Membrane Curvature in Endocytosis
We are interested in in vitro and cell-free reconstitution approaches to address fundamental questions in membrane biology, particularly those related to membrane compartmentalization, curvature generation, vesicular budding and tubulation.
During my PhD, I pioneered the use of patterned supported lipid bilayers to investigate membrane lateral organization and intracellular trafficking. Notably, I provided direct imaging evidence for lipid-dependent sorting of inner leaflet proteins (Wu et al., PNAS 2004) and demonstrated localized trafficking of recycling endosomes to sites of receptor cross-linking (Wu et al., JCS 2007). I also employed zero-mode waveguides and fluorescence correlation spectroscopy, in collaboration with the labs of Watt Webb and Harold Craighead, to measure the diffusion rates of membrane proteins at high resolution.
During my postdoctoral training, I developed a cell-free reconstitution system to study endocytic vesicle budding and fission. This was the first system that enabled real-time, dynamic analysis of endocytic pit formation at single-vesicle resolution (Wu et al., Nature Cell Biology 2010). We discovered F-BAR-dependnet tubulation is coupled with clathrin-coated pits but they are spatially segregated, which was demonstrated with both ultrastructural electron microscopy and super-resolution imaging. Using Stochastic Optical Reconstruction Microscopy (STORM), in collaboration with Bo Huang and Xiaowei Zhuang’s group, we achieved the highest spatial resolution at the time.
In my independent lab, we combined single-molecule imaging and cryo-electron tomography (in collaboration with Ruben Fernandez-Busnadiego) in the cell-free reconstitution system to reveal that the dynamic instability of clathrin assembly acts as a biochemical checkpoint for productive endocytosis (Chen Y et al., Journal of Cell Biology, 2019). Our findings have important implications for cargo sorting during endocytosis (Wu, 2021). Contrary to the traditional view of lock-and-key recognition based solely on binding affinity, our results suggest that the residence time of clathrin coat is a critical factor. This supports a kinetic proofreading model in which cargo selection and quality control depend on the temporal dynamics of the coat disassembly. It also explains why the endocytic reaction requires energy in cells while coat assembly in vitro does not require external energy.
Although the behavior of individual endocytic proteins at the single-vesicle level is well characterized, less is known about their coordination at the subcellular level. We discovered that endocytosis can occur in a synchronized manner in mast cells (Yang et al., Developmental Cell, 2017). This finding points to a non-autonomous regulation of vesicle budding, which intersects with our broader interest in subcellular pattern formation. Synchronized clathrin-mediated endocytosis occurs upstream of F-BAR protein assembly but depends on F-BAR–mediated positive feedback to achieve synchrony, similar to how coupled metronomes synchronize their motion. The formation of curvature waves requires the shallow curvature preference of the F-BAR domain and does not occur if the F-BAR domain is replaced by a high-curvature–preferring BAR domain (Wu et al., Nature Communications 2018). This provides unique insight into how different curvature-generating proteins contribute to endocytosis.
- Wu, Min; Holowka, David A.; Craighead, Harold G.; Baird, Barbara A. Visualization of Plasma Membrane Compartmentalization with Patterned Lipid Bilayers. Proc Natl Acad Sci U S A. 2004, 101(38):13798-803.
- Wu, Min; Huang, Bo; Graham, Morven; Raimondi, Andrea; Heuser, John E.; Zhuang, Xiaowei; De Camilli, Pietro. Coupling between Clathrin-dependent Endocytic Budding and F-BAR-dependent Tubulation in a Cell-free System. Nat Cell Biol. 2010, 12(9): 902-908.
- Yang, Yang; Xiong, Ding; Pipathsouk, Anne; Weiner, Orion D.; Wu, Min*. Clathrin assembly defines the onset and geometry of cortical patterning. Dev Cell. 2017, 43, 507-521.
- Wu, Zhanghan †; Su, Maohan †; Tong, Cheesan; Wu, Min*; Liu, Jian*. Membrane shape-mediated wave propagation of cortical protein dynamics. Nat. Comm. 2018, 9 (1), 136. (†co-first authors; * co-corresponding authors)
- Chen, Yan; Yong, Jeffery; Martinez-Sanchez, Antonio; Wu, Yumei; De Camilli, Pietro; Fernandez-Busnadiego, Ruben; Wu, Min*. Dynamic Instability of Clathrin Assembly Provides Proofreading Control for Endocytosis. The Journal of Cell Biology, 2019, 218(10):3200-3211
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