The rapid development of high throughput biotechnologies has led to an onslaught of data describing genetic perturbations and changes in mRNA and protein levels in the cell. Because each assay provides a one-dimensional snapshot of active signaling pathways, it has become desirable to perform multiple assays (e.g. mRNA expression and phospho-proteomics) to measure a single condition. However, as experiments expand to accommodate various cellular conditions, proper analysis and interpretation of these data have become more challenging. Here we introduce a novel approach called SAMNet, for Simultaneous Analysis of Multiple Networks, that is able to interpret diverse assays over multiple perturbations. The algorithm uses a constrained optimization approach to integrate mRNA expression data with upstream genes, selecting edges in the protein-protein interaction network that best explain the changes across all perturbations. The result is a putative set of protein interactions that succinctly summarizes the results from all experiments, highlighting the network elements unique to each perturbation. We evaluated SAMNet in both yeast and human datasets. The yeast dataset measured the cellular response to seven different transition metals, and the human dataset measured cellular changes in four different lung cancer models of Epithelial-Mesenchymal Transition (EMT), a crucial process in tumor metastasis. SAMNet was able to identify canonical yeast metal –processing genes unique to each commodity in the yeast dataset, as well as human genes such as β-catenin and TCF7L2/TCF4 that are required for EMT signaling but escaped detection in the mRNA and phospho-proteomic data. Moreover, SAMNet also highlighted drugs likely to modulate EMT, identifying a series of less canonical genes known to be affected by the BCR-ABL inhibitor imatinib (Gleevec), suggesting a possible influence of this drug on EMT.
While much is known about general controls over axon guidance of broad classes of projection neurons (those with long-distance axonal connections), molecular controls over specific axon targeting by distinct neuron subtypes are poorly understood. Corticospinal motor neurons (CSMN) are prototypical and clinically important cerebral cortex projection neurons; they are the brain neurons that degenerate in amyotrophic lateral sclerosis (ALS) and related motor neuron diseases, and their injury is central to the loss of motor function in spinal cord injury. Primary culture of purified immature murine CSMN has been recently established, using either fluorescence-activated cell sorting (FACS) or immunopanning, enabling a previously unattainable level of subtype-specific investigation, but the resulting number of CSMN is quite limiting for standard approaches to study axon guidance. We developed a microfluidic system specifically designed to investigate axon targeting of limited numbers of purified CSMN and other projection neurons in culture. The system contains two chambers for culturing target tissue explants, allowing for biologically revealing axonal growth “choice” experiments. This device will be uniquely enabling for investigation of controls over axon growth and neuronal survival of many types of neurons, particularly those available only in limited numbers.
Highly complex molecular networks, which play fundamental roles in almost all cellular processes, are known to be dysregulated in a number of diseases, most notably in cancer. As a consequence, there is a critical need to develop practical methodologies for constructing and analysing molecular networks at a systems level. Mathematical models built with continuous differential equations are an ideal methodology because they can provide a detailed picture of a network’s dynamics. To be predictive, however, differential equation models require that numerous parameters be known a priori and this information is almost never available. An alternative dynamical approach is the use of discrete logic-based models that can provide a good approximation of the qualitative behaviour of a biochemical system without the burden of a large parameter space. Despite their advantages, there remains significant resistance to the use of logic-based models in biology. Here, we address some common concerns and provide a brief tutorial on the use of logic-based models, which we motivate with biological examples.
The O6-methylguanine (O6MeG) DNA lesion is well known for its mutagenic, carcinogenic, and cytotoxic properties, and understanding how a cell processes such damage is of critical importance for improving current cancer therapy. Here we use human cells differing only in their O6MeG DNA methyltransferase (MGMT) or mismatch repair (MMR) status to explore the O6MeG/MMR-dependent molecular and cellular responses to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We find that O6MeG triggers MMR-dependent cell cycle perturbations in both the first and second cell cycle post treatment. At lower levels of damage, we show that a transient arrest in the second S-phase precedes survival and progression into subsequent cell cycles. However, at higher levels of damage, arrest in the second S-phase coincides with a cessation of DNA replication followed by initiation of apoptotic cell death. Further, we show that entry into apoptotic cell death is specifically from S-phase of the second cell cycle. Finally, we demonstrate the key role of an O6MeG/MMR-dependent multi-pathway, multi-time-scale signaling network activation, led by early ATM, H2AX, CHK1, and p53 phosphorylation and followed by greatly amplified late phosphorylation of the early pathway nodes along with activation of the CHK2 kinase and the stress-activated JNK kinase.
Encapsulations of cells in type-I collagen matrices are widely used three-dimensional (3D) in vitro models of wound healing and tissue morphogenesis and are common constructs for drug delivery and for in vivo implantation. As cells remodel the exogenous collagen scaffold, they also assemble a dense fibronectin (Fn) matrix that aids in tissue compaction; however, the spatio-temporal (re)organization of Fn and collagen in this setting has yet to be quantitatively investigated. Here, we utilized microfabricated tissue gauges (µTUGs) to guide the contraction of microscale encapsulations of fibroblasts within collagen gels. We combined this system with a Foerster Radius Energy Transfer (FRET) labeled biosensor of Fn conformation to probe the organization, conformation and remodeling of both the exogenous collagen and the cell-assembled Fn matrices. We show that within hours, compact Fn from culture media adsorbed to the collagen scaffold. Over the course of tissue remodeling, this Fn-coated collagen scaffold was compacted into a thin, sparsely populated core around which cells assembled a dense fibrillar Fn shell that was rich in both cell and plasma derived Fn. This resulted in two separate Fn populations with different conformations (compact/adsorbed and extended/fibrillar) in microtissues. Cell contractility and microtissue geometry cooperated to remodel these two populations, resulting in spatial gradients in Fn conformation. Together, these results highlight an important spatio-temporal interplay between two prominent extracellular matrix (ECM) molecules (Fn and collagen) and cellular traction forces, and will have implications for future studies of the force-mediated remodeling events that occur within collagen scaffolds either in 3D in vitro models or within surgical implants in vivo.
Investigation of biochemical cues in isolation or in combinations in cell culture systems is crucial for unraveling the mechanisms that govern neural development and repair. The most widely used experimental paradigms that elicit axon guidance in vitro utilize as the source of the gradient a pulsatile pipette, transfected cells, or a loaded gel, producing time-varying gradients of poor reproducibility which are not well suited for studying slow-growing mammalian cells. Although microfluidic device design have allowed for generating stable, complex gradients of diffusible molecules, the flow-induced shear forces in a microchannel has made it impossible to maintain viable mammalian neuronal cultures for sufficiently long times. In this paper, we describe axonal responses of mouse cortical neurons in a “neuron-benign” gradient-generator device based on an open chamber that can establish highly stable gradients of diffusible molecules for at least 6 hours with negligible shear stress, and also allows the neurons to thrive for at least 2 weeks. Except for the period when the gradient is on, the cells in the gradient are under the same conditions as the cells on the control surfaces, which ensure a consistent set of micro-environmental variables. The gradient stability and uniformity over the cell culture surface achieved by the device, together with our software platform for acquiring, post-processing and quantitatively analyzing the large number of images allowed us to extract valuable information even from small datasets. We report a directed response of primary mammalian neurons (from E14 embryonic mice cortex) to a diffusible gradient of netrin in vitro. We infer from our studies that a large majority (~73%) of the neurons that extend axons during the gradient application grow towards the netrin source, and our data analysis also indicates that netrin acts as a growth factor for this same population of neurons.
The success of magnetic nanoparticle (mNP)-based diagnostic and therapeutic techniques is dependent upon how the mNP are distributed in vivo. The potential efficacy and timing of a given magnetic nanoparticle treatment or diagnostic test is largely determined by the number of nanoparticles in each tissue and microscopic compartment: e.g., in the intravascular and extravascular spaces, in the interstitial space, cell surface and in cell cytoplasm. Techniques for monitoring these cell-level interactions generally require the harvesting and destruction of tissues or cells at each time point of interest. We present a method (magnetic spectroscopy of Brownian motion, MSB) for longitudinally monitoring nanoparticle binding to cell surface proteins and uptake by cancer cells in vitro using the harmonics of the magnetization produced by the nanoparticles. These harmonics can be measured rapidly and noninvasively without the need for nanoparticle modifications and without damaging the cells. We demonstrate sensitivity of this harmonic signal to the nanoparticles’ microenvironment and use this technique to monitor the nanoparticle binding activities of different cell lines.
Fluid dynamics play a fundamental role in the development of diabetic retinopathy, one of the leading causes of blindness in the Western world, affecting over 4 million people in the US alone. The disease is defined by microaneurysms, local expansions of capillaries that disturb the hemodynamic forces experienced by the endothelium leading to dysfunction, leakage and edema. Here we present a method to identify microaneurysms with a high risk of leakage based on a critical ratio of microaneurysm to vessel diameter. We derive this non-dimensional parameter from an analytical solution and generalize it using experimentally validated numerical methods. We show that this non-dimensional parameter defines the shear force experienced by endothelial cells, below which endothelial dysfunction is evident in vivo. Our results demonstrate the involvement of vWF in diabetic retinopathy, and explain a perceived disconnect between microaneurysm size and leakage. This method will allow experts to treat microaneurysms poising a high-risk of leakage, prior to edema, minimizing damage and saving vision.
Non-steroidal anti-inflammatory drugs (NSAIDs) play a significant role in the chemoprevention of cancer. We recently showed the chemopreventive response of a NSAID, 2-[(3-chloro-2- methylphenyl)amino]benzoic acid) known as tolfenamic acid (TA) in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in rats. Pre-clinical studies showed that TA inhibits Specificity protein (Sp) transcription factors and acts as an anti-cancer agent in several cancer models; however the pertinent mechanisms associated with its chemopreventive response in esophageal cancer are not known. Since the bioactivation of carcinogens through cytochrome P450 (CYP) is critical for the induction of cancer, we have studied the effect of TA on critical CYP isozymes in mouse liver samples. Athymic nude mice were treated with vehicle (corn oil) or TA (50mg/kg 3 times/wk) for 4 weeks. Protein extracts (whole cell lysates and microsomal fractions) were prepared from liver tissue and the expression of various CYP isozymes was determined by Western blot analysis. Rat (Sprague-Dawley) livers were harvested and primary hepatocyte cultures were treated with vehicle (DMSO) or TA (50 μM) and cell viability was assessed at 2 and 5 days post-treatment. TA caused remarkable decrease in the expression of CYP2E1 in both liver lysates and sub-cellular fraction, while its response on other tested isozymes was marginal. TA did not affect the body weight of animals (mice) and viability of rat hepatocytes. These results demonstrate that TA modulates the expression of CYP2E1 which is associated with the bioactivation of carcinogens without causing apparent toxicity. These data suggest that TA-induced inhibition of CYP2E1 attenuates the bioactivation of carcinogens potentially leading to the chemoprevention of NMBA-induced esophageal tumorigenesis in rats.
Esophageal cancer; chemoprevention; cytochrome P450; CYP2E1; NSAID; Tolfenamic acid
Human pluripotent stem cells (hPSCs) are of great interest in biology and medicine due to their ability to self-renew and differentiate into any adult or fetal cell type. Important efforts have identified biochemical factors, signaling pathways, and transcriptional networks that regulate hPSC biology. However, recent work investigating the effect of biophysical cues on mammalian cells and adult stem cells suggests that the mechanical properties of the microenvironment, such as stiffness, may also regulate hPSC behavior. While several studies have explored this mechanoregulation in mouse embryonic stem cells (mESCs), it has been challenging to extrapolate these findings and thereby explore their biomedical implications in hPSCs. For example, it remains unclear whether hPSCs can be driven down a given tissue lineage by providing tissue-mimetic stiffness cues. Here we address this open question by investigating the regulation of hPSC neurogenesis by microenvironmental stiffness. We find that increasing extracellular matrix (ECM) stiffness in vitro increases hPSC cell and colony spread area but does not alter self-renewal, in contrast to past studies with mESCs. However, softer ECMs with stiffnesses similar to that of neural tissue promote the generation of early neural ectoderm. This mechanosensitive increase in neural ectoderm requires only a short 5-day soft stiffness “pulse,” which translates into downstream increases in both total neurons as well as therapeutically relevant dopaminergic neurons. These findings further highlight important differences between mESCs and hPSCs and have implications for both the design of future biomaterials as well as our understanding of early embryonic development.
Cultured human lung cancer cell lines have been used extensively to dissect signaling pathways underlying cancer malignancy, including proliferation and resistance to chemotherapeutic agents. However, the ability of malignant cells to grow and metastasize in vivo is dependent upon specific cell-cell and cell-extracellular matrix (ECM) interactions, many of which are absent when cells are cultured on conventional tissue culture plastic. Previous studies have found that breast cancer cell lines show differential growth morphologies in three-dimensional (3D) gels of laminin-rich (lr) ECM, and that gene expression patterns associated with organized cell structure in 3D lrECM were associated with breast cancer patient prognosis. We show here that established lung cancer cell lines also can be classified by growth in lrECM into different morphological categories and that transcriptional alterations distinguishing growth on conventional tissue culture plastic from growth in 3D lrECM are reflective of tissue-specific differentiation. We further show that gene expression differences that distinguish lung cell lines that grow as smooth vs. branched structures in 3D lrECM can be used to stratify adenocarcinoma patients into prognostic groups with significantly different outcome, defining phenotypic response to 3D lrECM as a potential surrogate of lung cancer malignancy.
Microscale engineering plays a significant role in developing tools for biological applications by miniaturizing devices and providing controllable microenvironments for in vitro cell research. Miniaturized devices offer numerous benefits in comparison to their macroscale counterparts, such as lower use of expensive reagents, biomimetic environments, and the ability to manipulate single cells. Microscale electroporation is one of the main beneficiaries of microscale engineering as it provides spatial and temporal control of various electrical parameters. Microscale electroporation devices can be used to reduce limitations associated with the conventional electroporation approaches such as variations in the local pH, electric field distortion, sample contamination, and the difficulties in transfecting and maintaining the viability of desired cell types. Here, we present an overview of recent advances of the microscale electroporation methods and their applications in biology, as well as current challenges for its use for clinical applications. We categorize microscale electroporation into microchannel and microcapillary electroporation. Microchannel-based electroporation can be used for transfecting cells within microchannels under dynamic flow conditions in a controlled and high-throughput fashion. In contrast, microcapillary-based electroporation can be used for transfecting cells within controlled reaction chambers under static flow conditions. Using these categories we examine the use of microscale electroporation for clinical applications related to HIV-1, stem cells, cancer and other diseases and discuss the challenges in further advancing this technology for use in clinical medicine and biology.
Reciprocal mechanical interactions between cells and the extracellular matrix (ECM) are thought to play important instructive roles in branching morphogenesis. However, most studies to date have failed to characterize these interactions on a length scale relevant to cells, especially in three-dimensional (3D) matrices. Here we utilized two complementary methods, spatio-temporal image correlation spectroscopy (STICS) and laser optical tweezers-based active microrheology (AMR), to quantify endothelial cell (EC)-mediated deformations of individual ECM elements and the local ECM mechanical properties, respectively, during the process of capillary morphogenesis in a 3D cell culture model. In experiments in which the ECM density was systematically varied, STICS revealed that the rate at which ECs deformed individual ECM fibers on the microscale positively correlated with capillary sprouting on the macroscale. ECs expressing constitutively active V14-RhoA displaced individual matrix fibers at significantly faster rates and displayed enhanced capillary sprouting relative to wild-type cells, while those expressing dominant-negative N19-RhoA behaved in an opposite fashion. In parallel, AMR revealed a local stiffening of the ECM proximal to the tips of sprouting ECs. By quantifying the dynamic physical properties of the cell-ECM interface in both space and time, we identified a correlation linking ECM deformation rates and local ECM stiffening at the microscale with capillary morphogenesis at the macroscale.
When cancer cells spread away from the primary tumor, they often follow the trajectories of lymphatic vessels, nerves, white matter tracts, or other heterogeneous structures in tissues. To better understand this form of guided cell migration we designed a series of microfluidic devices that mechanically constrain migrating cancer cells inside microchannels with cross-section comparable to cell size. We observed unexpectedly fast and persistent movement in one direction for several hours of cancer cells of different types. The persistent motility occurs spontaneously, in the absence of external gradients, suggesting the presence of intrinsic mechanisms driving cancer cell motility that are induced in conditions of mechanical confinement. To probe the mechanisms responsible for this behavior, we exposed cancer cells inside channels to drugs targeting the microtubules, and measured a significant reduction in the average migration speed. Surprisingly, a small number of cells appeared not to be affected by the treatment and displayed fast and persistent migration, comparable to the untreated cells. The new matrix-free, 3D-confined motility assay replicates critical interactions that cancer cells would normally have inside tissues, is compatible with high-content, high-throughput analysis of cellular motility at single cell level, and could provide useful insights into the biology of cancer cell migratory phenotype.
A major challenge in molecular biology is interrogating the human transcriptome on a genome wide scale when only a limited amount of biological sample is available for analysis. Current methodologies using microarray technologies for simultaneously monitoring mRNA transcription levels require nanogram amounts of total RNA. To overcome the sample size limitation of current technologies, we have developed a method to probe the global gene expression in biological samples as small as 150 cells, or the equivalent of approximately 300 pg total RNA. The new method employs microfluidic devices for the purification of total RNA from mammalian cells and ultra-sensitive whole transcriptome amplification techniques. We verified that the RNA integrity is preserved through the isolation process, accomplished highly reproducible whole transcriptome analysis, and established high correlation between repeated isolations of 150 cells and the same cell culture sample. We validated the technology by demonstrating that the combined microfluidic and amplification protocol is capable of identifying biological pathways perturbed by stimulation, which are consistent with the information recognized in bulk-isolated samples.
Here we describe a microfluidic device that accurately reproduces the dynamics of vascular anastomosis, the process by which vascular sprouts connect to achieve perfusion during angiogenesis. The micro-device features two parallel endothelial cell-lined vessel analogues separated by a 300 µm wide collagenous matrix into which the vessels can sprout and form perfused bridging connections. By accurately recapitulating anastomosis in vitro, the device will enable a new generation of studies of the mechanisms of angiogenesis and provide a novel and practical platform for drug screening.
The phenotypic expression and function of stem cells are regulated by their integrated response to variable microenvironmental cues, including growth factors and cytokines, matrix-mediated signals, and cell-cell interactions. Recently, growing evidence suggests that matrix-mediated signals include mechanical stimuli such as strain, shear stress, substrate rigidity and topography, and these stimuli have a more profound impact on stem cell phenotypes than had previously been recognized, e.g. self-renewal and differentiation through the control of gene transcription and signaling pathways. Using a variety of cell culture models enabled by micro and nanoscale technologies, we are beginning to systematically and quantitatively investigate the integrated response of cells to combinations of relevant mechanobiological stimuli. This paper reviews recent advances in engineering physical stimuli for stem cell mechanobiology and discusses how micro- and nanoscale engineered platforms can be used to control stem cell niches environment and regulate stem cell fate and function.
biomechanical cues; stem cell; micro- and nanotechnology; surface topography
Regulating endothelial cell behavior is a key step in understanding and controlling neovascularization for both pro-angiogenic and anti-angiogenic therapeutic strategies. Here, we characterized the effects of a covalently immobilized peptide mimic of vascular endothelial growth factor, herein referred to as VEGF receptor-binding peptide (VR-BP), on human umbilical vein endothelial cell (HUVEC) behavior. Self assembled monolayer arrays presenting varied densities of covalently immobilized VR-BP and varied densities of the fibronectin-derived cell adhesion peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) were used to probe for changes in HUVEC attachment, proliferation and tubulogenesis. In a soluble form, VR-BP exhibited pro-angiogenic effects in agreement with previous studies, indicated by increases in HUVEC proliferation. However, when presented to cells in an insoluble context, covalently immobilized VR-BP inhibited several pro-angiogenic HUVEC behaviors, including attachment and proliferation, and also inhibited HUVEC response to soluble recombinant VEGF protein. Furthermore, substrates with covalently immobilized VR-BP also modulated HUVEC tubulogenesis when a matrigel overlay assay was used to provide cells with a pseudo-three dimensional environment. Taken together, these results demonstrate that the context in which ligands are presented to cell surface receptors strongly influences their effects, and that the same ligand can be an agonist or an antagonist depending on the manner of presentation to the cell.
self-assembled monolayers; RGD; VEGF; umbilical vein endothelial cells; array
Interleukin-12 (IL12) enhances anti-tumor immunity when delivered to the tumor microenvironment. However, local immunoregulatory elements dampen the efficacy of IL12. The identity of these local mechanisms used by tumors to suppress immunosurveillance represents a key knowledge gap for improving tumor immunotherapy. From a systems perspective, local suppression of anti-tumor immunity is a closed-loop system - where system response is determined by an unknown combination of external inputs and local cellular cross-talk. Here, we recreated this closed-loop system in vitro and combined quantitative high content assays, in silico model-based inference, and a proteomic workflow to identify the biochemical cues responsible for immunosuppression. Following an induction period, the B16 melanoma cell model, a transplantable model for spontaneous malignant melanoma, inhibited the response of a T helper cell model to IL12. This paracrine effect was not explained by induction of apoptosis or creation of a cytokine sink, despite both mechanisms present within the co-culture assay. Tumor-derived Wnt-inducible signaling protein-1 (WISP-1) was identified to exert paracrine action on immune cells by inhibiting their response to IL12. Moreover, WISP-1 was expressed in vivo following intradermal challenge with B16F10 cells and was inferred to be expressed at the tumor periphery. Collectively, the data suggest that (1) biochemical cues associated with epithelial-to-mesenchymal transition can shape anti-tumor immunity through paracrine action and (2) remnants of the immunoselective pressure associated with evolution in cancer include both sculpting of tumor antigens and expression of proteins that proactively shape anti-tumor immunity.
Brain derived neurotrophic factor (BDNF) plays a key role in the growth, development and maintenance of the central and peripheral nervous systems. Exogenous BDNF activates its membrane receptors at the axon terminal, and subsequently sends regulation signals to the cell body. To understand how BDNF signal propagates in neurons, it is important to follow the trafficking of BDNF after it is internalized at the axon terminal. Here we labeled BDNF with bright, photostable quantum dot (QD-BDNF) and followed the axonal transport of QD-BDNF in real time in hippocampal neurons. We showed that QD-BDNF was able to bind BDNF receptors and activate downstream signaling pathways. When QD-BDNF was applied to the distal axons of hippocampal neurons, it was observed to be actively transported toward the cell body at an average speed of 1.11 ± 0.05 μm/s. A closer examination revealed that QD-BDNF was transported by both discrete endosomes and multivesicular body-like structures. Our results showed that QD-BDNF could be used to track the movement of exogenous BDNF in neurons over long distances and to study the signaling organelles that contain BDNF.
Objectives are to examine the efficacy of new synthetic curcumin analogs EF31 in head and neck squamous cell carcinoma in vitro and in vivo, and study their pharmacokinetic and toxicologic effects in vivo. The synthesis of EF31 was described for the first time. Solubility of EF24, EF31 was compared using nephelometric analysis. Human head and neck squamous cell carcinoma Tu212 xenograft tumors were established in athymic nude mice and treated with EF31 i.p. once daily five days a week for about 5 – 6 weeks. The long term effect of EF31 on the NF-κB signaling system in the tumors was examined by Western blot analysis. EF31 at 25 mg/kg, i.p. inhibited tumor growth almost completely. Solubility of EF24 and EF31 are <10, 13 μg/mL or <32, 47 μM, respectively. The serum chemistry profiles of treated mice were within the limits of normal, it revealed a linear increase of Cmax. EF31 decreased the level of phosphorylation of NF-κB p65. In conclusion, the novel synthetic curcumin analogs EF31 is efficacious in inhibiting the growth of Tu212 xenograft tumors and may be useful for treating head and neck squamous cell carcinoma. The long term EF31 treatment inhibited NF-kB p65 phosphorylation in xenografts, implicating downregulation of cancer promoting transcription factors such as angiogenesis and metastasis.
synthetic curcumin analog; EF31; pharmacokinetic and toxicologic studies; NF-kB p65 phosphorylation
Cellular membranes are composed of hundreds of different lipids, ion channels, receptors and scaffolding complexes that act as signalling and trafficking platforms for processes fundamental to life. Cellular signalling and membrane trafficking are often regulated by peripheral proteins, which reversibly interact with lipid molecules in highly regulated spatial and temporal fashions. In most cases, one or more modular lipid-binding domain(s) mediate recruitment of peripheral proteins to specific cellular membranes. These domains, of which more than 10 have been identified since 1989, harbour structurally selective lipid-binding sites. Traditional in vitro and in vivo studies have elucidated how these domains coordinate their cognate lipids and thus how the parent proteins associate with membranes. Cellular activities of peripheral proteins and subsequent physiological processes depend upon lipid binding affinities and selectivity. Thus, the development of novel sensitive and quantitative tools is essential in furthering our understanding of the function and regulation of these proteins. As this field expands into new areas such as computational biology, cellular lipid mapping, single molecule imaging, and lipidomics, there is an urgent need to integrate technologies to detail the molecular architecture and mechanisms of lipid signalling. This review surveys emerging cellular and in vitro approaches for studying protein–lipid interactions and provides perspective on how integration of methodologies directs the future development of the field.
Collective cell invasion (CCI) through interstitial collagenous extracellular matrix (ECM) is crucial to the initial stages of branching morphogenesis, and a hallmark of tissue repair and dissemination of certain tumors. The collagenous ECM acts as a mechanical barrier against CCI. However, the physical nature of this barrier and how it is overcome by cells remains incompletely understood. To address these questions, we performed theoretical and experimental analysis of mammary epithelial branching morphogenesis in 3D type I collagen (collagen-I) gels. We found that the mechanical resistance of collagen-I is largely due to its elastic rather than its viscous properties. We also identified two strategies utilized by mammary epithelial cells that can independently minimize ECM mechanical resistance during CCI. First, cells adopt a narrow tube-like geometry during invasion, which minimizes the elastic opposition from the ECM as revealed by theoretical modeling of the most frequent invasive shapes and sizes. Second, the stiffness of the collagenous ECM is reduced at invasive fronts due to its degradation by matrix metalloproteinases (MMPs), as indicated by direct measurements of collagen-I microelasticity by atomic force microscopy. Molecular techniques further specified that the membrane-bound MMP14 mediates degradation of collagen-I at invasive fronts. Thus, our findings reveal that MMP14 is necessary to efficiently reduce the physical restraints imposed by collagen-I during branching morphogenesis, and help our overall understanding of how forces are balanced between cells and their surrounding ECM to maintain collective geometry and mechanical stability during CCI.
Advances in the fields of proteomics, molecular imaging, and therapeutics are closely linked to the availability of affinity reagents that selectively recognize their biological targets. Here we present a review of Iterative Peptide In Situ Click Chemistry (IPISC), a novel screening technology for designing peptide multiligands with high affinity and specificity. This technology builds upon in situ click chemistry, a kinetic target-guided synthesis approach where the protein target catalyzes the conjugation of two small molecules, typically through the azide–alkyne Huisgen cycloaddition. Integrating this methodology with solid phase peptide libraries enables the assembly of linear and branched peptide multiligands we refer to as Protein Catalyzed Capture Agents (PCC Agents). The resulting structures can be thought of as analogous to the antigen recognition site of antibodies and serve as antibody replacements in biochemical and cell-based applications. In this review, we discuss the recent progress in ligand design through IPISC and related approaches, focusing on the improvements in affinity and specificity as multiligands are assembled by target-catalyzed peptide conjugation. We compare the IPISC process to small molecule in situ click chemistry with particular emphasis on the advantages and technical challenges of constructing antibody-like PCC Agents.
Specific cell types are critically implicated in a variety of neuropathologies that exhibit region-specific susceptibility. Neuronal and glial function is impaired in a host of neurodegenerative diseases. Previous reports suggest that mass spectrometry imaging has the potential to resolve cell-specific enrichment in brain regions; however, individual ions cannot resolve glial and neuronal cells within the complex structure of brain tissue. Here, we utilized a matrix-free surface mass spectrometry approach, nanostructure initiator mass spectrometry (NIMS) to determine if multiple lipid ions can be used as a ‘fingerprint’ to discriminate between neuronal- and glial-enriched brain regions and between glial cells in different regions of the brain, such as the cortex and the cerebellum. This is accomplished by using established stains to define glial cell enrichment (GFAP) and NIMS imaging on adjacent serial sections to measure phospholipid distributions. Identifications were made using QTOF analysis of whole-brain extracts and comparison with previous reports. From these identifications, it was found that utilization of multiple lipids can indeed resolve glial and neuronal cell enriched brain regions in the imaged brain section. The resolution of brain regions and cell populations is greatly enhanced through application of multivariate statistical analysis (Nonnegative Matrix Factorization) of the 18 dominant potassium adducts of phospholipids. Strikingly, this analysis resolves other brain regions that are difficult to distinguish using conventional stains but known to have distinct physiological functions. For example, this method could accurately distinguish the frontal (or somatomotor) and dorsal (or retrosplenial) regions of the cortex, which have important functional differences, from each other and from the pons region. This work suggests that using this approach with inclusion of larger numbers of lipids has the potential to greatly improve our understanding of regional and cell type specific variation in the brain.