Scaffold protein SHANK3 regulates endothelial cell motility and tissue mechanics

This study reveals that the scaffolding protein SHANK3, previously known for its neuronal roles, is essential for maintaining endothelial barrier function and regulating collective cell motility and tissue mechanics, as its depletion disrupts vascular development in both zebrafish and mice.

The Gist

The Big Picture: A "Glue" Protein You've Never Heard Of

You've probably heard of SHANK3 in the news before. It's a famous protein in the world of brain science. Think of it like the mortar between bricks in a house. In the brain, this mortar holds neurons together so they can talk to each other. When this mortar is broken, it can lead to conditions like autism.

But this new study discovered something surprising: SHANK3 isn't just in the brain. It's also everywhere in your blood vessels, acting as a crucial "glue" for the cells that line your veins and arteries (called endothelial cells).

The researchers found that when you remove this protein, the blood vessels don't just fall apart; they actually start behaving like a chaotic crowd instead of an organized team.

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The Analogy: The Traffic Jam vs. The Mosh Pit

To understand what happens when SHANK3 is missing, imagine a busy city street.

• Normal Blood Vessels (With SHANK3): Imagine a well-organized parade. The cells (the people) are holding hands, moving in a coordinated line, and keeping a tight formation. They know exactly where to go, and the "street" (the vessel) stays smooth and strong. This is like a solid, organized crowd.
• Blood Vessels Without SHANK3: Now, imagine you pull the "holding hands" rule away. The people stop coordinating. Some run fast, some stand still, and they bump into each other. The line breaks up. The street becomes a chaotic mosh pit.

In scientific terms, the researchers found that without SHANK3:

1. The Cells Get Weird: They stretch out and become long and skinny instead of their usual neat, hexagonal shapes.
2. The Barrier Breaks: The "fence" the cells make to keep blood inside the vessel gets leaky.
3. The Viscosity Changes: This is the coolest part. The researchers measured how "thick" or "sticky" the tissue felt. Without SHANK3, the tissue became runny and fluid, like water, instead of thick and jelly-like. It lost its structural integrity.

The Experiment: What Happened When They Turned Off the Switch?

The scientists used two different "test tubes" to see what happens when they removed SHANK3:

1. The Flat Surface (The Petri Dish)
When they grew blood vessel cells on a flat glass slide and removed SHANK3, the cells started moving faster and more erratically. It was like taking the brakes off a car. They were zooming around, but they weren't working together.

2. The Real World (Zebrafish and Baby Mice)
This is where the plot twist happened.

• In a flat dish: The cells moved faster.
• In a living animal: The blood vessels grew slower and got stuck.

Why the difference?
Think of building a new road through a forest (which is what blood vessels do when they grow).

• In the flat dish, the road is already paved. The workers (cells) just need to run around. Without SHANK3, they run wild and fast, but they can't build anything new.
• In the living animal, the workers need to build the road from scratch. They need to hold hands tightly to pull themselves forward and push through the soil (the body tissue). Without SHANK3, they can't hold hands. They slip, they get lost, and the road (the blood vessel) never gets built properly.

In the baby mice and zebrafish, the lack of SHANK3 meant the blood vessels couldn't branch out correctly. They were short, stubby, and didn't reach their destinations.

Why Does This Matter?

This discovery connects two very different worlds: Brain Health and Heart/Blood Health.

1. Autism and Vision: We already know SHANK3 mutations cause autism. This study suggests that maybe some of the vision problems or eye issues seen in people with autism aren't just about the brain, but because the tiny blood vessels in the retina (the back of the eye) didn't develop correctly due to missing SHANK3.
2. Healing and Disease: Since SHANK3 helps blood vessels stay strong and organized, problems with this protein could play a role in diseases where blood vessels are leaky or don't heal well, like in diabetes or after a heart attack.

The Takeaway

SHANK3 is the "Team Captain" of your blood vessel cells.

• With the Captain: The team holds hands, moves together, builds strong roads, and keeps the fence secure.
• Without the Captain: The team turns into a chaotic mob. They run fast but go nowhere, the fence leaks, and the roads never get built.

This paper teaches us that the same protein that helps our brains think is also essential for our blood vessels to grow and stay strong. It's a reminder that our body's systems are all connected, and a single broken part can cause trouble in unexpected places.

Technical Summary

1. Problem Statement

SHANK3 is a well-characterized multidomain scaffolding protein primarily studied in the context of neuronal function, where its mutations are linked to autism spectrum disorder (ASD), Phelan-McDermid syndrome, and schizophrenia. While recent studies have hinted at SHANK3's role in non-neuronal tissues (e.g., cancer cell survival, intestinal barrier integrity, and blood-brain barrier regulation), its specific function in endothelial cells (ECs) and vascular development remains poorly understood. Given that angiogenesis relies on the precise coordination of cell migration, cell-cell junctions, and tissue mechanics, the authors sought to determine if SHANK3 plays a critical role in endothelial barrier function, collective cell motility, and vascular morphogenesis.

2. Methodology

The study employed a multi-faceted approach combining bioinformatics, cell biology, biophysics, and in vivo animal models:

• Bioinformatics: Analysis of single-cell RNA sequencing data (Tabula Sapiens) to map SHANK3 expression across human tissues and cell types.
• Cell Culture Models:
  • Human Umbilical Vein Endothelial Cells (HUVECs): Used for siRNA-mediated knockdown (two distinct siRNAs) and doxycycline-inducible shRNA depletion.
  • U2OS Cells: Used for proximity labeling (BioID) to identify SHANK3 interactors and "knock-sideways" assays to validate interactions.
• Proteomics: BioID (proximity biotinylation) coupled with Mass Spectrometry (MS) to map the SHANK3 interactome.
• Biophysical Assays:
  • Traction Force Microscopy (TFM): To measure cell-matrix forces.
  • Spheroid Assays: Wetting (spreading) and fusion assays to measure tissue viscosity and surface tension.
  • 3D Collagen Sprouting: To assess angiogenic sprouting in a 3D matrix.
  • Live-cell Imaging: Tracking of nuclear migration, velocity correlation functions, and T1 event frequency to analyze collective migration dynamics.
• In Vivo Models:
  • Zebrafish: CRISPR-Cas9 generated shank3b crispants (F0 mutants) to analyze intersegmental vessel (ISV) development.
  • Mice: Inducible endothelial-specific deletion of Shank3 (Shank3^iECKO) using Cdh5-CreERT2 in postnatal retinas to study angiogenic sprouting.
• Imaging: Confocal microscopy, spinning-disk microscopy, and whole-mount immunofluorescence for tissue staining (VE-cadherin, ZO-1, α-catenin, etc.).

3. Key Contributions

• Discovery of Endothelial SHANK3: Established that SHANK3 is widely and highly expressed in endothelial cells across diverse human tissues (spleen, heart, adipose, trachea) and localizes specifically to cell-cell junctions.
• Mechanistic Link to Tissue Mechanics: Demonstrated that SHANK3 is a critical regulator of endothelial tissue viscosity and mechanical integrity, linking a neuronal scaffold protein to vascular biomechanics.
• Dual Role in Migration: Revealed a paradoxical role where SHANK3 depletion increases migration speed in 2D monolayers (fluid-like state) but impairs coordinated sprouting and migration in 3D and in vivo environments.
• Interactome Mapping: Provided a comprehensive map of SHANK3 proximity interactors in endothelial contexts, highlighting connections to actin regulators and junctional proteins (e.g., ZO-1, VE-cadherin, α-catenin).

4. Key Results

A. Expression and Localization

• SHANK3 is highly expressed in endothelial cells, particularly in the spleen, heart, and adipose tissue.
• In HUVECs, SHANK3 localizes to cell-cell junctions, co-localizing with VE-cadherin, α-catenin, β-catenin, and notably ZO-1. It is found at both linear adherens junctions and protrusive, finger-like structures connecting to actin "aster" structures at the basal side.

B. The SHANK3 Interactome

• BioID screening identified 94 high-confidence interactors.
• 30.9% were cell junction proteins (e.g., ZO-1, p120-catenin, afadin) and 25.5% were cytoskeletal proteins.
• Validation confirmed interactions with ZO-1, WAVE2, ASAP1, and IRSp53.

C. Effects of SHANK3 Depletion in Vitro (2D & 3D)

• Morphology & Barrier: Depletion caused cell elongation, reduced circularity, and compromised barrier integrity (increased gaps in monolayers).
• Migration Dynamics:
  • 2D Monolayers: SHANK3-depleted cells exhibited increased migration speed and total displacement. However, this was accompanied by dynamic heterogeneity (spatial segregation of fast and slow cells) rather than coordinated collective migration.
  • Tissue Viscosity: Depleted spheroids spread faster (wetting assay) and fused more rapidly (fusion assay), indicating a reduction in effective tissue viscosity (transition toward a more fluid-like state).
  • Forces: Traction forces on the substrate were reduced, and junctional tension (measured by α-catenin force sensor) was altered, suggesting impaired force transmission.
• 3D Sprouting: In collagen gels, SHANK3-depleted spheroids formed more numerous but shorter sprouts (<100 µm) and fewer long sprouts, indicating a failure in coordinated elongation.

D. In Vivo Phenotypes

• Zebrafish (shank3b crispants): Showed delayed ISV sprouting, reduced endothelial cell migration speed, and a higher frequency of missing ISVs at 48 hours post-fertilization.
• Mouse Retina (Shank3^iECKO): While the overall radial expansion of the vascular plexus was normal, there was a significant reduction in sprout number, fewer branch points, shortened vessel skeleton length, and a loss of long sprouts at the angiogenic front. This mirrors the 3D in vitro phenotype.

5. Significance

• Novel Function of SHANK3: This study expands the functional scope of SHANK3 beyond the nervous system, identifying it as a crucial regulator of endothelial barrier function and vascular morphogenesis.
• Mechanotransduction in Vasculature: The findings suggest that SHANK3 is essential for maintaining the "solid-like" (jammed) state of endothelial monolayers required for coordinated collective migration during angiogenesis. Its loss leads to a "fluid-like" state where cells move individually but fail to form organized vascular networks.
• Clinical Implications:
  • Vascular Development: Provides a mechanistic explanation for potential vascular defects in patients with SHANK3 mutations.
  • ASD and Vision: The study highlights a potential link between SHANK3 mutations, vascular development in the retina, and the high prevalence of vision/eye disorders (e.g., reduced peripheral vision) observed in ASD patients.
  • Vascular Pathologies: The role of SHANK3 in barrier integrity suggests potential implications for vascular permeability in inflammatory or metastatic contexts.

In conclusion, SHANK3 acts as a mechanical scaffold in endothelial cells, ensuring the structural integrity of cell junctions and the coordinated tissue mechanics necessary for efficient angiogenic sprouting. Its depletion disrupts the balance between cell motility and tissue cohesion, leading to defective vascular development.