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Science Spotlight

Endophilin’s charged relationship with curved membranes

From the Bai lab, Basic Sciences Division

In life, as in art, the beautiful moves in curves. -Edward Bulwer-Lytton

The cell membrane is more than just a structural barrier. It’s also a dynamic transportation hub, constantly absorbing and budding off small bits of itself in the form of vesicles that move proteins in and out of the cell. The importance of this process is particularly notable in the nervous system – the fusion of intracellular synaptic vesicles with the cell membrane releases neurotransmitters that allow a neuron to send signals to another downstream neuron, supporting signal propagation in the nervous system. Because synaptic vesicles are consumed by fusion and become a part of the plasma membrane, to sustain communication, the neuron must regenerate synaptic vesicles via endocytosis, replenishing the pool for another round of signaling. Key to the formation of new vesicles is the generation of high-curvature regions in the cell membrane, a process in which so-called “curvature-sensing proteins” play important roles. The endophilin protein, for instance, promotes the formation of new synaptic vesicles. “Research on curvature-sensing mechanisms has intensified in recent years,” says Dr. Jihong Bai, Professor in Fred Hutch’s Basic Sciences Division. “However, while the list of known curvature-sensing proteins continues to grow, a significant knowledge gap exists between in vitro protein biochemistry and in vivo functions of curvature-sensing proteins.” In a new article in Developmental Cell, Dr. Bai’s group, led by research technician Lin Zhang and graduate student Yu Wang, examined the properties that specify the function of endophilin and other curvature sensing proteins in the neurons of the C. elegans worm.

“One class of curvature-sensing modules consists of amphipathic motifs [which have both hydrophilic and hydrophobic regions]…in solution, the amphipathic motifs are disordered. Upon contact with curved membranes, they fold into a helical structure with polar and nonpolar residues segregated into two faces of the helix,” the authors wrote. The amphipathic H0 motif of endophilin is essential for its curvature sensing: loss of this region impairs synaptic vesicle recycling in neurons. To understand whether amphipathic motifs act as generic or highly specified curvature sensors, the authors replaced endophilin’s H0 motif with the amphipathic helices of several other curvature-sensing proteins and examined how well these hybrid proteins functioned in promoting locomotion, which requires endophilin activity. Some hybrid proteins were non-functional, but several of the motifs they tested, including those with significant differences in sequence and native site of action, could effectively replace the H0 motif.

What allowed some amphipathic motifs to successfully replace endophilin H0 and some to fail puzzled the authors. For instance, they noted that two motifs with similar biochemical curvature sensing properties – from the NUP133 and KES1 proteins – “exhibited drastic differences in their ability to replace…H0 in vivo.” While both hybrid proteins localized to the synapse appropriately, direct measurements of neurotransmitter recycling and synaptic activity showed that the NUP133 hybrid proteins functioned normally while the KES1 hybrid protein exhibited slow and impaired function. Furthermore, high-resolution visualization of vesicles within the synapse using electron microscopy showed that the KES1 hybrid protein generated fewer small synaptic vesicles, but more large endosome-like vesicles, suggesting a potential trade-off in the creation of these two vesicular structures.

The group next asked what properties might distinguish the functionality of these two motifs. They observed an intriguing difference in electrical charge between these motifs – H0 and NUP133 both had a positive charge, while KES1 was neutral. Mutating the KES1 hybrid protein to be positively charged was sufficient to render it functional in synaptic recycling. Additionally, mutating the H0 motif to remove its positive charge disrupted endophilin’s function. Thus, the group concluded, endophilin is a more complex protein than previously appreciated. It appears to act as a dual sensor, recognizing both membrane curvature via its amphipathic motif and membrane charge via its ionic composition to promote synaptic vesicle formation.

Because vesicular trafficking impacts many key cellular functions, Dr. Bai is excited about the promise of this work to inform synthetic biology approaches to drive new cellular functions or treat diseases such as neurodegeneration. “Our work sets a precedent for elucidating and reprogramming the functional specificity of curvature-sensing motifs in vivo,” he explains. The next big question is, “can we design curvature sensors with specific functions from scratch?”

endophilin model
Endophilin acts as a dual-function sensor of membrane curvature and electrostatic charge to promote synaptic vesicle (SV) recycling. Image provided by Dr. Jihong Bai.

This work was supported by the National Institutes of Health.

Fred Hutch/UW Cancer Consortium member Jihong Bai contributed to this work.

Zhang L, Wang Y, Dong Y, Pant A, Liu Y, Masserman L, Xu Y, McLaughlin RN Jr, Bai J. The endophilin curvature-sensitive motif requires electrostatic guidance to recycle synaptic vesicles in vivo. Dev Cell. 2022 Mar 28;57(6):750-766.e5. doi: 10.1016/j.devcel.2022.02.021. Epub 2022 Mar 17. PMID: 35303431; PMCID: PMC8969179.