טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentVishnia Kalanit
SubjectStructure-Function Studies of BAR Domain Proteins
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Dganit Danino


Abstract

Many cellular activities such as organelle biogenesis, cell division, cell migration, cell trafficking, exocytosis, and endocytosis, crucially depend on dynamic membrane remodeling achieved by the interplay between lipids and proteins. Members of the Bin-Amphiphysin-Rvs167 (BAR) domain family proteins are key players in membrane remodeling. They can sense, stabilize and induce membrane curvature.

BAR domains are self-dimerized to form helical bundle homo-dimers and create elongated crescent or banana-shaped structures. The dimerization module forms a positively charged surface that associates with negatively charged phospholipids in the cellular membrane. BAR domain proteins can be categorized into 3 sub-groups: N-BAR, F-BAR, and I-BAR. These groups differ in their domain overall degree of curvature and its direction (concave/convex).

The exact function of some BAR domain proteins remains elusive. However, it is obvious that their interaction with membranes is a key factor in their functions.

We analyzed the interactions of BAR proteins, mainly Missing in Metastasis (MIM) and Nervous wreck (Nwk) with model membranes, as a function of lipid type and composition and parameters as fluidity and charge, by cryogenic-TEM, spectroscopy, and calorimetry methods. We focused on two model membranes: liposomes of a single lipid, phosphatidylserine (PS), and phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2)-containing liposomes. These lipids are negatively charged and enriched at the plasma membrane where MIM and Nwk function.

MIM is a classical I-BAR domain protein. It is involved in protrusion formation (like filopodia) in the plasma membrane of different cell types. We identify for the first time the ability of MIM to remodel PS liposomes to various structures: long and straight tubes, branched net of tubes, and unique structure of membrane tunnels (revealed by cryo-electron tomography). The interaction between MIM and PI(4,5)P2 resulted in similar structures. While literature highlights preferential binding of BAR domains to PI(4,5)P2, our data shows that MIM can bind and remodel numerous anionic lipid systems.

To date, there is almost no information on binding constants of BAR domains with membranes. Although challenging, we succeed in establishing a methodology relying on the isothermal titration calorimetry (ITC) method, where we can routinely quantify protein-lipid interactions of curvature remodeling proteins. We found that MIM has a high binding affinity to PS, yet, higher binding affinity to PI(4,5)P2-containing liposomes. This trend was verified with another method called the microscale thermophoresis (MST). Spectroscopy measurements showed a change in MIM secondary structure upon binding to PS liposomes, but not to PI(4,5)P2-containing liposomes.

Last, we performed high-resolution imaging of MIM tubes. The high-resolution analysis revealed that the protein is bound to the inner leaflet of the tube, creating a characteristic pattern. Computational analysis provided the first evidence for the high order helical organization of MIM inside the tube.

Nwk is an F-BAR protein, functioning in the nervous system. However, in cells, it is involved in protrusion formation rather than invaginations. Interactions of Nwk F-BAR and Nwk full length with PI(4,5)P2 containing liposomes resulted in gentle deformations to liposomes. Additionally, we found that the level of PI(4,5)P2 in the liposomes regulates Nwk full-length protein activity.