PyF2F: a robust and simplified fluorophore-to-fluorophore distance measurement tool for Protein interactions from Imaging Complexes after Translocation experiments.

Structural knowledge of protein assemblies in their physiological environment is paramount to understand cellular functions at the molecular level. Protein interactions from Imaging Complexes after Translocation (PICT) is a live-cell imaging technique for the structural characterization of macromolecular assemblies in living cells. PICT relies on the measurement of the separation between labelled molecules using fluorescence microscopy and cell engineering. Unfortunately, the required computational tools to extract molecular distances involve a variety of sophisticated software programs that challenge reproducibility and limit their implementation to highly specialized researchers. Here we introduce PyF2F, a Python-based software that provides a workflow for measuring molecular distances from PICT data, with minimal user programming expertise. We used a published dataset to validate PyF2F's performance.

In situ architecture of the ER-mitochondria encounter structure.

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.

The cellular slime mold Fonticula alba forms a dynamic, multicellular collective while feeding on bacteria.

Multicellularity evolved in fungi and animals, or the opisthokonts, from their common amoeboflagellate ancestor but resulted in strikingly distinct cellular organizations. The origins of this multicellularity divergence are not known. The stark mechanistic differences that underlie the two groups and the lack of information about ancestral cellular organizations limits progress in this field. We discovered a new type of invasive multicellular behavior in Fonticula alba, a unique species in the opisthokont tree, which has a simple, bacteria-feeding sorocarpic amoeba lifestyle. This invasive multicellularity follows germination dependent on the bacterial culture state, after which amoebae coalesce to form dynamic collectives that invade virgin bacterial resources. This bacteria-dependent social behavior emerges from amoeba density and allows for rapid and directed invasion. The motile collectives have animal-like properties but also hyphal-like search and invasive behavior. These surprising findings enrich the diverse multicellularities present within the opisthokont lineage and offer a new perspective on fungal origins.

Type-I myosins promote actin polymerization to drive membrane bending in endocytosis.

Clathrin-mediated endocytosis in budding yeast requires the formation of a dynamic actin network that produces the force to invaginate the plasma membrane against the intracellular turgor pressure. The type-I myosins Myo3 and Myo5 are important for endocytic membrane reshaping, but mechanistic details of their function remain scarce. Here, we studied the function of Myo3 and Myo5 during endocytosis using quantitative live-cell imaging and genetic perturbations. We show that the type-I myosins promote, in a dose-dependent way, the growth and expansion of the actin network, which controls the speed of membrane and coat internalization. We found that this myosin-activity is independent of the actin nucleation promoting activity of myosins, and cannot be compensated for by increasing actin nucleation. Our results suggest a new mechanism for type-I myosins to produce force by promoting actin filament polymerization.

Systematic Nanoscale Analysis of Endocytosis Links Efficient Vesicle Formation to Patterned Actin Nucleation.

Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially ordered recruitment according to function. WASP family proteins form a circular nanoscale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template optimizes force generation for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

Quantitative imaging of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is a process by which eukaryotic cells bend a small region of their plasma membrane to form a transport vesicle that carries specific cargo molecules into the cell. Endocytosis controls the composition of the plasma membrane, imports nutrients and regulates many signalling pathways. The roles of most of the proteins involved in endocytosis have been thoroughly characterised. However, how these proteins cooperate in the cell to drive the endocytic process is not well understood. Microscopy methods have been instrumental in describing the dynamics and the molecular mechanism of endocytosis. Here, we will review the challenges and the recent advances in visualising the endocytic machinery and we will reflect on how the integration of current imaging technologies can lead us toward a quantitative understanding of the molecular mechanisms of endocytosis.

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.

Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle.

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.

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.

Visualizing the functional architecture of the endocytic machinery.

Clathrin-mediated endocytosis is an essential process that forms vesicles from the plasma membrane. Although most of the protein components of the endocytic protein machinery have been thoroughly characterized, their organization at the endocytic site is poorly understood. We developed a fluorescence microscopy method to track the average positions of yeast endocytic proteins in relation to each other with a time precision below 1 s and with a spatial precision of ~10 nm. With these data, integrated with shapes of endocytic membrane intermediates and with superresolution imaging, we could visualize the dynamic architecture of the endocytic machinery. We showed how different coat proteins are distributed within the coat structure and how the assembly dynamics of N-BAR proteins relate to membrane shape changes. Moreover, we found that the region of actin polymerization is located at the base of the endocytic invagination, with the growing ends of filaments pointing toward the plasma membrane.

Composition profiling of inhomogeneous SiGe nanostructures by Raman spectroscopy.

In this work, we present an experimental procedure to measure the composition distribution within inhomogeneous SiGe nanostructures. The method is based on the Raman spectra of the nanostructures, quantitatively analyzed through the knowledge of the scattering efficiency of SiGe as a function of composition and excitation wavelength. The accuracy of the method and its limitations are evidenced through the analysis of a multilayer and of self-assembled islands.

Precise, correlated fluorescence microscopy and electron tomography of lowicryl sections using fluorescent fiducial markers.

The application of fluorescence and electron microscopy to the same specimen allows the study of dynamic and rare cellular events at ultrastructural detail. Here, we present a correlative microscopy approach, which combines high accuracy of correlation, high sensitivity for detecting faint fluorescent signals, as well as robustness and reproducibility to permit large dataset collections. We provide a step-by-step protocol that allows direct mapping of fluorescent protein signals into electron tomograms. A localization precision of <100 nm is achieved by using fluorescent fiducial markers which are visible both in fluorescence images and in electron tomograms. We explain the critical details of the procedure, give background information on the individual steps, present results from test experiments carried out during establishment of the method, as well as information about possible modifications to the protocol, such as its application to 2D electron micrographs. This simple, robust, and flexible method can be applied to a large variety of cellular systems, such as yeast cell pellets and mammalian cell monolayers, to answer a broad spectrum of structure-function related questions.

Unraveling the influence of endothelial cell density on VEGF-A signaling.

Vascular endothelial growth factor-A (VEGF) is the master determinant for the activation of the angiogenic program leading to the formation of new blood vessels to sustain solid tumor growth and metastasis. VEGF specific binding to VEGF receptor-2 (VEGFR-2) triggers different signaling pathways, including phospholipase C-γ (PLC-γ) and Akt cascades, crucial for endothelial proliferation, permeability, and survival. By combining biologic experiments, theoretical insights, and mathematical modeling, we found that: (1) cell density influences VEGFR-2 protein level, as receptor number is 2-fold higher in long-confluent than in sparse cells; (2) cell density affects VEGFR-2 activation by reducing its affinity for VEGF in long-confluent cells; (3) despite reduced ligand-receptor affinity, high VEGF concentrations provide long-confluent cells with a larger amount of active receptors; (4) PLC-γ and Akt are not directly sensitive to cell density but simply transduce downstream the upstream difference in VEGFR-2 protein level and activation; and (5) the mathematical model correctly predicts the existence of at least one protein tyrosine phosphatase directly targeting PLC-γ and counteracting the receptor-mediated signal. Our data-based mathematical model quantitatively describes VEGF signaling in quiescent and angiogenic endothelium and is suitable to identify new molecular determinants and therapeutic targets.

Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.

Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm. We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.