The contributions of the actin machinery to endocytic membrane bending and vesicle formation.

Branched and cross-linked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analyzed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thus temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our data show that initiation of membrane bending requires the actin network to be physically linked to the plasma membrane and to be optimally cross-linked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independent of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 s before scission, when the filament nucleation rate drops. From that time point on, invagination growth and vesicle scission are driven by an expansion of the actin network without a proportional increase of net actin amounts. The expansion is sensitive to the amount of filamentous actin and its cross-linking. Our results suggest that the mechanism by which actin reshapes the membrane changes during the progress of endocytosis, possibly adapting to varying force requirements.

The In Vivo Architecture of the Exocyst Provides Structural Basis for Exocytosis.

The structural characterization of protein complexes in their native environment is challenging but crucial for understanding the mechanisms that mediate cellular processes. We developed an integrative approach to reconstruct the 3D architecture of protein complexes in vivo. We applied this approach to the exocyst, a hetero-octameric complex of unknown structure that is thought to tether secretory vesicles during exocytosis with a poorly understood mechanism. We engineered yeast cells to anchor the exocyst on defined landmarks and determined the position of its subunit termini at nanometer precision using fluorescence microscopy. We then integrated these positions with the structural properties of the subunits to reconstruct the exocyst together with a vesicle bound to it. The exocyst has an open hand conformation made of rod-shaped subunits that are interlaced in the core. The exocyst architecture explains how the complex can tether secretory vesicles, placing them in direct contact with the plasma membrane.

Clathrin modulates vesicle scission, but not invagination shape, in yeast endocytosis.

In a previous paper (Picco et al., 2015), the dynamic architecture of the protein machinery during clathrin-mediated endocytosis was visualized using a new live imaging and particle tracking method. Here, by combining this approach with correlative light and electron microscopy, we address the role of clathrin in this process. During endocytosis, clathrin forms a cage-like coat around the membrane and associated protein components. There is growing evidence that clathrin does not determine the membrane morphology of the invagination but rather modulates the progression of endocytosis. We investigate how the deletion of clathrin heavy chain impairs the dynamics and the morphology of the endocytic membrane in budding yeast. Our results show that clathrin is not required for elongating or shaping the endocytic membrane invagination. Instead, we find that clathrin contributes to the regularity of vesicle scission and thereby to controlling vesicle size.

Detection and characterization of protein interactions in vivo by a simple live-cell imaging method.

Over the last decades there has been an explosion of new methodologies to study protein complexes. However, most of the approaches currently used are based on in vitro assays (e.g. nuclear magnetic resonance, X-ray, electron microscopy, isothermal titration calorimetry etc). The accurate measurement of parameters that define protein complexes in a physiological context has been largely limited due to technical constrains. Here, we present PICT (Protein interactions from Imaging of Complexes after Translocation), a new method that provides a simple fluorescence microscopy readout for the study of protein complexes in living cells. We take advantage of the inducible dimerization of FK506-binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain to translocate protein assemblies to membrane associated anchoring platforms in yeast. In this assay, GFP-tagged prey proteins interacting with the FRB-tagged bait will co-translocate to the FKBP-tagged anchor sites upon addition of rapamycin. The interactions are thus encoded into localization changes and can be detected by fluorescence live-cell imaging under different physiological conditions or upon perturbations. PICT can be automated for high-throughput studies and can be used to quantify dissociation rates of protein complexes in vivo. In this work we have used PICT to analyze protein-protein interactions from three biological pathways in the yeast Saccharomyces cerevisiae: Mitogen-activated protein kinase cascade (Ste5-Ste11-Ste50), exocytosis (exocyst complex) and endocytosis (Ede1-Syp1).

Reassessment of the role of plasma membrane domains in the regulation of vesicular traffic in yeast.

The Saccharomyces cerevisiae plasma membrane has been proposed to contain two stably distributed domains. One of these domains, known as MCC (membrane compartment of Can1) or eisosomes, consists of furrow-like membrane invaginations and associated proteins. The other domain, called MCP (membrane compartment of Pma1), consists of the rest of the membrane area surrounding the MCC patches. The role of this plasma membrane domain organization in endocytosis is under debate. Here we show by live-cell imaging that vesicular traffic is restricted to the MCP and the distribution of endocytic and exocytic sites within the MCP is independent of the MCC patch positions. Photobleaching experiments indicated that Can1 and Tat2, two MCC-enriched permeases, exchange quickly between the two domains. Total internal reflection fluorescence and epi-fluorescence microscopy showed that the enrichment of Can1 at the MCC persisted after addition of its substrate, whereas the enrichment of Tat2 disappeared within 90 seconds. The rates of stimulated endocytosis of Can1 as well as Tat2 were similar in both wild-type cells and pil1Δ cells, which lack the MCC. Thus, our data suggest that the enrichment of certain plasma membrane proteins in the MCC does not regulate the rate of their endocytosis.