Resolving Cellular Mechanisms through Cell Surface Architecture

A framework for mechanistic insights beyond protein abundance

Key principle:

Protein abundance defines which proteins are present.
Protein organisation reveals how those proteins are arranged to function.

Introduction

Cell surface proteins are central to immune cell biology, as they mediate nearly all
communication with the surrounding environment. Conventional single - cell proteomics
analysis allows the assessment of cell surface protein composition. However, measuring
protein abundance merely reveals what is present, but not how these proteins are organised
into functional systems. In fact, proteins rarely act alone but are dependent on interactions
with other proteins.

Cell surface architecture: the missing dimension

This higher - order protein organisation can be described as the cell surface architecture : the
presence of various proteins, their proximity to one another, and their organisation into
functional clusters and domains across the membrane.

The cell surface architecture is at the core of cellular function. It determines whether proteins
can physically interact, how efficiently signalling pathways are activated, and how cells
respond to external cues. Importantly, it is dynamic and can change independently of
expression, enabling cells to regulate function without altering protein levels . Two cells with
similar expression profiles may therefore exhibit a very different behaviour depending on
how their proteins are organised.

Figure legend: Expanding the central dogma. To fully comprehend how proteins carry out their
function, protein interactions and overall organization need to be profiled. This need to be done
at a high multiplex to capture complex structures like protein do mains.

Measuring cell surface architecture with the Proximity Network Assay (PNA)

Conventional single - cell protein profiling methods are limited to either supporting high -
multiplex analysis of protein abundance (such as spectral flow cytometry), or analyzing the
organization of a few selected markers (such as microscopy). The Proximity Network Assay
(PNA), was developed to address this limitation by enabling high-dimensional measurement
of protein organisation at the single-cell level.

Instead of only quantifying how much of a protein is present, PNA captures spatial
relationships between 155 markers by identifying which individual proteins are located in
proximity to each other across the cell surface. The identified proteins are reconstructed into a
network representation of the cell surface, a cell surface map, where each protein is linked based on its spatial associations.

Figure legend: The Pixelgen Proxiome Kit is based on the Proximity Network Assay, a
technology for nanoscale spatial analysis of immune cell proteins. Cells in suspension are
labelled with barcoded antibodies and amplified in situ by rolling circle amplifi cation (RCA).
Linker oligos are bound to the RCAs, connecting neighboring proteins. The connections are
read out by standard NGS. Single - cell surface maps, or Proximity Networks, are reconstructed
using the Pixelator analysis pipeline, generating spatial s tatistics for quantitative analysis of
protein organization across thousands of single cells.

From abundance to architecture: what PNA enables

By design, PNA simultaneously captures three key dimensions of the cell surface:

Protein abundance , describing how much of each protein is present
Protein clustering , reflecting how proteins organise into clusters
Protein colocalisation , revealing proximity relationships between different proteins

These features are derived from the same dataset, enabling researchers to directly compare
abundance and architectural properties within the same cells.

Biological examples: cell surface architecture reveals function

The biological relevance of the cell surface architecture becomes clear when assessed across
different immune systems, where protein organisation provides insight into functions that
cannot be captured by abundance measurements alone.

CAR T cells: optimising therapeutic products through architecture

Chimeric Antigen Receptor (CAR) T cells have revolutionised the treatment of several
haematological cancers, and significant efforts are now focused on improving CAR design and
extending these approaches to solid tumours. While much of this optimisation ha s historically
centred on receptor structure and expression levels, it is increasingly evident that how the
CAR is organised at the cell surface plays a critical role in determining its function.

For instance, Adrienne H Long et al. and Jian Chen et al. showed that CAR distribution and
clustering have a major impact on the overall function of a number of CAR T cells, including
GD2, EGFR and CSPG4 targeting cells. These cells presented spontaneous CAR clustering,
with enhanced tonic signalling and as a c onsequence reduced functional capacity. Altering the
membrane composition of these cells could change CAR organization and restore functional
capacity. Hence, understanding the architectural phenotype of current and next - generation
CARs is essential for developing optimal therapeutic products.

Figure legend: The network displays the
proxiome of the CD19 CAR. Although widely
distributed over the cell membrane, the
receptor organizes in a non - random fashion
displaying colocalization with key functional
proteins like ICAMs, coreceptors and the T
ce ll receptor (TCR), while segregating from
lipid rafts and tetraspanin proteins. Profiling
the organization of the CAR can reveal key
mechanistic regulation and biomarkers of
therapeutic response.

SLE B cells: organisation uncovers dysregulated signalling

In autoimmune disease, changes in protein organisation can provide critical insight into altered
signalling mechanisms. B cells are known to be dysregulated in systemic lupus erythematosus
(SLE), but so far, these dysfunctions have not been fully explained by changes in protein
abundance.

Petter Brodin et al. showed that SLE B cells display an altered organisation of the co - receptor
CD21. While abundance measurements demonstrated protein downregulation, spatial analysis
showed that CD21 exhibits increased clustering, suggesting that the receptor is redistribu ted
into distinct functional domains.

Figure legend: SLE B cells display
downregulation of the coreceptor CD21.
However, the receptor shows increased
clustering, possibly altering its signalling
capacity.

These observations indicate that receptor behaviour, and therefore signalling, can be altered
through changes in organisation even when expression levels are reduced.

This leads to an important mechanistic insight: Dysregulated immune responses in SLE may be
driven by altered cell surface architecture rather than changes in protein abundance alone.

Membrane composition: linking lipid content to function

Cell surface architecture is strongly influenced by the lipid content and physical properties of
the membrane. Proteins are embedded within this environment, and changes in lipid content
can reshape how proteins are distributed and interact.

Luca A. Andronico et al. recently showed that immune cells with different membrane
characteristics exhibit distinct functional advantages. These differences are associated not
only with variations in protein abundance, but also with changes in protein organisation and
spatial context .

This work establishes a direct connection between membrane composition, lipid content, cell
surface architecture, and cellular behaviour. Cells with different membrane states display
differences in functional properties such as migration and cytotoxicity.

This example highlights that cell surface architecture is not only defined by protein
interactions, but also by the biophysical environment in which those interactions occur, and
that it can have drastic effects on cellular function and disease development.

Figure legend: Luca. A. Andronico et al. showed that membrane order (delta GP) is different in
healthy donors, long - covid patients and chronic lymphocytic leukemia (CLL) patients.
Additionally, it affects cellular function and this is driven by alteration s by both protein
expression and organization.

Key takeaway

Protein abundance defines which proteins are present.
Protein organisation reveals how those proteins are arranged to function.

Cell surface architecture plays an essential role in regulating cellular function, both in health
and disease. The Proximity Network Assay enables high - dimensional analysis of this layer of
biology.

Explore further about the technology and its application here:
Protein Interactomics by Proximity Networks - Pixelgen Technologies

Preprint by Pixelgen, “Single - Cell Protein Interactomes by the Proximity Network Assay“
https://www.biorxiv.org/content/10.1101/2025.06.19.660329v1

Preprint by Erdinc Sezgin “Plasma membrane order maps functional diversity in immune cells”,
https://www.biorxiv.org/content/10.1101/2024.01.15.575649v3

GO BEYOND PROTEIN EXPRESSION

Measure more than which proteins are present. Map how cell surface proteins are organized, connected, and spatially arranged across single cells.

Pixelgen’s Proximity Network Assay brings nanoscale protein interactomics to high-throughput immune cell research, helping teams uncover biology that abundance-based methods can miss.

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