Fly Me to the Micron: Microtechnologies for Drosophila Research.

Multicellular model organisms, such as Drosophila melanogaster (fruit fly), are frequently used in a myriad of biological research studies due to their biological significance and global standardization. However, traditional tools used in these studies generally require manual handling, subjective phenotyping, and bulk treatment of the organisms, resulting in laborious experimental protocols with limited accuracy. Advancements in microtechnology over the course of the last two decades have allowed researchers to develop automated, high-throughput, and multifunctional experimental tools that enable novel experimental paradigms that would not be possible otherwise. We discuss recent advances in microtechnological systems developed for small model organisms using D. melanogaster as an example. We critically analyze the state of the field by comparing the systems produced for different applications. Additionally, we suggest design guidelines, operational tips, and new research directions based on the technical and knowledge gaps in the literature. This review aims to foster interdisciplinary work by helping engineers to familiarize themselves with model organisms while presenting the most recent advances in microengineering strategies to biologists.

Mutant huntingtin disrupts mitochondrial proteostasis by interacting with TIM23.

Mutant huntingtin (mHTT), the causative protein in Huntington's disease (HD), associates with the translocase of mitochondrial inner membrane 23 (TIM23) complex, resulting in inhibition of synaptic mitochondrial protein import first detected in presymptomatic HD mice. The early timing of this event suggests that it is a relevant and direct pathophysiologic consequence of mHTT expression. We show that, of the 4 TIM23 complex proteins, mHTT specifically binds to the TIM23 subunit and that full-length wild-type huntingtin (wtHTT) and mHTT reside in the mitochondrial intermembrane space. We investigated differences in mitochondrial proteome between wtHTT and mHTT cells and found numerous proteomic disparities between mHTT and wtHTT mitochondria. We validated these data by quantitative immunoblotting in striatal cell lines and human HD brain tissue. The level of soluble matrix mitochondrial proteins imported through the TIM23 complex is lower in mHTT-expressing cell lines and brain tissues of HD patients compared with controls. In mHTT-expressing cell lines, membrane-bound TIM23-imported proteins have lower intramitochondrial levels, whereas inner membrane multispan proteins that are imported via the TIM22 pathway and proteins integrated into the outer membrane generally remain unchanged. In summary, we show that, in mitochondria, huntingtin is located in the intermembrane space, that mHTT binds with high-affinity to TIM23, and that mitochondria from mHTT-expressing cells and brain tissues of HD patients have reduced levels of nuclearly encoded proteins imported through TIM23. These data demonstrate the mechanism and biological significance of mHTT-mediated inhibition of mitochondrial protein import, a mechanism likely broadly relevant to other neurodegenerative diseases.

Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis.

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of ß-catenin.

Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called two-dimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on ß-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of ß-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development.

Molecular Sieving on the Surface of a Nano-Armored Protein.

The molecular sieving properties of protein surface-attached polymers are the central features in how polymers extend therapeutic protein lifetimes in vivo. Yet, even after 30 years of research, permeation rates of molecules through polymer-surrounded protein surfaces are largely unknown. As a result, the generation of protein-polymer conjugates remains a stochastic process, unfacilitated by knowledge of structure-function-polymer architecture relationships. In this work, polymers are grown from the surface of avidin using atom transfer radical polymerization (ATRP) and used to determine how polymer length and density influence the binding kinetics of ligands as a function of ligand size and shape. The rate of binding is strongly dependent on the grafting density of polymers and the size of the ligand but interestingly, far less dependent on the length of the polymer. This study unveils a deeper understanding of relationship between polymer characteristics and binding kinetics, discovering important steps in rational design of protein-polymer conjugates.

Immuno-proteomics: Development of a novel reagent for separating antibodies from their target proteins.

Immunoprecipitation (IP) is a widely used technique for identifying the binding partners of the target proteins of specific antibodies. Putative binding targets and their partners are usually in much lower amounts than the antibodies used to capture these target proteins. Thus antigen identification using proteomics following IP is often confounded by the presence of an overwhelming amount of interfering antibody protein. Even covalently linking antibodies to beads is susceptible to antibody leaching during IP. To circumvent this interference, we describe here a reagent, called Biotin-CDM that reversibly tags all potential target proteins in a cell lysate with biotin. The presence of biotin coupled to the target proteins allows for a secondary separation step in which antibodies are washed away from the reversibly biotinylated target proteins by binding them to an Avidin-coupled matrix. The captured target proteins are released from the Avidin matrix by reversing the Biotin-CDM link, thus releasing a pool of target proteins ready for further proteomic analysis compatible with 2D-electrophoresis. Here, we describe the synthesis and characterization of Biotin-CDM. We also demonstrate Biotin-CDM's use for immunoprecipitation of a known antigen, as well as its use for capturing an array of proteins targeted by the autoantibodies found in the serum a patient suffering from rheumatoid arthritis. The use of this reagent allows one to combine immunoprecipitation and 2D-Difference gel electrophoresis, overcoming the current limitations of Serological Proteome Analysis (SERPA) in discovering autoantigens. This article is part of a Special Issue entitled: Medical Proteomics.

High dynamic range proteome imaging with the structured illumination gel imager.

A current challenge for proteomics is detecting proteins over the large concentration ranges found in complex biological samples such as whole-cell extracts. Currently, no unbiased, whole-proteome analysis scheme is capable of detecting the full range of cellular proteins. This is due in part to the limited dynamic range of the detectors used to sense proteins or peptides. We present a new technology, structured illumination (SI) gel imager, which detects fluorescently labeled proteins in electrophoretic gels over a 1 000 000-fold concentration range. SI uses computer-generated masks to attenuate the illumination of highly abundant proteins, allowing for long exposures of low-abundance proteins, thus avoiding detector saturation. A series of progressively masked gel images are assembled into a single, very high dynamic range image. We demonstrate that the SI imager can detect proteins over a concentration range of approximately 1 000 000-fold, making it a useful tool for comprehensive, unbiased proteome-wide surveys.

In-gel equilibration for improved protein retention in 2DE-based proteomic workflows.

The 2DE is a powerful proteomic technique, with excellent protein separation capabilities where intact proteins are spatially separated by pI and molecular weight. 2DE is commonly used in conjunction with MS to identify proteins of interest. Current 2DE workflow requires several manual processing steps that can lead to experimental variability and sample loss. One such step is the transition between first dimension IEF and second-dimension SDS-PAGE, which requires exchanging denaturants and the reduction and alkylation of proteins. This in-solution-based equilibration step has been shown to be rather inefficient, losing up to 30% of the original starting material through diffusion effects. We have developed a refinement of this equilibration step using agarose stacking gels poured on top of the second-dimension SDS-PAGE gel, referred to as in-gel equilibration. We show that in-gel equilibration is effective at reduction and alkylation in SDS-PAGE gels. Quantification of whole-cell extracts separated on 2DE gels shows that in-gel equilibration increases protein retention, decreased intergel variability, and simplifies 2DE workflow.

One-pot method for preparing DNA, RNA, and protein for multiomics analysis.

Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids. We demonstrate the utility of this method to compare the genomes, transcriptomes, and proteomes of four triple-negative breast cancer cell lines with different degrees of malignancy. These data show the involvement of both RNA and associated proteins, and protein-only dependent pathways that distinguish these cell lines. We also demonstrate the utility of this multiomics workflow for tissue analysis using mouse brain, liver, and lung tissue.

Assessing the critical period for Rho kinase activity during Drosophila ventral furrow formation.

BACKGROUND: Drosophila ventral furrow formation (VFF), which is the first morphogenetic event during embryo development, serves as a model for epithelial sheet folding. VFF can be subdivided into five cell shape changes: apical membrane flattening, apicobasal nuclear migration, apicobasal cell shortening, random apical constriction, and concerted apical constriction. These processes are generally believed to be driven by Rho kinase (Rok) activation of myosin II to stimulate the constriction of the apical actomyosin network. To test the role of Rok and its downstream target myosin II in VFF, timed injections of the Rok inhibitor, Y-27632, were performed. RESULTS: Embryos injected with Y-27632 before the concerted apical constriction phase of VFF were able to execute apicobasal nuclear migration and random apical constriction, but were unable to enter the concerted apical constriction phase. Embryos injected with Y-27632 during concerted apical constriction reverted to the transition point between random apical constriction and concerted apical constriction. Finally, embryos injected with Y-27632 upon the initiation of furrow ingression were able to complete VFF. CONCLUSIONS: Together these results suggest a critical period for Rok activity and presumably myosin II activation during the initiation of the concerted apical constriction phase of VFF.

Microfluidics for understanding model organisms.

New microfluidic systems for whole organism analysis and experimentation are catalyzing biological breakthroughs across many fields, from human health to fundamental biology principles. This perspective discusses recent microfluidic tools to study intact model organisms to demonstrate the tremendous potential for these integrated approaches now and into the future. We describe these microsystems' technical features and highlight the unique advantages for precise manipulation in areas including immobilization, automated alignment, sorting, sensory, mechanical and chemical stimulation, and genetic and thermal perturbation. Our aim is to familiarize technologically focused researchers with microfluidics applications in biology research, while providing biologists an entrée to advanced microengineering techniques for model organisms.

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

LCVM infection generates tumor antigen-specific immunity and inhibits growth of nonviral tumors.

Antibodies and T cells specific for tumor-associated antigens (TAA) are found in individuals without cancer but with a history of infections and are associated with lowered cancer risk. We hypothesized that those immune responses were generated to transiently abnormally expressed self-antigens on infected cells (disease-associated antigens, DAA) and later on tumor cells as TAA. We tested this hypothesis in mice with a history of infection with lymphocytic choriomeningitis virus (LCMV) Armstrong strain (Arm) that causes acute infection when injected intraperitoneally or CL-13 strain that establishes chronic infection when injected intravenously. Both elicited antibodies and T cells that recognized DAA/TAA on infected cells and on mouse tumors. When challenged with those tumors, Arm-experienced mice controlled tumors better than CL-13-experienced mice or infection-naïve mice. We characterized 7 DAA/TAA that were targets of LCMV-elicited antitumor immunity. We then vaccinated mice with tumor-derived gp96, a heat shock protein that binds a variety of TAA peptides, including those expressed on virus-infected cells as DAA. Tumor-gp96 vaccine induced DAA/TAA-specific immunity. When challenged with Cl-13, the mice showed lower viral copy numbers both early (day 7) and late (day 70) in infection. DAA/TAA may be immunogenic and safe candidates to develop vaccines to control both infections and cancer.

Wolbachia action in the sperm produces developmentally deferred chromosome segregation defects during the Drosophila mid-blastula transition.

Wolbachia, a vertically transmitted endosymbiont infecting many insects, spreads rapidly through uninfected populations by a mechanism known as cytoplasmic incompatibility (CI). In CI, a paternally delivered modification of the sperm leads to chromatin defects and lethality during and after the first mitosis of embryonic development in multiple species. However, whether CI-induced defects in later stage embryos are a consequence of the first division errors or caused by independent defects remains unresolved. To address this question, we focused on ~1/3 of embryos from CI crosses in Drosophila simulans that develop apparently normally through the first and subsequent pre-blastoderm divisions before exhibiting mitotic errors during the mid-blastula transition and gastrulation. We performed single embryo PCR and whole genome sequencing to find a large percentage of these developed CI-derived embryos bypass the first division defect. Using fluorescence in situ hybridization, we find increased chromosome segregation errors in gastrulating CI-derived embryos that had avoided the first division defect. Thus, Wolbachia action in the sperm induces developmentally deferred defects that are not a consequence of the first division errors. Like the immediate defect, the delayed defect is rescued through crosses to infected females. These studies inform current models on the molecular and cellular basis of CI.

Proteomic analysis reveals CCT is a target of Fragile X mental retardation protein regulation in Drosophila.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that is required for the translational regulation of specific target mRNAs. Loss of FMRP causes Fragile X syndrome (FXS), the most common form of inherited mental retardation in humans. Understanding the basis for FXS has been limited because few in vivo targets of FMRP have been identified and mechanisms for how FMRP regulates physiological targets are unclear. We have previously demonstrated that Drosophila FMRP (dFMRP) is required in early embryos for cleavage furrow formation. In an effort to identify new targets of dFMRP-dependent regulation and new effectors of cleavage furrow formation, we used two-dimensional difference gel electrophoresis and mass spectrometry to identify proteins that are misexpressed in dfmr1 mutant embryos. Of the 28 proteins identified, we have identified three subunits of the Chaperonin containing TCP-1 (CCT) complex as new direct targets of dFMRP-dependent regulation. Furthermore, we found that the septin Peanut, a known effector of cleavage, is a likely conserved substrate of fly CCT and is mislocalized in both cct and in dfmr1 mutant embryos. Based on these results we propose that dFMRP-dependent regulation of CCT subunits is required for cleavage furrow formation and that at least one of its substrates is affected in dfmr1- embryos suggesting that dFMRP-dependent regulation of CCT contributes to the cleavage furrow formation phenotype.

pHMA, a pH-sensitive GFP reporter for cell engulfment, in Drosophila embryos, tissues, and cells.

Engulfment of apoptotic cells by phagocytosis ensures the removal of unwanted and defective cells. We developed a genetically encoded marker for cell engulfment, pHMA, which consists of the pH-Sensitive derivative of GFP, pHluorin, fused to the actin-binding domain of Moesin. In healthy cells of Drosophila embryos and cultured cells, pHMA resides at the cell cortex. In dying cells, pHMA loses its cortical localization and reports a modest decrease in pH. In embryos, the dying cells lose their apical contacts, then move basally and are ultimately engulfed by neighboring cells or macrophages. The cell corpse material is strongly acidified soon after engulfment and persists in the phagocytic cell for several hours. Changes in the pHMA signal correlate well with increases or decreases in apoptosis. These data show that pHMA is a useful reporter for cell engulfment and can be used in screening for mutations that affect cell engulfment.

Identification of Cell Surface Molecules That Determine the Macrophage Activation Threshold Associated With an Early Stage of Malignant Transformation.

The ability of immune cells to sense changes associated with malignant transformation as early as possible is likely to be important for the successful outcome of cancer immunosurveillance. In this process, the immune system faces a trade-off between elimination of cells harboring premalignant or malignant changes, and autoimmune pathologies. We hypothesized that the immune system has therefore evolved a threshold for the stage of transformation from normal to fully malignant cells that first provides a threat (danger) signal requiring a response. We co-cultured human macrophages with a unique set of genetically related human cell lines that recapitulate successive stages in breast cancer development: MCF10A (immortalized, normal); MCFNeoT (benign hyperplasia); MCFT1 (atypical hyperplasia); MCFCA1 (invasive cancer). Using cytokines-based assays, we found that macrophages were inert towards MCF10A and MCFNeoT but were strongly activated by MCFT1 and MCFCA1 to produce inflammatory cytokines, placing the threshold for recognition between two premalignant stages, the earlier stage MCFNeoT and the more advanced MCFT1. The cytokine activation threshold paralleled the threshold for enhanced phagocytosis. Using proteomic and transcriptomic approaches, we identified surface molecules, some of which are well-known tumor-associated antigens, that were absent or expressed at low levels in MCF10A and MCFNeoT but turned on or over-expressed in MCFT1 and MCFCA1. Adding antibodies specific for two of these molecules, Annexin-A1 and CEACAM1, inhibited macrophage activation, supporting their role as cancer "danger signals" recognized by macrophages.

Inflammation-Induced Abnormal Expression of Self-molecules on Epithelial Cells: Targets for Tumor Immunoprevention.

Tumor-associated antigens (TAA) are self-molecules abnormally expressed on tumor cells, which elicit humoral and cellular immunity and are targets of immunosurveillance. Immunity to TAAs is found in some healthy individuals with no history of cancer and correlates positively with a history of acute inflammatory and infectious events and cancer risk reduction. This suggests a potential role in cancer immunosurveillance for the immune memory elicited against disease-associated antigens (DAA) expressed on infected and inflamed tissues that are later recognized on tumors as TAAs. To understand probable sources for DAA generation, we investigated in vitro the role of inflammation that accompanies both infection and carcinogenesis. After exposure of normal primary breast epithelial cells to proinflammatory cytokines IL1ß, IL6, and TNFα, or macrophages producing these cytokines, we saw transient overexpression of well-known TAAs, carcinoembryonic antigen and Her-2/neu, and overexpression and hypoglycosylation of MUC1. We documented inflammation-induced changes in the global cellular proteome by 2D difference gel electrophoresis combined with mass spectrometry and identified seven new DAAs. Through gene profiling, we showed that the cytokine treatment activated NF-κB and transcription of the identified DAAs. We tested three in vitro-identified DAAs, Serpin B1, S100A9, and SOD2, and found them overexpressed in premalignant and malignant breast tissues as well as in inflammatory conditions of the colon, stomach, and liver. This new category of TAAs, which are also DAAs, represent a potentially large number of predictable, shared, immunogenic, and safe antigens to use in preventative cancer vaccines and as targets for cancer therapies.

Difference gel electrophoresis.

Difference gel electrophoresis (DIGE) was invented to circumvent the inherent variability of 2-DE. This variability is a natural consequence of separating thousands of proteins over a large space, such as a 15 x 20 cm slab of polyacrylamide gel. The originators of 2-DE envisioned being able to compare cancerous cells and normal cells to understand what makes these cells different. Gel-to-gel variability made this an extremely difficult task. We reasoned that if both samples could be run on the same gel, then the inherent variability would be obviated. Thus, we created matched sets of fluorescent dyes that allows one to compare two or three protein samples on a single gel. In the 12 years since the description of DIGE first appeared in Electrophoresis, this founding paper has been cited over 660 times. This review highlights some of the improvements and applications of DIGE. We hope these examples are illustrative of what has been done and where the field is headed.

High-throughput mechanotransduction in Drosophila embryos with mesofluidics.

Developing embryos create complexity by expressing genes to coordinate movement which generates mechanical force. An emerging theory is that mechanical force can also serve as an input signal to regulate developmental gene expression. Experimental methods to apply mechanical stimulation to whole embryos have been limited, mainly to aspiration, indentation, or moving a coverslip; these approaches stimulate only a few embryos at a time and require manual alignment. A powerful approach for automation is microfluidic devices, which can precisely manipulate hundreds of samples. However, using microfluidics to apply mechanical stimulation has been limited to small cellular systems, with fewer applications for larger scale whole embryos. We developed a mesofluidic device that applies the precision and automation of microfluidics to the Drosophila embryo: high-throughput automatic alignment, immobilization, compression, real-time imaging, and recovery of hundreds of live embryos. We then use twist:eGFP embryos to show that the mechanical induction of twist depends on the dose and duration of compression. This device allows us to quantify responses to compression, map the distribution of ectopic twist, and measure embryo stiffness. For building mesofluidic devices, we describe modifications on ultra-thick photolithography, derive an analytical model that predicts the deflection of sidewalls, and discuss parametric calibration. This "mesomechanics" approach combines the high-throughput automation and precision of microfluidics with the biological relevance of live embryos to examine mechanotransduction. These analytical models facilitate the design of future devices to process multicellular organisms such as larvae, organoids, and mesoscale tissue samples.