Living cells as a biological analog of optical tweezers - a non-invasive microrheology approach.

Microrheology, the study of fluids on micron length-scales, promises to reveal insights into cellular biology, including mechanical biomarkers of disease and the interplay between biomechanics and cellular function. Here a minimally-invasive passive microrheology technique is applied to individual living cells by chemically binding a bead to the surface of a cell, and observing the mean squared displacement of the bead at timescales ranging from milliseconds to 100s of seconds. Measurements are repeated over the course of hours, and presented alongside analysis to quantify changes in the cells' low-frequency elastic modulus, G0', and the cell's dynamics over the time window ∼10-2 s to 10 s. An analogy to optical trapping allows verification of the invariant viscosity of HeLa S3 cells under control conditions and after cytoskeletal disruption. Stiffening of the cell is observed during cytoskeletal rearrangement in the control case, and cell softening when the actin cytoskeleton is disrupted by Latrunculin B. These data correlate with conventional understanding that integrin binding and recruitment triggers cytoskeletal rearrangement. This is, to our knowledge, the first time that cell stiffening has been measured during focal adhesion maturation, and the longest time over which such stiffening has been quantified by any means. STATEMENT OF SIGNIFICANCE: Here, we present an approach for studying mechanical properties of live cells without applying external forces or inserting tracers. Regulation of cellular biomechanics is crucial to healthy cell function. For the first time in literature, we can non-invasively and passively quantify cell mechanics during interactions with functionalised surface. Our method can monitor the maturation of adhesion sites on the surface of individual live cells without disrupting the cell mechanics by applying forces to the cell. We observe a stiffening response in cells over tens of minutes after a bead chemically binds. This stiffening reduces the deformation rate of the cytoskeleton, although the internal force generation increases. Our method has potential for applications to study mechanics during cell-surface and cell-vesicle interactions.

A mechanoresponsive nano-sized carrier achieves intracellular release of drug on external ultrasound stimulus.

Control over intracellular release of therapeutic compounds incorporated into nano-carriers will open new possibilities for targeted treatments of various diseases including cancer, and viral and bacterial infections. Here we report our study on mechanoresponsive nano-sized liposomes which, following internalization by cells, achieve intracellular delivery of encapsulated cargo on application of external ultrasound stimulus. This is demonstrated in a bespoke cell reporter system designed to assess free drug in cytoplasm. Biophysical analyses show that drug release is attributable to the action of a mechanoresponsive spiropyran-based compound embedded in the liposomal lipid membrane. Exposure to external ultrasound stimulus results in opening of the molecular structure of the embedded spiropyran, a consequent increase in liposomal lipid membrane fluidity, and size-dependent release of encapsulated model drugs, all pointing to lipid bilayer perturbation. The study hence illustrates feasibility of the proposed concept where intracellular drug release from mechanoresponsive liposomes can be triggered on demand by external ultrasound stimulus.

Synthesis and Antibacterial Evaluation of New Pyrazolo[3,4-d]pyrimidines Kinase Inhibitors.

Pyrazolo[3,4-d]pyrimidines represent an important class of heterocyclic compounds well-known for their anticancer activity exerted by the inhibition of eukaryotic protein kinases. Recently, pyrazolo[3,4-d]pyrimidines have become increasingly attractive for their potential antimicrobial properties. Here, we explored the activity of a library of in-house pyrazolo[3,4-d]pyrimidines, targeting human protein kinases, against Staphylococcus aureus and Escherichia coli and their interaction with ampicillin and kanamycin, representing important classes of clinically used antibiotics. Our results represent a first step towards the potential application of dual active pyrazolo[3,4-d]pyrimidine kinase inhibitors in the prevention and treatment of bacterial infections in cancer patients.

A multi-phenotypic imaging screen to identify bacterial effectors by exogenous expression in a HeLa cell line.

We present a high-content screen (HCS) for the simultaneous analysis of multiple phenotypes in HeLa cells expressing an autophagy reporter (mcherry-LC3) and one of 224 GFP-fused proteins from the Crohn's Disease (CD)-associated bacterium, Adherent Invasive E. coli (AIEC) strain LF82. Using automated confocal microscopy and image analysis (CellProfiler), we localised GFP fusions within cells, and monitored their effects upon autophagy (an important innate cellular defence mechanism), cellular and nuclear morphology, and the actin cytoskeleton. This data will provide an atlas for the localisation of 224 AIEC proteins within human cells, as well as a dataset to analyse their effects upon many aspects of host cell morphology. We also describe an open-source, automated, image-analysis workflow to identify bacterial effectors and their roles via the perturbations induced in reporter cell lines when candidate effectors are exogenously expressed.

The LRR and RING domain protein LRSAM1 is an E3 ligase crucial for ubiquitin-dependent autophagy of intracellular Salmonella Typhimurium.

Several species of pathogenic bacteria replicate within an intracellular vacuolar niche. Bacteria that escape into the cytosol are captured by the autophagic pathway and targeted for lysosomal degradation, representing a defense against bacterial exploitation of the host cytosol. Autophagic capture of Salmonella Typhimurium occurs predominantly via generation of a polyubiquitin signal around cytosolic bacteria, binding of adaptor proteins, and recruitment of autophagic machinery. However, the components mediating bacterial target selection and ubiquitination remain obscure. We identify LRSAM1 as the E3 ligase responsible for anti-Salmonella autophagy-associated ubiquitination. LRSAM1 localizes to several intracellular bacterial pathogens and generates the bacteria-associated ubiquitin signal; these functions require LRSAM1's leucine-rich repeat and RING domains, respectively. Using cells from LRSAM1-deficient individuals, we confirm that LRSAM1 is required for ubiquitination associated with intracellular bacteria but dispensable for ubiquitination of aggregated proteins. LRSAM1 is therefore a bacterial recognition protein and ubiquitin ligase that defends the cytoplasm from invasive pathogens.

Tribbles 2 (Trib2) is a novel regulator of toll-like receptor 5 signaling.

BACKGROUND: Toll-like receptors (TLRs) are expressed by a variety of cells, including intestinal epithelia. However, the full spectrum of regulators modulating innate responses via TLRs has not been delineated. Tribbles (Trib) have been identified as a highly conserved family of kinase-like proteins. We sought to clarify the role of Trib2 in the TLR signaling pathway. METHODS: Trib2 mRNA and protein levels were analyzed by quantitative polymerase chain reaction (PCR) and western blot, respectively. Immunohistochemical staining was used to determine the expression of Trib2 in human tissue. Involvement of Trib2 in nuclear factor kappa B (NF-κB) pathways was assessed in epithelial cells by NF-κB reporter assay. Proteins that interacted with Trib2 were identified by mass spectrometry and confirmed by immunoprecipitation. The domain essential for Trib2 function was mapped using truncated constructs. RESULTS: Trib2 expression is decreased in active inflamed tissue from patients with inflammatory bowel disease (IBD). Trib2 is expressed in human and mouse colonic epithelium as well as immune cells, and its expression in epithelium is inducible in a ligand-dependent manner by TLR5 ligand stimulation. Trib2 inhibits TLR5-mediated activation of NF-κB downstream of TRAF6. Trib2 selectively modulates mitogen-activated protein kinase (MAPK) pathways p38 and Jun N-terminal kinase (JNK) but not p44/p42 (ERK1/2). NF-κB2 (p100) was identified as a Trib2 binding partner in regulating the TLR5 signaling pathway that leads to inhibition of NF-κB activity. Residues 158-177 in the Trib2 kinase-like domain are required for Trib2 function. CONCLUSIONS: These observations indicate that Trib2 is a novel regulator in the TLR5 signaling pathway and altered expression of Trib2 may play a role in IBD.

Crohn disease: a current perspective on genetics, autophagy and immunity.

Crohn disease (CD) is a chronic and debilitating inflammatory condition of the gastrointestinal tract. Prevalence in Western populations is 100-150/100,000 and somewhat higher in Ashkenazi Jews. Peak incidence is in early adult life, although any age can be affected and a majority of affected individuals progress to relapsing and chronic disease. Medical treatments rely significantly on empirical corticosteroid therapy and immunosuppression, and intestinal resectional surgery is frequently required. Thus, 80% of patients with CD come to surgery for refractory disease or complications. It is hoped that an improved understanding of pathogenic mechanisms, for example by studying the genetic basis of CD and other forms of inflammatory bowel diseases (IBD), will lead to improved therapies and possibly preventative strategies in individuals identified as being at risk.

Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity.

In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for "nucleic acid-binding" function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)-dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.

A systems biology viewpoint on autophagy in health and disease.

PURPOSE OF REVIEW: The field of autophagy is rapidly expanding to encompass many important areas of cell biology, physiology and disease. Recent discoveries and tools allow the connection of the autophagy pathway to other cellular signals and processes, thus beginning a systematic approach to elucidation of autophagy components, functions and connections. RECENT FINDINGS: We outline recent discoveries illustrating the role of autophagy in Parkinson's disease, inflammatory bowel disease (IBD) and cancer. Recently important details of the mechanisms by which autophagy operates in these contexts have been elucidated. We illustrate how autophagy can be triggered by diverse stimuli and how cell fate is determined by the responses to many signals and stresses. We discuss the known links between autophagy and apoptosis and present a working model of the current interactions between autophagy components, apoptosis and cell cycle control at different stages of autophagic vesicle progression. SUMMARY: Autophagy represents not only an essential metabolic process, but a hub which responds to diverse stresses and signals to aid cell survival or control cell fate. There are currently many known links between autophagy and disease states, and the pace of discovery appears to be accelerating. Thus an understanding of autophagy is likely to be crucial to current and future approaches to therapy. Here we give a systems biology view of the autophagy field and how it is being connected to other pathways, such as apoptosis and responses to reactive oxygen damage.

Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly.

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome-wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD-associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3-positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non-pathogenic, environmental, commensal, or pathogenic strains involved in gastro enteritis. Together these findings demonstrate a central role for autophagy restraining Adherent-Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation.

Autophagy at the gut interface: mucosal responses to stress and the consequences for inflammatory bowel diseases.

Autophagy is a conserved homeostatic process by which cells degrade and recycle cytoplasmic contents and organelles. Recently, autophagy has come to prominence as a factor in many disease states, including inflammatory bowel diseases. In this review we explore the recent discoveries in autophagy and how these relate to the special conditions experienced by the gut mucosa. We will pay particular attention to autophagy as an innate immune process and its role in the development and education of the adaptive immune system.

A novel hybrid yeast-human network analysis reveals an essential role for FNBP1L in antibacterial autophagy.

Autophagy is a conserved cellular process required for the removal of defective organelles, protein aggregates, and intracellular pathogens. We used a network analysis strategy to identify novel human autophagy components based upon the yeast interactome centered on the core yeast autophagy proteins. This revealed the potential involvement of 14 novel mammalian genes in autophagy, several of which have known or predicted roles in membrane organization or dynamics. We selected one of these membrane interactors, FNBP1L (formin binding protein 1-like), an F-BAR-containing protein (also termed Toca-1), for further study based upon a predicted interaction with ATG3. We confirmed the FNBP1L/ATG3 interaction biochemically and mapped the FNBP1L domains responsible. Using a functional RNA interference approach, we determined that FNBP1L is essential for autophagy of the intracellular pathogen Salmonella enterica serovar Typhimurium and show that the autophagy process serves to restrict the growth of intracellular bacteria. However, FNBP1L appears dispensable for other forms of autophagy induced by serum starvation or rapamycin. We present a model where FNBP1L is essential for autophagy of intracellular pathogens and identify FNBP1L as a differentially used molecule in specific autophagic contexts. By using network biology to derive functional biological information, we demonstrate the utility of integrated genomics to novel molecule discovery in autophagy.

The cytoskeletal scaffold Shank3 is recruited to pathogen-induced actin rearrangements.

The common gastrointestinal pathogens enteropathogenic Escherichia coli (EPEC) and Salmonella typhimurium both reorganize the gut epithelial cell actin cytoskeleton to mediate pathogenesis, utilizing mimicry of the host signaling apparatus. The PDZ domain-containing protein Shank3, is a large cytoskeletal scaffold protein with known functions in neuronal morphology and synaptic signaling, and is also capable of acting as a scaffolding adaptor during Ret tyrosine kinase signaling in epithelial cells. Using immunofluorescent and functional RNA-interference approaches we show that Shank3 is present in both EPEC- and S. typhimurium-induced actin rearrangements and is required for optimal EPEC pedestal formation. We propose that Shank3 is one of a number of host synaptic proteins likely to play key roles in bacteria-host interactions.

On the level: IRGM gene function is all about expression.

Crohn disease is a complex, multigenic, chronic inflammatory bowel disease of uncertain etiology. Recent advances in genetics, including high-throughput single-nucleotide polymorphism typing platforms and deep sequencing technologies have begun to shed light upon disease predisposition and pathogenesis. Autophagy is emerging as a key player in both innate and adaptive immunity, as well as tissue homeostasis and development in the gut. Here we describe our recent studies into the Crohn disease-associated Immunity-Related GTPase family, M (IRGM) gene and our discovery of a large risk-conferring upstream deletion. We discuss the effects of this deletion upon expression levels of IRGM alleles and how tissue-specific expression might be affected by the promoter polymorphism. In addition, we comment upon the potential roles of IRGM in autophagy of intracellular pathogens, and the challenges ahead for further elucidating IRGM function.

Impaired autophagy of an intracellular pathogen induced by a Crohn's disease associated ATG16L1 variant.

The genetic risk factors predisposing individuals to the development of inflammatory bowel disease are beginning to be deciphered by genome-wide association studies. Surprisingly, these new data point towards a critical role of autophagy in the pathogenesis of Crohn's disease. A single common coding variant in the autophagy protein ATG16L1 predisposes individuals to the development of Crohn's disease: while ATG16L1 encoding threonine at amino acid position 300 (ATG16L1*300T) confers protection, ATG16L1 encoding for alanine instead of threonine (ATG16L1*300A, also known as T300A) mediates risk towards the development of Crohn's disease. Here we report that, in human epithelial cells, the Crohn's disease-associated ATG16L1 coding variant shows impairment in the capture of internalized Salmonella within autophagosomes. Thus, we propose that the association of ATG16L1*300A with increased risk of Crohn's disease is due to impaired bacterial handling and lowered rates of bacterial capture by autophagy.