Dynamic myosin 10 coupling to DCC and β1 integrin is mediated by intrinsically disordered regions during filopodial transport and patterning

This study reveals that Myosin 10 utilizes intrinsically disordered regions to employ distinct binding mechanisms—combining preformed recognition elements with dynamic weak interactions—to differentially engage DCC and β1 integrin cargos, thereby enabling tunable multivalent binding that regulates their spatial patterning and competitive occupancy within filopodia.

The Gist

The Big Picture: The Cell's Delivery Truck and Its Packages

Imagine a cell as a busy city. Inside this city, there are tiny, microscopic delivery trucks called Myosin 10 (Myo10). Their job is to drive along the cell's "highways" (which are made of actin filaments) to deliver important packages to specific locations, like the tips of finger-like projections called filopodia. These projections are like the cell's feelers, used to sense the environment, find a path, or grab onto surfaces.

The problem? The trucks need to know which packages to pick up. In this study, the researchers looked at two very different types of packages:

1. DCC: A rigid, structured package that acts like a GPS for neurons (helping them grow in the right direction).
2. Beta-1 Integrin: A flexible, sticky package that acts like a grappling hook, helping the cell stick to surfaces.

The big question was: How does the truck know which package to grab, and how does it keep them from falling off while driving?

The Secret Weapon: The "Fuzzy" Rope

The researchers discovered that the truck (Myo10) doesn't use a rigid clamp to hold these packages. Instead, it uses a special part of its tail made of Intrinsically Disordered Regions (IDRs).

Think of IDRs not as solid metal hooks, but as fuzzy, stretchy bungee cords.

• The DCC Package (The GPS): This package has a specific "key" (called the P3 motif) that fits perfectly into a lock on the truck. Once it clicks in, the rest of the DCC package is a long, floppy tail. The study found that this floppy tail wraps around the truck like a bungee cord. Even if the truck hits a bump or the road shakes, the bungee cord stretches and snaps back, keeping the package attached. It's a "fuzzy" grip that is actually very strong because it has many weak points that all work together.
• The Integrin Package (The Grappling Hook): This package is different. It doesn't have a strong "key" that locks in tightly. It just kind of hangs on with a few weak, temporary touches. It's like trying to hold a wet bar of soap with one finger. It's much easier to lose.

The Traffic Jam: Who Gets the Truck?

The researchers found something fascinating happens when both packages are in the cell at the same time.

Imagine the truck is driving down the highway.

• DCC is holding on tight with its bungee cord.
• Integrin is holding on weakly.

If the truck is busy and can only carry one, DCC wins. Because DCC holds on so much better (thanks to that bungee cord effect), it pushes the Integrin off the truck.

The Result:

• DCC stays on the truck all the way to the very tip of the cell's finger (the filopodium tip). This is crucial for telling the cell where to grow next.
• Integrin, having been bumped off, falls off the truck earlier. It ends up stuck to the side of the cell or the bottom of the finger, acting as an anchor to hold the cell in place, rather than guiding it forward.

Why This Matters

This study explains a clever biological trick: The cell uses "messy" and "flexible" parts of proteins to control traffic.

1. Flexibility is Strength: The "disordered" (messy) parts of the DCC protein aren't a flaw; they are a feature. They act like shock absorbers, allowing the package to stay attached even when the cell is moving or being pulled.
2. Prioritization: By having one package (DCC) hold on tighter than the other (Integrin), the cell can decide what is most important at any given moment. If the cell needs to grow a new path, it keeps the GPS (DCC) on the truck. If it needs to stop and stick, the grappling hook (Integrin) gets left behind to do its job.

Summary Analogy

Think of the cell as a construction site.

• Myo10 is the crane.
• DCC is a heavy, valuable blueprint wrapped in a bungee cord.
• Integrin is a bag of sand held by a rubber band.

When the crane moves, the bungee cord keeps the blueprint safe and secure, even if the crane sways. The rubber band holding the sand is weak; if the crane moves too fast or if the blueprint gets in the way, the sand falls off. The crane naturally prioritizes the blueprint because it's easier to keep hold of. This ensures the construction crew (the cell) knows exactly where to build next, while the sand (the anchor) stays where it was dropped to keep the site stable.

This research shows us that in biology, sometimes being "messy" and "flexible" is the smartest way to get the job done.

Technical Summary

1. Problem Statement

Myosin 10 (Myo10) is an unconventional motor protein essential for filopodia formation, neuronal guidance, and cancer progression. It transports diverse cargo receptors, notably Deleted in Colorectal Cancer (DCC) and β1 integrin, to filopodial tips. While the structural domains involved (the Myo10 M4F domain and specific motifs on the cargo tails) are partially known, the molecular mechanisms governing:

• How Myo10 achieves high-affinity binding with cargo despite weak individual interactions.
• The specific role of Intrinsically Disordered Regions (IDRs) in the cargo tails.
• How Myo10 selectively coordinates or switches between different cargo types (DCC vs. integrin) during transport.
• The spatial organization of these complexes within the thin geometry of filopodia.

remained unclear. Traditional models often assume rigid, high-affinity binding, but the presence of long IDRs in cargo tails suggests a more dynamic, "fuzzy" interaction mechanism that is not fully understood.

2. Methodology

The authors employed a multi-modal approach combining structural biophysics, mass spectrometry, and advanced super-resolution microscopy:

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Used to map binding interfaces and monitor conformational changes (disorder-to-order transitions) upon complex formation. Experiments were conducted on isolated M4F domains, cytoplasmic cargo tails (DCC and β1 integrin), and single-chain constructs (M4F linked to cargo) to increase effective local concentration.
• Cross-Linking Mass Spectrometry (XL-MS): Utilized a DSSO crosslinker to capture transient and weak interactions between M4F and the disordered regions of DCC, identifying "hotspots" of interaction that HDX might miss.
• Live-Cell Imaging (TIRF Microscopy): U2OS cells were transfected with HaloTag-Myo10 and EGFP-tagged DCC (including various P-motif deletion mutants: ΔP1, ΔP2, ΔP3). This allowed for real-time tracking of cargo recruitment and co-movement during filopodial initiation and elongation.
• Super-Resolution Microscopy (DNA-PAINT): Provided ~5 nm localization precision to map the 3D spatial distribution of Myo10, DCC, and endogenous β1 integrin along the filopodial shaft and tip.
• Quantitative Analysis: Protein concentrations were estimated from DNA-PAINT data to calculate binding occupancy relative to known dissociation constants ($K_D$). Pearson correlation coefficients were used to quantify cargo-Motor co-localization dynamics.

3. Key Contributions & Results

A. Mechanisms of IDR-Mediated Binding

The study reveals two distinct mechanisms by which Myo10 engages its cargo:

1. Disorder-to-Order Transition (DCC): The P3 motif of the DCC cytoplasmic tail undergoes a conformational shift from a disordered state to a weakly helical structure upon binding the Myo10 M4F domain. HDX-MS showed a ~10-fold increase in protection (stability) for the P3 region.
2. "Fuzzy" Binding (DCC & Integrin):
   • DCC: While P3 orders, the N-terminal and P1/P2 regions of DCC remain disordered but contribute to binding via multiple weak, transient interactions ("fuzzy" binding). XL-MS confirmed crosslinks between disordered DCC regions and multiple sites on M4F.
   • β1 Integrin: The integrin tail binds via NPxY motifs that remain disordered upon binding. HDX showed only minor stabilization of the M4F domain, indicating a lack of rigid structural ordering.

B. Binding Interfaces and Competition

• Shared vs. Unique Sites: Both DCC and β1 integrin bind to a common surface on the Myo10 M4F domain (residues ~1994-2000). However, they also engage distinct secondary sites: DCC interacts with the C-terminal helix, while integrin engages a unique site (residues ~1557-1567).
• Competitive Binding: Because DCC has a higher affinity (lower $K_D$) and utilizes a stabilizing helical transition, it can out-compete β1 integrin for Myo10 binding when both are present.
  • Evidence: In co-expression experiments, high levels of DCC caused active β1 integrin to redistribute from the filopodial tip to the shaft/basal surface, whereas DCC remained tightly coupled to Myo10 at the tip.

C. Spatial Patterning and Dynamics

• DCC Organization: DNA-PAINT revealed that DCC and Myo10 are tightly colocalized and distributed uniformly around the filopodial circumference. They often appear as a 2:1 complex (Myo10:DCC), suggesting stable association during transport.
• Integrin Organization: In contrast, β1 integrin is frequently dissociated from Myo10 along the shaft and is enriched on the basal surface (closest to the coverslip).
• Role of IDRs as "Bungee Cords": The authors propose that the disordered regions of DCC act as elastic linkers. They allow the cargo to remain attached to the motor even under mechanical stress (impulses) during filopodial growth. If one interaction breaks, others can reform, maintaining complex integrity. In contrast, the more brittle, rigid connection of integrin leads to frequent detachment.

D. Functional Mutagenesis

• DCC Mutants: Deletion of the disordered P1 and P2 motifs (ΔP1ΔP2) did not abolish binding but significantly delayed the recruitment of DCC to Myo10 at the onset of filopodial formation and reduced the correlation of movement during transport. This confirms that IDRs are crucial for the kinetics and stability of the initial cargo capture.
• Myo10 Mutants: Mutations in the integrin-binding sites of M4F (Myo10-7mut) significantly reduced active β1 integrin localization at filopodial tips, confirming the functional relevance of the identified binding sites.

4. Significance

• Mechanistic Insight into Transport: The paper challenges the static "lock-and-key" model of motor-cargo transport. It demonstrates that regulated disorder is a functional feature, allowing motors like Myo10 to use "tunable, multivalent binding strategies" to coordinate diverse signaling cargos.
• Cargo Selection Logic: It provides a physical basis for how cells prioritize signaling: Myo10 preferentially transports DCC (high affinity, stable fuzzy binding) over integrin (lower affinity, brittle binding). This competition dictates the spatial patterning of receptors, influencing whether integrins are transported to the tip for guidance or deposited on the shaft for adhesion.
• Biological Implications: These findings elucidate how neuronal pathfinding (via DCC/Netrin) and cell adhesion/invasion (via Integrin) are mechanistically coordinated. The "bungee cord" mechanism of IDRs may be a general principle for maintaining protein complex integrity in dynamic cellular environments subject to mechanical forces.
• Cancer and Development: Given the roles of Myo10, DCC, and integrin in tumor progression and axon guidance, understanding these competitive binding dynamics offers new potential targets for modulating cell migration and growth cone behavior.

In summary, the study establishes that Intrinsically Disordered Regions are not merely flexible linkers but active mediators of binding affinity, selectivity, and mechanical resilience, enabling Myo10 to dynamically sort and transport distinct cargos to specific cellular locations.