Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria.

Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection.

Peptidoglycan crosslinking relaxation promotes Helicobacter pylori's helical shape and stomach colonization.

The mechanisms by which bacterial cells generate helical cell shape and its functional role are poorly understood. Helical shape of the human pathogen Helicobacter pylori may facilitate penetration of the thick gastric mucus where it replicates. We identified four genes required for helical shape: three LytM peptidoglycan endopeptidase homologs (csd1-3) and a ccmA homolog. Surrounding the cytoplasmic membrane of most bacteria, the peptidoglycan (murein) sacculus is a meshwork of glycan strands joined by peptide crosslinks. Intact cells and isolated sacculi from mutants lacking any single csd gene or ccmA formed curved rods and showed increased peptidoglycan crosslinking. Quantitative morphological analyses of multiple-gene deletion mutants revealed each protein uniquely contributes to a shape-generating pathway. This pathway is required for robust colonization of the stomach in spite of normal directional motility. Our findings suggest that the coordinated action of multiple proteins relaxes peptidoglycan crosslinking, enabling helical cell curvature and twist.

MicroRNA silencing for cancer therapy targeted to the tumour microenvironment.

MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.

MicroRNAs as modulators of longevity and the aging process.

MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally repress translation or induce mRNA degradation of target transcripts through sequence-specific binding. miRNAs target hundreds of transcripts to regulate diverse biological pathways and processes, including aging. Many microRNAs are differentially expressed during aging, generating interest in their use as aging biomarkers and roles as regulators of the aging process. In the invertebrates Caenorhabditis elegans and Drosophila, a number of miRNAs have been found to both positive and negatively modulate longevity through canonical aging pathways. Recent studies have also shown that miRNAs regulate age-associated processes and pathologies in a diverse array of mammalian tissues, including brain, heart, bone, and muscle. The review will present an overview of these studies, highlighting the role of individual miRNAs as biomarkers of aging and regulators of longevity and tissue-specific aging processes.

A microRNA feedback loop regulates global microRNA abundance during aging.

Expression levels of many microRNAs (miRNAs) change during aging, notably declining globally in a number of organisms and tissues across taxa. However, little is known about the mechanisms or the biological relevance for this change. We investigated the network of genes that controls miRNA transcription and processing during C. elegans aging. We found that miRNA biogenesis genes are highly networked with transcription factors and aging-associated miRNAs. In particular, miR-71, known to influence life span and itself up-regulated during aging, represses alg-1/Argonaute expression post-transcriptionally during aging. Increased ALG-1 abundance in mir-71 loss-of-function mutants led to globally increased miRNA expression. Interestingly, these mutants demonstrated widespread mRNA expression dysregulation and diminished levels of variability both in gene expression and in overall life span. Thus, the progressive molecular decline often thought to be the result of accumulated damage over an organism's life may be partially explained by a miRNA-directed mechanism of age-associated decline.

A novel small molecule that disrupts a key event during the oocyte-to-embryo transition in C. elegans.

The complex cellular events that occur in response to fertilization are essential for mediating the oocyte-to-embryo transition. Here, we describe a comprehensive small-molecule screen focused on identifying compounds that affect early embryonic events in Caenorhabditis elegans We identify a single novel compound that disrupts early embryogenesis with remarkable stage and species specificity. The compound, named C22, primarily impairs eggshell integrity, leading to osmotic sensitivity and embryonic lethality. The C22-induced phenotype is dependent upon the upregulation of the LET-607/CREBH transcription factor and its candidate target genes, which primarily encode factors involved in diverse aspects of protein trafficking. Together, our data suggest that in the presence of C22, one or more key components of the eggshell are inappropriately processed, leading to permeable, inviable embryos. The remarkable specificity and reversibility of this compound will facilitate further investigation into the role and regulation of protein trafficking in the early embryo, as well as serve as a tool for manipulating the life cycle for other studies such as those involving aging.

High-throughput screening in the C. elegans nervous system.

The nematode Caenorhabditis elegans is widely used as a model organism in the field of neurobiology. The wiring of the C. elegans nervous system has been entirely mapped, and the animal's optical transparency allows for in vivo observation of neuronal activity. The nematode is also small in size, self-fertilizing, and inexpensive to cultivate and maintain, greatly lending to its utility as a whole-animal model for high-throughput screening (HTS) in the nervous system. However, the use of this organism in large-scale screens presents unique technical challenges, including reversible immobilization of the animal, parallel single-animal culture and containment, automation of laser surgery, and high-throughput image acquisition and phenotyping. These obstacles require significant modification of existing techniques and the creation of new C. elegans-based HTS platforms. In this review, we outline these challenges in detail and survey the novel technologies and methods that have been developed to address them.

Helicobacter pylori strains vary cell shape and flagellum number to maintain robust motility in viscous environments.

The helical shape of the human stomach pathogen Helicobacter pylori has been suggested to provide mechanical advantage for penetrating the viscous stomach mucus layer. Using single-cell tracking and quantitative morphology analysis, we document marked variation in cell body helical parameters and flagellum number among H. pylori strains leading to distinct and broad speed distributions in broth and viscous gastric mucin media. These distributions reflect both temporal variation in swimming speed and morphologic variation within the population. Isogenic mutants with straight-rod morphology showed 7-21% reduction in speed and a lower fraction of motile bacteria. Mutational perturbation of flagellum number revealed a 19% increase in speed with 4 versus 3 median flagellum number. Resistive force theory modeling incorporating variation of both cell shape and flagellum number predicts qualitative speed differences of 10-30% among strains. However, quantitative comparisons suggest resistive force theory underestimates the influence of cell body shape on speed for helical shaped bacteria.

Anthranilate fluorescence marks a calcium-propagated necrotic wave that promotes organismal death in C. elegans.

For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters--not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals--e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death.

Flow cytometry-based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter pylori morphology.

The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification programme involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has ld-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.

Multiple peptidoglycan modification networks modulate Helicobacter pylori's cell shape, motility, and colonization potential.

Helical cell shape of the gastric pathogen Helicobacter pylori has been suggested to promote virulence through viscosity-dependent enhancement of swimming velocity. However, H. pylori csd1 mutants, which are curved but lack helical twist, show normal velocity in viscous polymer solutions and the reason for their deficiency in stomach colonization has remained unclear. Characterization of new rod shaped mutants identified Csd4, a DL-carboxypeptidase of peptidoglycan (PG) tripeptide monomers and Csd5, a putative scaffolding protein. Morphological and biochemical studies indicated Csd4 tripeptide cleavage and Csd1 crosslinking relaxation modify the PG sacculus through independent networks that coordinately generate helical shape. csd4 mutants show attenuation of stomach colonization, but no change in proinflammatory cytokine induction, despite four-fold higher levels of Nod1-agonist tripeptides in the PG sacculus. Motility analysis of similarly shaped mutants bearing distinct alterations in PG modifications revealed deficits associated with shape, but only in gel-like media and not viscous solutions. As gastric mucus displays viscoelastic gel-like properties, our results suggest enhanced penetration of the mucus barrier underlies the fitness advantage conferred by H. pylori's characteristic shape.

Mechanism of shape determination in motile cells.

The shape of motile cells is determined by many dynamic processes spanning several orders of magnitude in space and time, from local polymerization of actin monomers at subsecond timescales to global, cell-scale geometry that may persist for hours. Understanding the mechanism of shape determination in cells has proved to be extremely challenging due to the numerous components involved and the complexity of their interactions. Here we harness the natural phenotypic variability in a large population of motile epithelial keratocytes from fish (Hypsophrys nicaraguensis) to reveal mechanisms of shape determination. We find that the cells inhabit a low-dimensional, highly correlated spectrum of possible functional states. We further show that a model of actin network treadmilling in an inextensible membrane bag can quantitatively recapitulate this spectrum and predict both cell shape and speed. Our model provides a simple biochemical and biophysical basis for the observed morphology and behaviour of motile cells.

MicroRNA predictors of longevity in Caenorhabditis elegans.

Neither genetic nor environmental factors fully account for variability in individual longevity: genetically identical invertebrates in homogenous environments often experience no less variability in lifespan than outbred human populations. Such variability is often assumed to result from stochasticity in damage accumulation over time; however, the identification of early-life gene expression states that predict future longevity would suggest that lifespan is least in part epigenetically determined. Such "biomarkers of aging," genetic or otherwise, nevertheless remain rare. In this work, we sought early-life differences in organismal robustness in unperturbed individuals and examined the utility of microRNAs, known regulators of lifespan, development, and robustness, as aging biomarkers. We quantitatively examined Caenorhabditis elegans reared individually in a novel apparatus and observed throughout their lives. Early-to-mid-adulthood measures of homeostatic ability jointly predict 62% of longevity variability. Though correlated, markers of growth/muscle maintenance and of metabolic by-products ("age pigments") report independently on lifespan, suggesting that graceful aging is not a single process. We further identified three microRNAs in which early-adulthood expression patterns individually predict up to 47% of lifespan differences. Though expression of each increases throughout this time, mir-71 and mir-246 correlate with lifespan, while mir-239 anti-correlates. Two of these three microRNA "biomarkers of aging" act upstream in insulin/IGF-1-like signaling (IIS) and other known longevity pathways, thus we infer that these microRNAs not only report on but also likely determine longevity. Thus, fluctuations in early-life IIS, due to variation in these microRNAs and from other causes, may determine individual lifespan.

Functional trajectories during innate spinal cord repair.

Adult zebrafish are capable of anatomical and functional recovery following severe spinal cord injury. Axon growth, glial bridging and adult neurogenesis are hallmarks of cellular regeneration during spinal cord repair. However, the correlation between these cellular regenerative processes and functional recovery remains to be elucidated. Whereas the majority of established functional regeneration metrics measure swim capacity, we hypothesize that gait quality is more directly related to neurological health. Here, we performed a longitudinal swim tracking study for 60 individual zebrafish spanning 8 weeks of spinal cord regeneration. Multiple swim parameters as well as axonal and glial bridging were integrated. We established rostral compensation as a new gait quality metric that highly correlates with functional recovery. Tensor component analysis of longitudinal data supports a correspondence between functional recovery trajectories and neurological outcomes. Moreover, our studies predicted and validated that a subset of functional regeneration parameters measured 1 to 2 weeks post-injury is sufficient to predict the regenerative outcomes of individual animals at 8 weeks post-injury. Our findings established new functional regeneration parameters and generated a comprehensive correlative database between various functional and cellular regeneration outputs.

Mutations in the nucleotide binding pocket of MreB can alter cell curvature and polar morphology in Caulobacter.

The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. Another subset have variable cell widths, with wide cell bodies and actively growing thin extensions of the cell poles that concentrate fluorescent MreB. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Further, we find that a mutation to glycine of two conserved aspartic acid residues that are important for nucleotide hydrolysis in other members of the actin superfamily abolishes robust midcell recruitment of MreB but supports a normal rate of growth. These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape.

High temporal resolution measurements of movement reveal novel early-life physiological decline in C. elegans.

Age-related physiological changes are most notable and best-studied late in life, while the nature of aging in early- or middle-aged individuals has not been explored as thoroughly. In C. elegans, many studies of movement vs. age generally focus on three distinct phases: sustained, youthful movement; onset of rapidly progressing impairment; and gross immobility. We investigated whether this first period of early-life adult movement is a sustained "healthy" level of high function followed by a discrete "movement catastrophe"-or whether there are early-life changes in movement that precede future physiological declines. To determine how movement varies during early adult life, we followed isolated individuals throughout life with a previously unachieved combination of duration and temporal resolution. By tracking individuals across the first six days of adulthood, we observed declines in movement starting as early as the first two days of adult life, as well as high interindividual variability in total daily movement. These findings suggest that movement is a highly dynamic behavior early in life, and that factors driving movement decline may begin acting as early as the first day of adulthood. Using simulation studies based on acquired data, we suggest that too-infrequent sampling in common movement assays limits observation of early-adult changes in motility, and we propose feasible strategies and a framework for designing assays with increased sensitivity for early movement declines.

Developmental biomarkers of aging in Caenorhabditis elegans.

The developmental process of the nematode Caenorhabditis elegans is famously invariant; however, these animals have surprisingly variable lifespans, even in extremely homogenous environments. Inter-individual differences in muscle-function decline, accumulation of lipofuscin in the gut, internal growth of food bacteria, and ability to mobilize heat-shock responses all appear to be predictive of a nematode's remaining lifespan; whether these are causal, or mere correlates of individual decline and death, has yet to be determined. Moreover, few "upstream" causes of inter-individual variability have been identified. It may be the case that variability in lifespan is entirely due to stochastic damage accumulation; alternately, perhaps such variability has a developmental origin and/or genes involved in developmental canalization also act to buffer phenotypic heterogeneity later in life. We review these two hypotheses with an eye toward whether they can be experimentally differentiated.

Global, cell non-autonomous gene regulation drives individual lifespan among isogenic C. elegans.

Across species, lifespan is highly variable among individuals within a population. Even genetically identical Caenorhabditis elegans reared in homogeneous environments are as variable in lifespan as outbred human populations. We hypothesized that persistent inter-individual differences in expression of key regulatory genes drives this lifespan variability. As a test, we examined the relationship between future lifespan and the expression of 22 microRNA promoter::GFP constructs. Surprisingly, expression of nearly half of these reporters, well before death, could effectively predict lifespan. This indicates that prospectively long- vs. short-lived individuals have highly divergent patterns of transgene expression and transcriptional regulation. The gene-regulatory processes reported on by two of the most lifespan-predictive transgenes do not require DAF-16, the FOXO transcription factor that is a principal effector of insulin/insulin-like growth factor (IGF-1) signaling. Last, we demonstrate a hierarchy of redundancy in lifespan-predictive ability among three transgenes expressed in distinct tissues, suggesting that they collectively report on an organism-wide, cell non-autonomous process that acts to set each individual's lifespan.

Emergence of large-scale cell morphology and movement from local actin filament growth dynamics.

Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actin-remodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged "canoe" shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features--cell shape, leading-edge shape, filamentous actin (F-actin) distribution, cell speed, and directional persistence--that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes--which had VASP highly enriched at their leading edges and migrated fast with straight trajectories--to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and behavior phenotypes as large-scale consequences of kinetic contributions of VASP to actin filament growth and protection from capping at the leading edge. This work shows that the local effects of actin-remodeling proteins on cytoskeletal dynamics and organization can manifest as global modifications of the shape and behavior of migrating cells and that mathematical modeling can elucidate these large-scale cell behaviors from knowledge of detailed multiscale protein interactions.