Multiscale imaging of basal cell dynamics in the functionally mature mammary gland.

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Modified horseshoe crab peptides target and kill bacteria inside host cells.

Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell-penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two ß-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogs [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell-penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

Uropathogenic Escherichia coli employs both evasion and resistance to subvert innate immune-mediated zinc toxicity for dissemination.

Toll-like receptor (TLR)-inducible zinc toxicity is a recently described macrophage antimicrobial response used against bacterial pathogens. Here we investigated deployment of this pathway against uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections. Primary human macrophages subjected EC958, a representative strain of the globally disseminated multidrug-resistant UPEC ST131 clone, to zinc stress. We therefore used transposon-directed insertion site sequencing to identify the complete set of UPEC genes conferring protection against zinc toxicity. Surprisingly, zinc-susceptible EC958 mutants were not compromised for intramacrophage survival, whereas corresponding mutants in the nonpathogenic E. coli K-12 strain MG1655 displayed significantly reduced intracellular bacterial loads within human macrophages. To investigate whether the intramacrophage zinc stress response of EC958 reflected the response of only a subpopulation of bacteria, we generated and validated reporter systems as highly specific sensors of zinc stress. Using these tools we show that, in contrast to MG1655, the majority of intramacrophage EC958 evades the zinc toxicity response, enabling survival within these cells. In addition, EC958 has a higher tolerance to zinc than MG1655, with this likely being important for survival of the minor subset of UPEC cells exposed to innate immune-mediated zinc stress. Indeed, analysis of zinc stress reporter strains and zinc-sensitive mutants in an intraperitoneal challenge model in mice revealed that EC958 employs both evasion and resistance against zinc toxicity, enabling its dissemination to the liver and spleen. We thus demonstrate that a pathogen of global significance uses multiple mechanisms to effectively subvert innate immune-mediated zinc poisoning for systemic spread.

LLAMA: a robust and scalable machine learning pipeline for analysis of large scale 4D microscopy data: analysis of cell ruffles and filopodia.

BACKGROUND: With recent advances in microscopy, recordings of cell behaviour can result in terabyte-size datasets. The lattice light sheet microscope (LLSM) images cells at high speed and high 3D resolution, accumulating data at 100 frames/second over hours, presenting a major challenge for interrogating these datasets. The surfaces of vertebrate cells can rapidly deform to create projections that interact with the microenvironment. Such surface projections include spike-like filopodia and wave-like ruffles on the surface of macrophages as they engage in immune surveillance. LLSM imaging has provided new insights into the complex surface behaviours of immune cells, including revealing new types of ruffles. However, full use of these data requires systematic and quantitative analysis of thousands of projections over hundreds of time steps, and an effective system for analysis of individual structures at this scale requires efficient and robust methods with minimal user intervention. RESULTS: We present LLAMA, a platform to enable systematic analysis of terabyte-scale 4D microscopy datasets. We use a machine learning method for semantic segmentation, followed by a robust and configurable object separation and tracking algorithm, generating detailed object level statistics. Our system is designed to run on high-performance computing to achieve high throughput, with outputs suitable for visualisation and statistical analysis. Advanced visualisation is a key element of LLAMA: we provide a specialised tool which supports interactive quality control, optimisation, and output visualisation processes to complement the processing pipeline. LLAMA is demonstrated in an analysis of macrophage surface projections, in which it is used to i) discriminate ruffles induced by lipopolysaccharide (LPS) and macrophage colony stimulating factor (CSF-1) and ii) determine the autonomy of ruffle morphologies. CONCLUSIONS: LLAMA provides an effective open source tool for running a cell microscopy analysis pipeline based on semantic segmentation, object analysis and tracking. Detailed numerical and visual outputs enable effective statistical analysis, identifying distinct patterns of increased activity under the two interventions considered in our example analysis. Our system provides the capacity to screen large datasets for specific structural configurations. LLAMA identified distinct features of LPS and CSF-1 induced ruffles and it identified a continuity of behaviour between tent pole ruffling, wave-like ruffling and filopodia deployment.

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus.

Cyclotides are plant-derived peptides characterized by an ∼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

Broadening the spectrum of fluorescent protein tools for use in the encapsulated human fungal pathogen Cryptococcus neoformans.

Green fluorescent protein (GFP) and its counterparts are modern molecular biology research tools indispensable in many experimental systems. Within fungi, researchers studying Saccharomyces cerevisiae and other model ascomycetes have access to a wide variety of fluorescent proteins. Unfortunately, many of these tools have not crossed the phylum divide into the Basidiomycota, where only GFP S65T, Venus, Ds-Red, and mCherry are currently available. To address this, we searched the literature for potential candidates to be expressed in the human fungal pathogen Cryptococcus neoformans and identified a suite of eight more modern fluorescent proteins that span the visible spectrum. A single copy of each fluorophore was heterologously expressed in Safe Haven 1 and their fluorescence intensities compared in this encapsulated yeast. mTurquoise2, mTFP1, Clover, mNeonGreen, mRuby3, and Citrine were highly visible under the microscope, whereas Superfolder GFP and mMaroon1 were not. Expressed fluorophores did not impact growth or virulence as demonstrated by an in vitro spotting assay and murine inhalation model, respectively.

Intertidal gobies acclimate rate of luminance change for background matching with shifts in seasonal temperature.

Rate of colour change and background matching capacity are important functional traits for avoiding predation and hiding from prey. Acute changes in environmental temperature are known to impact the rate at which animals change colour, and therefore may affect their survival. Many ectotherms have the ability to acclimate performance traits such as locomotion, metabolic rate and growth rate with changes in seasonal temperature. However, it remains unclear how other functional traits that are directly linked to behaviour and survival respond to long-term changes in temperature (within an individual's lifetime). We assessed whether the rate of colour change is altered by long-term changes in temperature (seasonal variation) and if rate of colour change can acclimate to seasonal thermal conditions. We used an intertidal rock-pool goby Bathygobius cocosensis, to test this and exposed individuals to representative seasonal mean temperatures (16 or 31°C, herein referred to cold- and warm-exposed fish respectively) for 9 weeks and then tested their rate of luminance change when placed on white and black backgrounds at acute test temperatures 16 and 31°C. We modelled rate of luminance change using the visual sensitives of a coral trout Plectropmus leopardus to determine how well gobies matched their backgrounds in terms of luminance contrast to a potential predator. After exposure to long-term seasonal conditions, the warm-exposed fish had faster rates of luminance change and matched their background more closely when tested at 31 than at 16°C. Similarly, the cold-exposed fish had faster rates of luminance change and matched their backgrounds more closely at 16°C than at 31°C. This demonstrates that rate of luminance change can be adjusted to compensate for long-term changes in seasonal temperature. This is the first study to show that animals can acclimate rate of colour change for background matching to seasonal thermal conditions. We also show that rapid changes in acute temperature reduce background matching capabilities. Stochastic changes in climate are likely to affect the frequency of predator-prey interactions which may have substantial knock-on effects throughout ecosystems.

Distinct Roles for APPL1 and APPL2 in Regulating Toll-like Receptor 4 Signaling in Macrophages.

Macrophages are activated by contact with pathogens to mount innate immune defenses against infection. Toll-like receptor 4 (TLR4) at the macrophage surface recognizes and binds bacterial lipopolysaccharide (LPS), setting off signaling and transcriptional events that lead to the secretion of pro- and anti-inflammatory cytokines; these in turn control inflammatory and antimicrobial responses. Although the complex regulatory pathways downstream of TLR4 have been extensively studied, further molecules critical for modulating the resulting cytokine outputs remain to be characterized. Here we establish potential roles for APPL1 and 2 signaling adaptors as regulators of LPS/TLR4-induced signaling, transcription, and cytokine secretion. APPL1 and 2 are differentially localized to distinct signaling-competent membrane domains on the surface and in endocytic compartments of LPS-activated macrophages. By depleting cells of each adaptor respectively we show separate and opposing functions for APPL1 and 2 in Akt and MAPK signaling. Specifically, APPL2 has a dominant role in nuclear translocation of NF-KB p65 and it serves to constrain the secretion of pro- and anti-inflammatory cytokines. The APPLs, and in particular APPL2, are thus revealed as adaptors with important capacity to modulate inflammatory responses mounted by LPS/TLR4 during infection.

Small GTPase Rab8a-recruited Phosphatidylinositol 3-Kinase γ Regulates Signaling and Cytokine Outputs from Endosomal Toll-like Receptors.

LPS-mediated activation of Toll-like receptor 4 (TLR4) in macrophages results in the coordinated release of proinflammatory cytokines, followed by regulatory mediators, to ensure that this potentially destructive pathway is tightly regulated. We showed previously that Rab8a recruits PI3Kγ for Akt-dependent signaling during TLR4 activation to limit the production of the proinflammatory cytokines IL-6 and IL-12p40 while enhancing the release of the regulatory/anti-inflammatory cytokine IL-10. Here we broaden the array of immune receptors controlled by Rab8a-PI3Kγ and further define the Rab-mediated membrane domains required for signaling. With CRISPR/Cas9-mediated gene editing to stably knock out and recover Rab8a in macrophage cell lines, we match Akt signaling profiles with cytokine outputs, confirming that Rab8a is a novel regulator of the Akt/mammalian target of rapamycin (mTOR) pathway downstream of multiple TLRs. Upon developing a Rab8a activation assay, we show that TLR3 and 9 agonists also activate Rab8a. Live-cell imaging reveals that Rab8a is first recruited to the plasma membrane and dorsal ruffles, but it is retained during collapse of ruffles to form macropinosomes enriched for phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), suggesting that the macropinosome is the location where Rab8a is active. We pinpoint macropinosomes as the sites for Rab8-mediated biasing of inflammatory signaling responses via inducible production of anti-inflammatory cytokines. Thus, Rab8a and PI3Kγ are positioned in multiple TLR pathways, and this signaling axis may serve as a pharmacologically tractable target during infection and inflammation.

On linear models and parameter identifiability in experimental biological systems.

A key problem in the biological sciences is to be able to reliably estimate model parameters from experimental data. This is the well-known problem of parameter identifiability. Here, methods are developed for biologists and other modelers to design optimal experiments to ensure parameter identifiability at a structural level. The main results of the paper are to provide a general methodology for extracting parameters of linear models from an experimentally measured scalar function - the transfer function - and a framework for the identifiability analysis of complex model structures using linked models. Linked models are composed by letting the output of one model become the input to another model which is then experimentally measured. The linked model framework is shown to be applicable to designing experiments to identify the measured sub-model and recover the input from the unmeasured sub-model, even in cases that the unmeasured sub-model is not identifiable. Applications for a set of common model features are demonstrated, and the results combined in an example application to a real-world experimental system. These applications emphasize the insight into answering "where to measure" and "which experimental scheme" questions provided by both the parameter extraction methodology and the linked model framework. The aim is to demonstrate the tools' usefulness in guiding experimental design to maximize parameter information obtained, based on the model structure.

Ras-Related Protein Rab5a Regulates Complement C5a Receptor Trafficking, Chemotaxis, and Chemokine Secretion in Human Macrophages.

Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits ß-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with ß-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or ß-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-ß-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs.

Quantifying Regulated Mitochondrial Fission in Macrophages.

Mitochondria have co-evolved with eukaryotic cells for more than a billion years, becoming an important cog in their machinery. They are best known for being tasked with energy generation through the production of adenosine triphosphate, but they also have roles in several other cellular processes, for example, immune and inflammatory responses. Mitochondria have important functions in macrophages, key innate immune cells that detect pathogens and drive inflammation. Mitochondrial activity is influenced by the highly dynamic nature of the mitochondrial network, which alternates between interconnected tubular and fragmented forms. The dynamic balance between this interconnected fused network and fission-mediated mitochondrial fragmentation modulates inflammatory responses such as production of cytokines and mitochondrial reactive oxygen species. Here we describe methods to differentiate mouse bone marrow cells into macrophages and the use of light microscopy, electron microscopy, flow cytometry, and Western blotting to quantify regulated mitochondrial dynamics in these differentiated macrophages.

Host glycocalyx captures HIV proximal to the cell surface via oligomannose-GlcNAc glycan-glycan interactions to support viral entry.

Here, we present ultrastructural analyses showing that incoming HIV are captured near the lymphocyte surface in a virion-glycan-dependent manner. Biophysical analyses show that removal of either virion- or cell-associated N-glycans impairs virus-cell binding, and a similar glycan-dependent relationship is observed between purified HIV envelope (Env) and primary T cells. Trimming of N-glycans from either HIV or Env does not inhibit protein-protein interactions. Glycan arrays reveal HIV preferentially binds to N-acetylglucosamine and mannose. Interfering with these glycan-based interactions reduces HIV infectivity. These glycan interactions are distinct from previously reported glycan-lectin and non-specific electrostatic charge-based interactions. Specific glycan-glycan-mediated attachment occurs prior to virus entry and enhances efficiency of infection. Binding and fluorescent imaging data support glycan-glycan interactions as being responsible, at least in part, for initiating contact between HIV and the host cell, prior to viral Env-cellular CD4 engagement.

Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells.

Peptides are being developed as targeted anticancer drugs to modulate cytosolic protein-protein interactions involved in cancer progression. However, their use as therapeutics is often limited by their low cell membrane permeation and/or inability to reach cytosolic targets. Conjugation to cell penetrating peptides has been successfully used to improve the cytosolic delivery of high affinity binder peptides, but cellular uptake does not always result in modulation of the targeted pathway. To overcome this limitation, we developed "angler peptides" by conjugating KD3, a noncell permeable but potent and specific peptide inhibitor of p53:MDM2 and p53:MDMX interactions, with a set of cyclic cell-penetrating peptides. We examined their binding affinity for MDM2 and MDMX, the cell entry mechanism, and role in reactivation of the p53 pathway. We identified two angler peptides, cTAT-KD3 and cR10-KD3, able to activate the p53 pathway in cancer cells. cTAT-KD3 entered cells via endocytic pathways, escaped endosomes, and activated the p53 pathway in breast (MCF7), lung (A549), and colon (HCT116) cancer cell lines at concentrations in the range of 1-12 µM. cR10-KD3 reached the cytosol via direct membrane translocation and activated the p53 pathway at 1 µM in all the tested cell lines. Our work demonstrates that nonpermeable anticancer peptides can be delivered into the cytosol and inhibit intracellular cancer pathways when they are conjugated with stable cell penetrating peptides. The mechanistic studies suggest that direct translocation leads to less toxicity, higher cytosol delivery at lower concentrations, and lower dependencies on the membrane of the tested cell line than occurs for an endocytic pathway with endosomal escape. The angler strategy can rescue high affinity peptide binders identified from high throughput screening and convert them into targeted anticancer therapeutics, but investigation of their cellular uptake and cell death mechanisms is essential to confirming modulation of the targeted cancer pathways.

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis.

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair, and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation, and migration. Extracellular signal-regulated kinase (Erk) is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response, however, involves a Vegfr-dependent mechanism that initiates concomitantly with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.

SCIMP is a spatiotemporal transmembrane scaffold for Erk1/2 to direct pro-inflammatory signaling in TLR-activated macrophages.

Immune cells are armed with Toll-like receptors (TLRs) for sensing and responding to pathogens and other danger cues. The role of extracellular-signal-regulated kinases 1/2 (Erk1/2) in TLR signaling remains enigmatic, with both pro- and anti-inflammatory functions described. We reveal here that the immune-specific transmembrane adaptor SCIMP is a direct scaffold for Erk1/2 in TLR pathways, with high-resolution, live-cell imaging revealing that SCIMP guides the spatial and temporal recruitment of Erk2 to membrane ruffles and macropinosomes for pro-inflammatory TLR4 signaling. SCIMP-deficient mice display defects in Erk1/2 recruitment to TLR4, c-Fos activation, and pro-inflammatory cytokine production, with these effects being phenocopied by Erk1/2 signaling inhibition. Our findings thus delineate a selective role for SCIMP as a key scaffold for the membrane recruitment of Erk1/2 kinase to initiate TLR-mediated pro-inflammatory responses in macrophages.

High-speed squeeze: Light-sheet imaging of zebrafish neutrophils.

Discussion of lattice light sheet microscopy used for high resolution 3D imaging of neutrophil behaviors in zebrafish larvae.

Automated Analysis of Cell Surface Ruffling: Ruffle Quantification Macro.

Cell surface protrusions include F-actin rich, wave-like ruffles that are erected transiently in response to stimuli and during cell migration. Macrophages are innate immune cells that ruffle constitutively and more dramatically in cells activated by pathogens. Dorsal ruffles and their resulting macropinosomes are key sites for environmental sampling, pathogen detection and immune signaling. Quantitative assessment of ruffling is important for assessing pathogen responses in macrophages and for analysis of growth factor responses in other cell types but automated and quantitative methods are lacking, and rely on manual and qualitative assessments. Here we present an automated ImageJ macro for quantifying dorsal cell surface protrusions from 3D microscope images. The assay presented here is suitable for high-throughput screening applications to detect drug, pathogen, or growth factor induced changes in cell ruffling by measuring ruffle area and intensity and providing normalized values in an easy to read combined spreadsheet.

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold.

Cyclotides are macrocyclic peptides with exceptionally stable structures and have been reported to penetrate cells, making them promising scaffolds for the delivery of inhibitory peptides to target intracellular proteins. However, their cellular uptake and cytosolic localization have been poorly understood until now, which has limited their therapeutic potential. In this study, the recently developed chloroalkane penetration assay was combined with established assays to characterize the cellular uptake and cytosolic delivery of the prototypic cyclotide, kalata B1. We show that kalata B1 enters the cytosol at low efficiency. A structure-activity study of residues in loop 6 showed that some modifications, such as increasing cationic residue content, did not affect delivery efficiency, whereas others, including introducing a single hydrophobic amino acid, did significantly improve cytosolic delivery. Our results provide a foundation for the further development of a structurally unique class of scaffolds for the delivery of therapeutic cargoes into cells.