X Wing Diagram

[Kenneth Larson]

Awiring diagram is a simple visual representation of the physical connections and physical layout of an electrical system or circuit. It shows how the electrical wires are interconnected and can also show where fixtures and components may be connected to the system.

SmartDraw comes with pre-made wiring diagram templates. Customize hundreds of electrical symbols and quickly drop them into your wiring diagram. Special control handles around each symbol allow you to quickly resize or rotate them as necessary.

To draw a wire, simply click on the Draw Lines option on the left hand side of the drawing area. If you right click on a line, you can change the line's color or thickness and add or remove arrowheads as necessary. Drag a symbol onto the line and it will insert itself and snap into place. Once connected, it will remain connected even if you move the wire.

If you need additional symbols, click the arrow next to the visible library to bring up a drop down menu and select More. You'll be able to search for additional symbols and open any relevant libraries.

Click on Set Line Hops in the SmartPanel to show or hide line hops at crossover points. You can also change the size and shape of your line hops. Select Show Dimensions to show the length of your wires or size of your component.

A schematic shows the plan and function for an electrical circuit, but is not concerned with the physical layout of the wires. Wiring diagrams show how the wires are connected and where they should located in the actual device, as well as the physical connections between all the components.

Unlike a pictorial diagram, a wiring diagram uses abstract or simplified shapes and lines to show components. Pictorial diagrams are often photos with labels or highly-detailed drawings of the physical components.

Most symbols used on a wiring diagram look like abstract versions of the real objects they represent. For example, a switch will be a break in the line with a line at an angle to the wire, much like a light switch you can flip on and off. A resistor will be represented with a series of squiggles symbolizing the restriction of current flow. An antenna is a straight line with three small lines branching off at its end, much like a real antenna.

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The human immune system is composed of a distributed network of cells circulating throughout the body, which must dynamically form physical associations and communicate using interactions between their cell-surface proteomes1. Despite their therapeutic potential2, our map of these surface interactions remains incomplete3,4. Here, using a high-throughput surface receptor screening method, we systematically mapped the direct protein interactions across a recombinant library that encompasses most of the surface proteins that are detectable on human leukocytes. We independently validated and determined the biophysical parameters of each novel interaction, resulting in a high-confidence and quantitative view of the receptor wiring that connects human immune cells. By integrating our interactome with expression data, we identified trends in the dynamics of immune interactions and constructed a reductionist mathematical model that predicts cellular connectivity from basic principles. We also developed an interactive multi-tissue single-cell atlas that infers immune interactions throughout the body, revealing potential functional contexts for new interactions and hubs in multicellular networks. Finally, we combined targeted protein stimulation of human leukocytes with multiplex high-content microscopy to link our receptor interactions to functional roles, in terms of both modulating immune responses and maintaining normal patterns of intercellular associations. Together, our work provides a systematic perspective on the intercellular wiring of the human immune system that extends from systems-level principles of immune cell connectivity down to mechanistic characterization of individual receptors, which could offer opportunities for therapeutic intervention.

Our quantitative wiring diagram, if truly systematic to the point of approaching completeness, should make it possible to derive a reductionist model that explains how circulating immune cells associate with each other solely from receptor-binding mechanisms and physics-based formulas. We built a coarse yet principled mathematical model that integrates quantitative proteomics expression, binding kinetics and published cell parameters to summarize the contributions of individual protein interactions to a given cellular interaction (Fig. 2f and Supplementary Equations). Using equations based on the law of mass action, the model then computes how the overall probability of binding between two cell types emerges from the distinct spectrum of cell-surface receptors that connect them (Extended Data Fig. 7). Although greatly simplistic, our model could still infer the relative frequencies at which human immune cells physically interact with sufficient precision to be consistent with published empirical measurements25 (Fig. 2g and Extended Data Fig. 7d).

The immune system is distinctive for being a distributed system. It is not fixed to a single localized organ in the body, but rather is made up of numerous specialized cell types that must adaptably organize their intercellular connections to respond to pathogens and other threats wherever they may appear. We provide a systematic and quantitative view of the cell-surface proteins that enable immune cells to dynamically wire their interactions. The receptor interactions that we report in our network each merit further individualized study to characterize their full roles in health and disease. Of particular note are our discovery of HLA-E and HLA-F (but probably not HLA-G) as endogenous non-tumour ligands for the immune checkpoint receptor VISTA; the ability of vasorin to act as a receptor for Jagged ligands; and immunoglobulin family receptors binding members of the amyloid precursor protein family. Notably, members of this same family of non-classical MHC class I molecules that includes HLA-E and HLA-F have previously been identified as key ligands for maintaining innate immune quiescence34, which our findings extend to raise the possibility that they may similarly act as regulators of adaptive immune quiescence through VISTA35. Our functional screening on blood immune cells further points to pathways worth greater consideration; for example, a role of SLITRK4 in lymphocyte responses. While we were preparing this study, independent groups have provided supporting evidence for several of the interactions that we characterized here17,18,36, including PVR as the ligand for the formerly orphan KIR2DL5A and CD146 as an adhesive ligand for CNTN1.

More broadly, the integrated approaches that we pioneered here for disentangling the immune system provide a framework for future systematic investigations. Using our high-throughput biochemical method for interaction screening (SAVEXIS) and the strategies that we describe here to characterize interactions by combinations of multiplex cellular assays and genomics datasets, a range of other cellular communities in the human body could similarly be quantitatively mapped. To our knowledge, our study is among the first to systematically map and model how the collective actions of individual receptor molecules through physical laws could explain and predict cellular connectivity on a scale as large as the circulating immune system. Our analysis and the methods that we developed provide a template for future studies looking at physical cell wiring networks in detail. From these combined approaches, we may finally begin to disentangle cellular circuits in immunity and beyond, bridging from individual protein molecules to multicellular behaviour.

The full library of constructs for recombinant expression was assembled by a combination of cloning and gene synthesis. For cloned sequences, cDNA templates (OriGene) were amplified by PCR with primers delineating the extracellular domain. Overhangs on the primers introduced NotI and AscI restriction sites, which enabled ligation into the appropriate vector backbone. All assembled inserts not produced de novo by synthesis were verified by Sanger sequencing. Constructs that were not cloned from existing DNA templates were ordered as synthetic DNA (Twist Biosciences and Thermo Fisher Scientific GeneArt). Synthesized codons were optimized for human cell expression. Optimal Kozak consensus nucleotide sequences were included in all constructs, which occasionally required mutating the second amino acid of the endogenous signal peptide to alanine. Plasmids were prepared to transfection-grade quantity using midiprep or maxiprep kits (for example, Invitrogen K210007).

The cell-surface proteomes of blood immune cells were defined by two sources. First, the full dataset of a previous high-resolution proteomics survey of 28 leukocyte populations in resting and activated states (for 44 cell types and states total, covering all major categories)1 was merged against a previously established manually curated list of every cell-surface protein in the human genome54,55. The cell-surface proteome list was manually reviewed to verify that each protein does not have publications measuring its localization that contradict a presence on the cell surface. Every protein that was detected was included regardless of how low the expression counts were, with the exception of highly polymorphic proteins such as HLA-A. Second, we added all proteins with a designated CD number as of the 10th human cell differentiation molecule workshop56. The amino acid sequences and topologies57 of these proteins were manually inspected to determine the extracellular regions and in which structural class the protein belonged (out of type I single-pass/GPI-anchored, type II single-pass, multi-pass, and proteins that function as obligate dimers such as integrins). Proteins that lacked a single contiguous extracellular region of at least 20 amino acids after signal peptide processing were excluded. Similarly, multi-pass proteins without a clear contiguous extracellular domain to express were excluded as incompatible with our recombinant expression system. Constructs were produced as synthetic DNA sequences by Twist Biosciences or Thermo Fisher Scientific GeneArt, optimizing codons for human cell expression. As previously described, proteins were cloned into pTT3 expression vectors matching the intended topology49. Single-pass proteins with N-terminal extracellular domains retained their endogenous signal peptides in cases in which a well-annotated or SignalP-predicted signal peptide could be found. Otherwise, an exogenous signal peptide based on the mouse kappa antibody secretion sequence was inserted. Proteins with N-terminal domains had tags attached to their C terminus, whereas the inverted design was used for type II C-terminal proteins58. All proteins were produced as fusions with an established recombinant linker comprising domains 3 and 4 of rat CD4 in place of the original transmembrane sequence (termed rCD4)59, along with a biotin-acceptor site for covalent modification and a hexahistidine tag for purification purposes as described14. In the case of proteins that exist as dimeric complexes, including integrins, HLA-related molecules, CD1, CD8, GPIb, CD79 and CD94 family NK receptors, we relied on a previously determined design in which one chain would lack any tags, thus directing the purification of full complexes, particularly in cases in which the tagged chain is not secreted on its own60. Proteins with an intrinsic multimerizing ability owing to intermolecular disulfide bonds, including the TNF-superfamily CD27 and HVEM trimers or B7-family ICOS and CD28 dimers, were expressed with the appropriate cysteine residues intact and allowed to form functional complexes after cell secretion.

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