Seeing beyond the blot: A critical look at assumptions and raw data interpretation in Western blotting.

Rapid advancements in technology refine our understanding of intricate biological processes, but a crucial emphasis remains on understanding the assumptions and sources of uncertainty underlying biological measurements. This is particularly critical in cell signaling research, where a quantitative understanding of the fundamental mechanisms governing these transient events is essential for drug development, given their importance in both homeostatic and pathogenic processes. Western blotting, a technique developed decades ago, remains an indispensable tool for investigating cell signaling, protein expression, and protein-protein interactions. While improvements in statistical analysis and methodology reporting have undoubtedly enhanced data quality, understanding the underlying assumptions and limitations of visual inspection in Western blotting can provide valuable additional information for evaluating experimental conclusions. Using the example of agonist-induced receptor post-translational modification, we highlight the theoretical and experimental assumptions associated with Western blotting and demonstrate how raw blot data can offer clues to experimental variability that may not be fully captured by statistical analyses and reported methodologies. This article is not intended as a comprehensive technical review of Western blotting. Instead, we leverage an illustrative example to demonstrate how assumptions about experimental design and data normalization can be revealed within raw data and subsequently influence data interpretation.

ß-arrestin mediates communication between plasma membrane and intracellular GPCRs to regulate signaling.

It has become increasingly apparent that G protein-coupled receptor (GPCR) localization is a master regulator of cell signaling. However, the molecular mechanisms involved in this process are not well understood. To date, observations of intracellular GPCR activation can be organized into two categories: a dependence on OCT3 cationic channel-permeable ligands or the necessity of endocytic trafficking. Using CXC chemokine receptor 4 (CXCR4) as a model, we identified a third mechanism of intracellular GPCR signaling. We show that independent of membrane permeable ligands and endocytosis, upon stimulation, plasma membrane and internal pools of CXCR4 are post-translationally modified and collectively regulate EGR1 transcription. We found that ß-arrestin-1 (arrestin 2) is necessary to mediate communication between plasma membrane and internal pools of CXCR4. Notably, these observations may explain that while CXCR4 overexpression is highly correlated with cancer metastasis and mortality, plasma membrane localization is not. Together these data support a model where a small initial pool of plasma membrane-localized GPCRs are capable of activating internal receptor-dependent signaling events.

Are the biomedical sciences ready for synthetic biology?

The ability to construct a functional system from its individual components is foundational to understanding how it works. Synthetic biology is a broad field that draws from principles of engineering and computer science to create new biological systems or parts with novel function. While this has drawn well-deserved acclaim within the biotechnology community, application of synthetic biology methodologies to study biological systems has potential to fundamentally change how biomedical research is conducted by providing researchers with improved experimental control. While the concepts behind synthetic biology are not new, we present evidence supporting why the current research environment is conducive for integration of synthetic biology approaches within biomedical research. In this perspective we explore the idea of synthetic biology as a discovery science research tool and provide examples of both top-down and bottom-up approaches that have already been used to answer important physiology questions at both the organismal and molecular level.

Clathrin Heavy Chain Knockdown Impacts CXCR4 Signaling and Post-translational Modification.

Recent research has implicated endocytic pathways as important regulators of receptor signaling. However, the role of endocytosis in regulating chemokine CXC receptor 4 (CXCR4) signaling remains largely unknown. In the present work we systematically investigate the impact of clathrin knockdown on CXCR4 internalization, signaling, and receptor post-translational modification. Inhibition of clathrin-mediated endocytosis (CME) significantly reduced CXCR4 internalization. In contrast to other receptors, clathrin knockdown increased CXCL12-dependent ERK1/2 signaling. Simultaneous inhibition of CME and lipid raft disruption abrogated this increase in ERK1/2 phosphorylation suggesting that endocytic pathway compensation can influence signaling outcomes. Interestingly, using an antibody sensitive to CXCR4 post-translational modification, we also found that our ability to detect CXCR4 was drastically reduced upon clathrin knockdown. We hypothesize that this effect was due to differences in receptor post-translational modification as total CXCR4 protein and mRNA levels were unchanged. Lastly, we show that clathrin knockdown reduced CXCL12-dependent cell migration irrespective of an observed increase in ERK1/2 phosphorylation. Altogether, this work supports a complex model by which modulation of endocytosis affects not only receptor signaling and internalization but also receptor post-translational modification.

A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly.

The linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.

Detection of multi-drug resistant Escherichia coli in the urban waterways of Milwaukee, WI.

Urban waterways represent a natural reservoir of antibiotic resistance which may provide a source of transferable genetic elements to human commensal bacteria and pathogens. The objective of this study was to evaluate antibiotic resistance of Escherichia coli isolated from the urban waterways of Milwaukee, WI compared to those from Milwaukee sewage and a clinical setting in Milwaukee. Antibiotics covering 10 different families were utilized to determine the phenotypic antibiotic resistance for all 259 E. coli isolates. All obtained isolates were determined to be multi-drug resistant. The E. coli isolates were also screened for the presence of the genetic determinants of resistance including ermB (macrolide resistance), tet(M) (tetracycline resistance), and ß-lactamases (bla OXA, bla SHV, and bla PSE). E. coli from urban waterways showed a greater incidence of antibiotic resistance to 8 of 17 antibiotics tested compared to human derived sources. These E. coli isolates also demonstrated a greater incidence of resistance to higher numbers of antibiotics compared to the human derived isolates. The urban waterways demonstrated a greater abundance of isolates with co-occurrence of antibiotic resistance than human derived sources. When screened for five different antibiotic resistance genes conferring macrolide, tetracycline, and ß-lactam resistance, clinical E. coli isolates were more likely to harbor ermB and bla OXA than isolates from urban waterway. These results indicate that Milwaukee's urban waterways may select or allow for a greater incidence of multiple antibiotic resistance organisms and likely harbor a different antibiotic resistance gene pool than clinical sources. The implications of this study are significant to understanding the presence of resistance in urban freshwater environments by supporting the idea that sediment from urban waterways serves as a reservoir of antibiotic resistance.

Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.

Diet-induced obesity alters skeletal muscle fiber types of male but not female mice.

Skeletal muscles are highly plastic tissues capable dramatic remodeling in response to use, disuse, disease, and other factors. Growing evidence suggests that adipose tissues exert significant effects on the basic fiber-type composition of skeletal muscles. In the current study, we investigated the long-term effects of a high-fat diet and subsequent obesity on the muscle fiber types in C57 BLK/6J mice. Litters of mice were randomly assigned to either a high-fat diet or a control group at the time of weaning, and were maintained on this diet for approximately 1 year. Single fibers were harvested from the soleus and plantaris muscles, and fiber types were determined using SDS-PAGE. The high-fat diet mice were significantly heavier than the control mice (39.17 ± 2.7 g vs. 56.87 ± 3.4 g; P < 0.0003), but muscle masses were not different. In male mice, the high-fat diet was associated with a significantly lower proportion of slow, type I fibers in the soleus muscle (40.4 ± 3.5% vs. 29.33 ± 2.6%; P < 0.0165). Moreover, the proportion of type I fibers in the soleus of male mice was inversely proportional to the relative fatness of the male mice (P < 0.003; r (2) = 0.65), but no association was observed in female mice. In male mice, the decline in type I fibers was correlated with an increase in type I/IIA hybrid fibers, suggesting that the type I fibers were transformed primarily into these hybrids. The reported trends indicate that type I fibers are most susceptible to the effects of obesity, and that these fiber-type changes can be sex specific.