Yidi Sun

A Pathway for Association of Receptors, Adaptors, and Actin during Endocytic Internalization

In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition. Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility. Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions. Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly. In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes. Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization. Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved.

The mechanochemistry of endocytosis.

An integrated theoretical model reveals how the chemical and the mechanical aspects of endocytosis are coordinated coherently in yeast cells, driving progression through the endocytic pathway and ensuring efficient vesicle scission in vivo.

PtdIns(4,5)P2 turnover is required for multiple stages during clathrin- and actin-dependent endocytic internalization

The lipid phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) appears to play an important role in endocytosis. However, the timing of its formation and turnover, and its specific functions at different stages during endocytic internalization, have not been established. In this study, Sla2 ANTH-GFP and Sjl2-3GFP were expressed as functional fusion proteins at endogenous levels to quantitatively explore PtdIns(4,5)P2 dynamics during endocytosis in yeast. Our results indicate that PtdIns(4,5)P2 levels increase and decline in conjunction with coat and actin assembly and disassembly, respectively. Live-cell image analysis of endocytic protein dynamics in an sjl1Δ sjl2Δ mutant, which has elevated PtdIns(4,5)P2 levels, revealed that the endocytic machinery is still able to assemble and disassemble dynamically, albeit nonproductively. The defects in the dynamic behavior of the various endocytic proteins in this double mutant suggest that PtdIns(4,5)P2 turnover is required for multiple stages during endocytic vesicle formation. Furthermore, our results indicate that PtdIns(4,5)P2 turnover may act in coordination with the Ark1/Prk1 protein kinases in stimulating disassembly of the endocytic machinery.

Quantitative analysis of yeast internal architecture using soft X‐ray tomography

We used soft X‐ray tomography (SXT)—a high‐resolution, quantitative imaging technique—to measure cell size and organelle volumes in yeasts. Cell size is a key factor in initiating cell division in yeasts, whereas the number and volume of the organelles have a profound impact on the function and viability of a cell. Consequently, determining these cell parameters is fundamentally important in understanding yeast biology. SXT is well suited to this type of analysis. Specimens are imaged in a near‐native state, and relatively large numbers of cells can be readily analysed. In this study, we characterized haploid and diploid strains of Saccharomyces cerevisiae at each of the key stages in the cell cycle and determined the relationships that exist cellular and organelle volumes. We then compared these results with SXT data obtained from Schizosaccharomyces pombe, the three main phenotypes displayed by the opportunistic yeast pathogen Candida albicans and from a coff1‐22 mutant strain of S. cerevisiae. This comparison revealed that volumetric ratios were invariant, irrespective of yeast strain, ploidy or morphology, leading to the conclusion these volumetric ratios are common in all yeasts. Copyright

Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function

Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae. ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro. Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo. Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic. A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation. An Arp2 suppressor mutant increased Arp2/3 nucleation activity. Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors. These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture.

Determinants of endocytic membrane geometry, stability, and scission.

During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.

Mechanochemical crosstalk during endocytic vesicle formation

Membrane curvature has emerged as a key regulatory factor in endocytic vesicle formation. From a theoretical perspective, we summarize recent progress in understanding how membrane curvature and biochemical pathways are coupled and orchestrated during the coherent process of endocytic vesicle formation. We mainly focus on clathrin-mediated and actin-mediated endocytosis in yeast and in mammalian cells. We further speculate on how mechanochemical feedback could modulate other membrane-remodeling processes.

Analysis of yeast endocytic site formation and maturation through a regulatory transition point

ETOC: During yeast endocytic site formation, Ede1p (yeast Eps15), but not clathrin light chain, is important for the recruitment of most other early-arriving proteins to endocytic sites. Cargo and clathrin light chain may play roles in regulating the transition of endocytic sites out of the “intermediate coat” stage of endocytosis.

Analyzing the Birth and Propagation of Two Distinct Prions, [PSI+] and [Het-s]y, in Yeast

De novo formation of two structurally and compositionally distinct yeast prions, [PSI+] and [Het-s]y, is similar starting with formation of a peripheral ring and then internal ring, and then daughters propagate the prions as dots. The ring formation, but not dot propagation, requires an endocytic protein Sla2.

The functions of anionic phospholipids during clathrin-mediated endocytosis site initiation and vesicle formation

Summary Anionic phospholipids PI(4,5)P2 and phosphatidylserine (PS) are enriched in the cytosolic leaflet of the plasma membrane where endocytic sites form. In this study, we investigated the roles of PI(4,5)P2 and PS in clathrin-mediated endocytosis (CME) site initiation and vesicle formation in Saccharomyces cerevisiae. Live-cell imaging of endocytic protein dynamics in an mss4ts mutant, which has severely reduced PI(4,5)P2 levels, revealed that PI(4,5)P2 is required for endocytic membrane invagination but is less important for endocytic site initiation. We also demonstrated that, in various deletion mutants of genes encoding components of the Rcy1-Ypt31/32 GTPase pathway, endocytic proteins dynamically assemble not only on the plasma membrane but also on intracellular membrane compartments, which are likely derived from early endosomes. In rcy1&Dgr; cells, fluorescent biosensors indicated that PI(4,5)P2 only localized to the plasma membrane while PS localized to both the plasma membrane and intracellular membranes. Furthermore, we found that polarized endocytic patch establishment is defective in the PS-deficient cho1&Dgr; mutant. We propose that PS is important for directing endocytic proteins to the plasma membrane and that PI(4,5)P2 is required to facilitate endocytic membrane invagination.