MHV), a coronavirus, cause acute and chronic demyelinating encephalomyelitis with similarities to the human disease multiple sclerosis. Here, using a lineage-tracking system, we show that some cells, primarily oligodendrocytes (OLs) and oligodendrocyte precursor cells (OPCs), survive the acute MHV infection, are associated with regions of demyelination, and persist in the central nervous system (CNS) for at least 150 d. These surviving OLs express major histocompatibility complex (MHC) class I and other genes associated with an inflammatory response. Notably, the extent of inflammatory cell infiltration was variable, dependent on anatomic location within the CNS, and without obvious correlation with numbers of surviving cells. We detected more demyelination in regions with larger numbers of T cells and microglia/macrophages compared to those with fewer infiltrating cells. Conversely, in regions with less inflammation, these previously infected OLs more rapidly extended processes, consistent with normal myelinating function. Together, these results show that OLs are inducers as well as targets of the host immune response and demonstrate how a CNS infection, even after resolution, can induce prolonged inflammatory changes with CNS region-dependent impairment in remyelination.

Pseudomonas putida is a widely used host for metabolic engineering and synthetic biology. However, the use of P. putida has been hampered by the availability of a limited set of expression vectors for producing heterologous proteins. To widen the scope of expression vectors for gene co-expression studies, a previously established dual-inducible expression vector pRG_Duet2 developed for Corynebacterium glutamicum has been modified for use in P. putida. This expression vector, named pRGPDuo2, harbors two origins of replication, colE1 for replication in E. coli and pRO1600 for replication in P. putida. Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are individually controlled by inducible promoters Ptac or PtetR/tetA. Functional validation of pRGPDuo2 was confirmed by the co-expression of genes for the fluorescent proteins namely, superfolder green fluorescent protein (sfGFP), and red fluorescent protein (RFP). Moreover, the strength of the fluorescence signal was dependent on the inducer concentrations present in the culture medium. The expression vector pRGPDuo2 is an attractive addition to the existing repertoire of expression plasmids for expression profiling and adds to the tools available for P. putida metabolic engineering.

Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.

Genetically encoded fluorescent protein-based kinase biosensors are a central tool for illumination of the kinome. The adaptability and versatility of biosensors have allowed for spatiotemporal observation of real-time kinase activity in living cells and organisms. In this review, we highlight various types of kinase biosensors, along with their burgeoning applications in complex biological systems. Specifically, we focus on kinase activity reporters used in neuronal systems and whole animal settings. Genetically encoded kinase biosensors are key for elucidation of the spatiotemporal regulation of protein kinases, with broader applications beyond the Petri dish.

Phosphoinositides constitute a critical family of lipids that regulate numerous cellular processes. Phosphatidylinositol 4,5-bisphosphate (PIP2) is arguably the most important plasma membrane phosphoinositide and is involved in regulating diverse processes. It is also the precursor of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is critical for growth factor signaling, as well as membrane polarization and dynamics. Studying these lipids remains challenging, because of their compartmentalized activities and location-dependent signaling profiles. Here, we introduce several new genetically encoded fluorescent biosensors, including FRET-based and dimerization-dependent fluorescent protein (ddFP)-based biosensors, that enable real-time monitoring of PIP2 levels in live cells. In addition, we developed a red fluorescent biosensor for 3-phosphoinositides that can be co-imaged with the green PIP2 indicator. Simultaneous visualization of the dynamics of PIP2 and 3-phosphoinositides in the same cell shows that plasma membrane PIP3 formation upon EGF stimulation is coupled to a decrease in the local pool of PIP2.

Emerging literature suggests that after a stroke, the peri-infarct region exhibits dynamic changes in excitability. In rodent stroke models, treatments that enhance excitability in the peri-infarct cerebral cortex promote motor recovery. This increase in cortical excitability and plasticity is opposed by increases in tonic GABAergic inhibition in the peri-infarct zone beginning three days after a stroke in a mouse model. Maintenance of a favorable excitatory-inhibitory balance promoting cerebrocortical excitability could potentially improve recovery. Brevetoxin-2 (PbTx-2) is a voltage-gated sodium channel (VGSC) gating modifier that increases intracellular sodium ([Na+]i), upregulates N-methyl-D-aspartate receptor (NMDAR) channel activity and engages downstream calcium (Ca2+) signaling pathways. In immature cerebrocortical neurons, PbTx-2 promoted neuronal structural plasticity by increasing neurite outgrowth, dendritogenesis and synaptogenesis. We hypothesized that PbTx-2 may promote excitability and structural remodeling in the peri-infarct region, leading to improved functional outcomes following a stroke. We tested this hypothesis using epicortical application of PbTx-2 after a photothrombotic stroke in mice. We show that PbTx-2 enhanced the dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. PbTx-2 also produced a robust improvement of motor recovery. These results suggest a novel pharmacologic approach to mimic activity-dependent recovery from stroke.

Like cervical cancer, anal cancer is caused by human papillomavirus (HPV). HPV is the most common sexually transmitted agent and is found in the anal canal of almost all HIV-positive men who have sex with men (MSM). Rates of HPV anal cancer are disproportionately higher in this population. Although the nanovalent HPV vaccine is efficacious in protecting against oncogenic HPV types, a substantial proportion of MSM remains unvaccinated and anal HPV infection continues to be an important public health burden. Therefore, it is important to identify strategies to prevent HPV infection. We report on two promising and interlinked strategies: (1) the development of a cell-based Renilla luminescence reporter assay using HPV-16 pseudovirions that encapsidate SV40-driven Renilla luminescence reporter expression plasmid and (2) use of this assay for high-throughput screening (HTS) of FDA- and internationally approved drugs to identify those that could be repurposed to prevent HPV infection. We conducted a screen of 1906 drugs. The assay was valid with a Z' of 0.67 ± 0.04, percent coefficient of variance of 10.0, and signal-to-background noise window of 424.0 ± 8.0. Five drugs were chosen for further analyses based on selection parameters of ≥77.0% infection of HPV-16 pseudovirion-driven Renilla expression with

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

Naturally occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type-the astrocyte-has rarely been studied. It is presently unclear whether astrocytes are subject to significant developmental death, and if so, how it occurs. Here, we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia engulf astrocytes during the death period to promote their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally occurring cell death and demonstrate its importance for the formation and integrity of the retinal gliovascular network.

In response to proteotoxic stress, chloroplasts communicate with the nuclear gene expression system through a chloroplast unfolded protein response (cpUPR). We isolated Chlamydomonas reinhardtii mutants that disrupt cpUPR signaling and identified a gene encoding a previously uncharacterized cytoplasmic protein kinase, termed Mars1-for mutant affected in chloroplast-to-nucleus retrograde signaling-as the first known component in cpUPR signal transmission. Lack of cpUPR induction in MARS1 mutant cells impaired their ability to cope with chloroplast stress, including exposure to excessive light. Conversely, transgenic activation of cpUPR signaling conferred an advantage to cells undergoing photooxidative stress. Our results indicate that the cpUPR mitigates chloroplast photodamage and that manipulation of this pathway is a potential avenue for engineering photosynthetic organisms with increased tolerance to chloroplast stress.

Microfluidic devices have been increasingly used for low-volume liquid handling operations. However, laboratory automation of such delicate devices has lagged behind due to the lack of world-to-chip (macro-to-micro) interfaces. In this paper, we have presented the first pipette-free robotic-microfluidic interface using a microfluidic-embedded container cap, referred to as a microfluidic cap-to-dispense (μCD), to achieve a seamless integration of liquid handling and robotic automation without any traditional pipetting steps. The μCD liquid handling platform offers a generic and modular way to connect the robotic device to standard liquid containers. It utilizes the high accuracy and high flexibility of the robotic system to recognize, capture and position; and then using microfluidic adaptive printing it can achieve high-precision on-demand volume distribution. With its modular connectivity, nanoliter processability, high adaptability, and multitask capacity, μCD shows great potential as a generic robotic-microfluidic interface for complete pipette-free liquid handling automation.

A protein-fragment complementation assay (PCA) for detecting and localizing intracellular protein-protein interactions (PPIs) was built by bisection of miniSOG, a fluorescent flavoprotein derived from the light, oxygen, voltage (LOV)-2 domain of Arabidopsis phototropin. When brought together by interacting proteins, the fragments reconstitute a functional reporter that permits tagged protein complexes to be visualized by fluorescence light microscopy (LM), and then by standard as well as "multicolor" electron microscopy (EM) via the photooxidation of 3-3'-diaminobenzidine and its derivatives.

Despite the proliferation of proteins that can form filaments or phase-separated condensates, it remains unclear how this behavior is distributed over biological networks. We have found that 60 of the 440 yeast metabolic enzymes robustly form structures, including 10 that assemble within mitochondria. Additionally, the ability to assemble is enriched at branch points on several metabolic pathways. The assembly of enzymes at the first branch point in de novo purine biosynthesis is coordinated, hierarchical, and based on their position within the pathway, while the enzymes at the second branch point are recruited to RNA stress granules. Consistent with distinct classes of structures being deployed at different control points in a pathway, we find that the first enzyme in the pathway, PRPP synthetase, forms evolutionarily conserved filaments that are sequestered in the nucleus in higher eukaryotes. These findings provide a roadmap for identifying additional conserved features of metabolic regulation by condensates/filaments.

Self-associating split fluorescent proteins (FPs) are split FPs whose two fragments spontaneously associate to form a functional FP. They have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Recently developments have expanded the palette of self-associating split FPs beyond the original split GFP1-10/11. However, these new ones have suffered from suboptimal fluorescence signal after complementation. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living C. elegans, demonstrating its broad applications.

Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR-TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose-binding protein with a circularly permuted variant of the monomeric fluorescent protein T-Sapphire. Here, we provide more details on iGlucoSnFR-TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2-10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4-2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7-2.5 mM in cortical neurons from awake mice.

For thirsty animals, fluid intake provides both satiation and pleasure of drinking. How the brain processes these factors is currently unknown. Here, we identified neural circuits underlying thirst satiation and examined their contribution to reward signals. We show that thirst-driving neurons receive temporally distinct satiation signals by liquid-gulping-induced oropharyngeal stimuli and gut osmolality sensing. We demonstrate that individual thirst satiation signals are mediated by anatomically distinct inhibitory neural circuits in the lamina terminalis. Moreover, we used an ultrafast dopamine (DA) sensor to examine whether thirst satiation itself stimulates the reward-related circuits. Interestingly, spontaneous drinking behavior but not thirst drive reduction triggered DA release. Importantly, chemogenetic stimulation of thirst satiation neurons did not activate DA neurons under water-restricted conditions. Together, this study dissected the thirst satiation circuit, the activity of which is functionally separable from reward-related brain activity.

As a morphogen, Sonic Hedgehog (Shh) mediates signaling at a distance from its sites of synthesis. After secretion, Shh must traverse a distance through the extracellular matrix (ECM) to reach the target cells and activate the Hh response. ECM proteins, in particular, the heparan sulfate proteoglycans (HSPGs) of the glypican family, have both negative and positive effects on Shh signaling, all attributed to their ability to bind Shh. Using mouse embryonic stem cell-derived mosaic tissues with compartments that lack the glycosyltransferases Exostosin1 and Exostosin2, or the HSPG core protein Glypican5, we show that Shh accumulates around its source cells when they are surrounded by cells that have a mutated ECM. This accumulation of Shh is correlated with an increased noncell autonomous Shh response. Our results support a model in which Shh presented on the cell surface accumulates at or near ECM that lacks HSPGs, possibly due to the absence of these Shh sequestering molecules. Stem Cells 2019;37:899-909.

Brn3b (Pou4f2) is a class-4 POU domain transcription factor known to play central roles in the development of different neuronal populations of the Central Nervous System, including retinal ganglion cells (RGCs), the neurons that connect the retina with the visual centers of the brain. Here, we have used CRISPR-based genetic engineering to generate a Brn3b-mCherry reporter mouse without altering the endogenous expression of Brn3b. In our mouse line, mCherry faithfully recapitulates normal Brn3b expression in the retina, the optic tracts, the midbrain tectum, and the trigeminal ganglia. The high sensitivity of mCherry also revealed novel expression of Brn3b in the neuroectodermal cells of the optic stalk during early stages of eye development. Importantly, the fluorescent intensity of Brn3b-mCherry in our reporter mice allows for noninvasive live imaging of RGCs using Scanning Laser Ophthalmoscopy (SLO), providing a novel tool for longitudinal monitoring of RGCs.

Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white-opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.

Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Förster's resonance energy transfer (FRET) measurements, FP-tagged proteins can be used to study protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombic dipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and incoherently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy is distributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The interpretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weakly coupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typically associated with strong excitonic coupling, is limited by the FP β-barrel structure. Furthermore, physiological temperatures promote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasing times that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolution has shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitonic coupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by monitoring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause a change in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopy revealed Davydov splitting when the yellow FP VenusA206 dimerizes, and a novel approach combining photon antibunching and fluorescence correlation spectroscopy was used to confirm that the two fluorophores in a VenusA206 homodimer behave as a single-photon emitter. We conclude that excitonic coupling between VenusA206 fluorophores is possible at physiological temperatures.

Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.

Excitatory interneurons account for the majority of neurons in the superficial dorsal horn, but despite their presumed contribution to pain and itch, there is still limited information about their organisation and function. We recently identified 2 populations of excitatory interneuron defined by expression of gastrin-releasing peptide (GRP) or substance P (SP). Here, we demonstrate that these cells show major differences in their morphological, electrophysiological, and pharmacological properties. Based on their somatodendritic morphology and firing patterns, we propose that the SP cells correspond to radial cells, which generally show delayed firing. By contrast, most GRP cells show transient or single-spike firing, and many are likely to correspond to the so-called transient central cells. Unlike the SP cells, few of the GRP cells had long propriospinal projections, suggesting that they are involved primarily in local processing. The 2 populations also differed in responses to neuromodulators, with most SP cells, but few GRP cells, responding to noradrenaline and 5-HT; the converse was true for responses to the μ-opioid agonist DAMGO. Although a recent study suggested that GRP cells are innervated by nociceptors and are strongly activated by noxious stimuli, we found that very few GRP cells receive direct synaptic input from TRPV1-expressing afferents, and that they seldom phosphorylate extracellular signal-regulated kinases in response to noxious stimuli. These findings indicate that the SP and GRP cells differentially process somatosensory information.

During autophagy, actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in nonstarved cells, cytoplasmic JMY colocalizes with STRAP, a regulator of JMY's nuclear functions, on nonmotile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY's de novo actin nucleation activity via a cryptic actin-binding sequence near JMY's N terminus, and STRAP inhibits JMY's ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3- and JMY-dependent actin network formation on membranes and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY's actin assembly activities in trans during autophagy.

Atypical protein kinase C (aPKC) isozymes are unique in the PKC superfamily in that they are not regulated by the lipid second messenger diacylglycerol, which has led to speculation about whether a different second messenger acutely controls their function. Here, using a genetically encoded reporter that we designed, aPKC-specific C kinase activity reporter (aCKAR), we found that the lipid mediator sphingosine 1-phosphate (S1P) promoted the cellular activity of aPKC. Intracellular S1P directly bound to the purified kinase domain of aPKC and relieved autoinhibitory constraints, thereby activating the kinase. In silico studies identified potential binding sites on the kinase domain, one of which was validated biochemically. In HeLa cells, S1P-dependent activation of aPKC suppressed apoptosis. Together, our findings identify a previously undescribed molecular mechanism of aPKC regulation, a molecular target for S1P in cell survival regulation, and a tool to further explore the biochemical and biological functions of aPKC.

BACKGROUND:Activation of resident microglia accompanies every known form of neurodegeneration, but the involvement of peripheral monocytes that extravasate and rapidly transform into microglia-like macrophages within the central nervous system during degeneration is far less clear. METHODS:Using a combination of in vivo ocular imaging, flow cytometry, and immunohistochemistry, we investigated the response of infiltrating cells in a light-inducible mouse model of photoreceptor degeneration. RESULTS:Within 24 h, resident microglia became activated and began migrating to the site of degeneration. Retinal expression of CCL2 increased just prior to a transient period of CCR2+ cell extravasation from the retinal vasculature. Proliferation of microglia and monocytes occurred concurrently; however, there was no indication of proliferation in either population until 72-96 h after neurodegeneration began. Eliminating CCL2-CCR2 signaling blocked monocyte recruitment, but did not alter the extent of retinal degeneration. CONCLUSIONS:These results demonstrate that the immune response to photoreceptor degeneration includes both resident microglia and monocytes, even at very early times. Surprisingly, preventing monocyte infiltration did not block neurodegeneration, suggesting that in this model, degeneration is limited by cell clearance from other phagocytes or by the timing of intrinsic cell death programs. These results show monocyte involvement is not limited to disease states that overwhelm or deplete the resident microglial population and that interventions focused on modulating the peripheral immune system are not universally beneficial for staving off degeneration.