A switch in clathrin turnover controls endocytic coat size and organisation

Problem
Clathrin-mediated endocytosis (CME) is a conserved mechanism for nutrient uptake and signal regulation, yet the mechanical link between clathrin biochemistry and membrane curvature generation remains unclear. While clathrin is known to assemble into polyhedral structures, it is debated whether it directly drives membrane deformation or merely stabilizes shapes acquired by other means. Furthermore, the role of clathrin's dynamic assembly—specifically its turnover and stability—during the endocytic process is not fully understood. Previous studies suggest clathrin is not strictly required for membrane bending but is essential for the timing and robustness of the process. This study investigates how the dynamic properties of the clathrin lattice regulate the temporal progression, size, and organization of the endocytic coat.

Methodology
The researchers utilized Saccharomyces cerevisiae as a model system due to its stereotypical endocytic timeline. Key methodological approaches included:

• Two-Colour Fluorescence Recovery After Photobleaching (FRAP): To distinguish between protein growth (assembly) and turnover (exchange), the authors developed a simultaneous two-colour FRAP assay. They endogenously tagged clathrin light chain (Clc1) and heavy chain (Chc1) with tandem sfGFP and mCherry. By photobleaching only mCherry and monitoring both GFP (total amount) and mCherry (turnover) signals, they could separate coat growth from subunit exchange.
• Genetic Manipulation: Mutant strains were generated to disrupt specific regulatory mechanisms:
  • swa2-ΔJ: Deletion of the J-domain of Swa2 (yeast auxilin homolog), preventing recruitment of the Hsc70 ATPase uncoating machinery.
  • chc1Δtail: Truncation of the clathrin heavy chain tail, preventing ATPase binding.
  • sla1Δ and sla1AAA: Deletion of the late-coat protein Sla1 and a point mutation (LLDLQ to AAALQ) abolishing Sla1's clathrin-binding motif, respectively.
  • Double mutants (swa2-ΔJ sla1Δ) were created to study the combined effect of uncontrolled turnover and lack of stabilization.
• Imaging Techniques: Total Internal Reflection Fluorescence (TIRF) microscopy was used to visualize endocytic sites and perform FRAP. Correlative Light and Electron Microscopy (CLEM) was employed to correlate fluorescence signals with ultrastructural details of membrane deformation and actin networks.
• Quantitative Analysis: Centroid tracking of coat markers (Pan1, Sac6) and fluorescence intensity quantification were used to measure internalization speed, coat size, and protein accumulation.

Key Contributions and Results
The study reveals that clathrin dynamics undergo a regulated "switch" from a high-turnover state to a stable state, which is critical for endocytic progression.

1. Biphasic Clathrin Dynamics: Clathrin exhibits two distinct phases during coat assembly.

   • Early Phase: The clathrin lattice is highly dynamic, with rapid turnover (mobile fraction ~45%, half-time ~1.4–1.7 s). This turnover is driven by the Swa2/ATPase uncoating machinery.
   • Late Phase: Prior to the onset of membrane deformation (budding), clathrin turnover ceases, and the lattice becomes stable. This stabilization is mediated by the late-coat protein Sla1, which binds directly to clathrin.
   • Independence from Adaptors: While clathrin dynamics are regulated, the turnover of clathrin adaptors (e.g., Yap1801) remains independent of clathrin's own turnover, suggesting distinct functional layers within the coat.

2. Regulation of Coat Size and Timing:

   • Early Turnover: In the absence of Swa2-mediated turnover (swa2-ΔJ), the early phase is prolonged and variable. This leads to the accumulation of excessive early and late coat proteins, disrupting the precise timing of the transition to the late phase.
   • Late Stabilization: In the absence of Sla1-mediated stabilization (sla1Δ or sla1AAA), clathrin continues to turnover during the late phase. This results in delayed internalization and increased variability in membrane deformation.

3. Consequences of Uncontrolled Assembly:

   • In the double mutant (swa2-ΔJ sla1Δ), where both early turnover and late stabilization are compromised, endocytic coats grow abnormally large, exceeding the diffraction limit.
   • These oversized coats fail to deform the membrane. Instead of invaginating, the actin network pulls the edges of the coat inward while the center remains flat, leading to a "buckling" effect and detachment from the membrane.
   • CLEM analysis confirmed that in these mutants, large actin networks assemble without successful membrane curvature, resulting in failed vesicle budding.

Significance
The paper claims that the dynamic properties of the clathrin lattice are not merely a byproduct of assembly but are critical regulators of the mechanical and temporal aspects of endocytosis. The findings propose a model where:

• Controlled Turnover: Early-phase turnover prevents the uncontrolled accumulation of coat proteins, ensuring the coat reaches an optimal size.
• Stabilization: Late-phase stabilization creates a rigid template that allows the actin network to effectively transmit force to the membrane, overcoming turgor pressure to drive vesicle budding.
• Mechanical Link: The transition from a flexible, dynamic lattice to a solid, stable one is the essential step that enables the actin machinery to successfully deform the membrane.

The authors conclude that clathrin acts as a "switch" controlling coat size and organization, and that the regulation of its assembly state is fundamental to the robustness of endocytic vesicle formation. This challenges the view of clathrin solely as a static structural scaffold, highlighting its active role in the mechanics of membrane remodeling.