Design principles for ensuring robustness and efficiency in clathrin-mediated endocytosis revealed by mathematical modeling and quantitative imaging
Clathrin-mediated endocytosis (CME) is a highly conserved pathway in eukaryotes responsible for internalization of cell surface receptors and associated cargo, regulation of biochemical signaling pathways, and maintaining plasma membrane composition via the deformation and pinching off of a vesicle from the plasma membrane. Decades of work by the scientific community have served to identify over 60 different proteins that are directly involved in this process and to determine the relative timing and putative function of a large number of these proteins. One principal conclusion that could be drawn from this large body of work is that CME is both highly robust to perturbations as well as quite efficient in terms of the fraction of nascent endocytic sites that ultimately successfully internalize. The general principles that underlie this robustness and efficiency remained unclear as I began my graduate studies.
To investigate the mechanical robustness of CME, I performed continuum modeling of the shape progression of endocytic sites in response to a ``protein coat'' that can vary in size, stiffness, and spontaneous curvature. I demonstrated that elevated tension in the membrane stalls the progression of curvature, confirming the physical basis for similar observations made in experimental studies. Additionally, I found that the shape evolution of the membrane should undergo a snapthrough instability at physiologically relevant values of membrane tension, which might serve as both an energy and a kinetic barrier to membrane deformation. This instability can be avoided by either increasing the stiffness of the coat or by the action of external forces on the membrane, simulating the proposed role for actin polymerization at endocytic sites. Combining both increased coat stiffness and force from actin polymerization allows for vesiculation even under high membrane tension conditions. These results demonstrate that cells ensure the robustness of CME by employing redundant but complementary mechanisms for deforming the plasma membrane.
To explore how the high efficiency of CME is achieved, I sought to determine how progression through the pathway is regulated. Thus, I undertook a systematic imaging study in which I performed two-color live cell total internal reflection fluorescence (TIRF) microscopy on over two dozen strains of the budding yeast, Saccharomyces cerevisiae. Each strain expressed both a GFP-tagged endocytic protein and a red fluorophore-tagged reporter protein which were chosen so as to span the entirety of the CME pathway. Using automated tracking and association software, I extracted the fluorescence intensity over time of over 10,000 endocytic sites. This dataset not only recapitulates the canonical timeline for CME in budding yeast, but also reveals that there is substantially more variability in the timing and abundance of proteins in pathway than had previously been appreciated. Taking advantage of this inherent variability, I sought meaningful statistical correlations in the data that could give insight into the regulation of progression through the pathway. The lifetimes of proteins early in the endocytic pathway are highly correlated with one another, as are those of the late endocytic proteins. However, the lifetimes of the early proteins have no detectable correlation with their own abundances nor with the lifetimes or abundances of late-stage proteins. Taken together, these data quantitatively substantiate the existence of a regulatory transition point between early- and late-stage CME, as had previously been proposed in the absence of entirely conclusive evidence. Additional evidence indicates that progression through this point is regulated by the presence of cargo. This result suggests that cells not only optimize for efficiency in CME on the basis of internalization efficiency but also on a cost-benefit basis by setting a cargo requirement before membrane deformation is initiated.
These insights into the design principles for robustness and efficiency of CME build upon the foundations of past efforts to identify and characterize individual proteins in the endocytic pathway by expanding the focus to a systems level understanding of the process. Additionally, these results demonstrate the synergy of quantitative analysis and modeling in biology, which will become even more important as the tools for generating quantitative data in biology become ever more powerful and easy to use.