Quantifying the relationship between membrane-curving proteins and nanoscale membrane topology
Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cell Biology
First Advisor
Bewersdorf, Joerg
Abstract
The endoplasmic reticulum (ER) is the site of many critical cellular functions. Its unique sub-structures, including tubules and sheets, are responsible for different functions. Perturbations of ER structure have been implicated in the health of cells and the disease state of tissues, emphasizing the importance of maintaining the ER’s morphology. Yet, how cells maintain ER morphology is still poorly understood. Moreover, the analytical tools needed to enable the thorough investigation of the responsible processes have been severely lacking. This thesis addresses these problems by developing novel analysis tools and applying them to better understand the nanoscale organization of ER-shaping proteins and how they affect local ER structure, the results of which has led to a paradigm shift in ER morphology. First, I describe the shrink-wrapping algorithm. This is a novel tool that creates accurate 3D surfaces from single-molecule localization microscopy data (SMLM). While tools that can create surfaces from a set of points existed prior to shrink wrapping, none of them account for the unique features of noisy SMLM data. Shrink wrapping accounts for the localization precision of each point in a point attraction force that pulls the surface towards points in a manner that is weighted by their localization precisions. This force is balanced by a curvature force that penalizes curvatures on the surface that are biophysically unrealistic. This tool laid the foundation for my quantitative investigation of the nanoscale organization of reticulon 4 (Rtn4) and how that affects local ER structure. I discovered that Rtn4 organizes into linear-shaped oligomers, containing ~5 copies of the protein, that enrich at the sides of tubules and preferentially orient parallel to tubule axes. The local density of Rtn4 strongly correlates with the intensity of the local membrane curvature, and knocking out Rtn4 results in significantly larger tubule diameters. This strongly suggests that Rtn4 plays a significant role in determining curvature intensity in the ER in a concentration-dependent manner. These results challenge a long-standing model in the field of Rtn4 organization and culminate in a highly refined model of ER morphology as it relates to Rtn4 oligomers.
Recommended Citation
Fuentes, Lukas Allen, "Quantifying the relationship between membrane-curving proteins and nanoscale membrane topology" (2023). Yale Graduate School of Arts and Sciences Dissertations. 896.
https://elischolar.library.yale.edu/gsas_dissertations/896
