Load adaptation by endocytic actin networks.

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feed back on assembly. By manipulating membrane tension and Arp2/3 complex activity, we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions.

Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features.

Conserved proteins drive clathrin-mediated endocytosis (CME), which from yeast to humans involves a burst of actin assembly. To gain mechanistic insights into this process, we performed a side-by-side quantitative comparison of CME in two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae is more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast. In both yeasts, accumulation of ~70 WASp molecules activates the Arp2/3 complex to drive membrane invagination. In contrast to budding yeast, WASp-mediated actin nucleation plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, although the assembly of two zones of actin filaments is specific for fission yeast and not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations with broad implications that are expected to extend from yeast to humans.

Functional Redundancy of Septin Homologs in Dendritic Branching.

Septins are cytoskeletal GTPases present in nonpolar heteromeric complexes that assemble in a palindromic fashion from two to eight subunits. Mammalian septins function in several fundamental cellular processes at the membrane-cytoskeleton interface including dendritic branching in neurons. Sequence homology divides the 13 mammalian septin genes into four homology groups. Experimental findings suggest that septin function is redundant among septins from one homology group. This is best understood for the isoforms of the SEPT2 group, which form a homodimer at the center of septin complexes. In vitro, all SEPT2-group septins form recombinant hexameric complexes with two copies of SEPT6 and SEPT7. However, it remains unclear to what extent homologs septins can substitute for each other in specific cellular processes. Here, we use the experimental paradigm of dendritic branching in hippocampal rat neurons to ask, to what extent septins of the SEPT2-group are functionally redundant. Dendritic branching is significantly reduced when SEPT5 is downregulated. In neurons expressing SEPT5-shRNA, simultaneously expressed SEPT2-GFP, and SEPT4-GFP colocalize with SEPT7 at dendritic spine necks and rescue dendritic branching. In contrast, SEPT1-GFP is diffusely distributed in the cytoplasm in SEPT5 downregulated neurons and cannot rescue dendritic branching. Our findings provide a basis for the study of septin-specific functions in cells.

Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast.

Single-molecule localization-based superresolution microscopy methods allow the resolution of cellular structures in the range of tens of nanometers. However, these techniques are of limited use in current yeast labeling protocols, owing to problems with structural preservation. Here we describe an optimized sample preparation protocol that enables single-molecule localization microscopy at high resolution combined with improved structural preservation in Saccharomyces cerevisiae. The protocol uses small binders called nanobodies and an enzymatic labeling strategy to deliver organic dyes to the target protein. These small binders readily penetrate through the yeast cell wall and thus eliminate the requirement for its prior degradation, and they allow structural preservation. In addition, the small size of the binders reduces the distance of the dye to the target protein, and thus it reduces the localization error. The preparation of S. cerevisiae cells for superresolution imaging takes 2-4 h to perform. Researchers should have skills in yeast molecular biology, immunolabeling techniques and access to a microscope equipped for single-molecule imaging.

Absolute Arrangement of Subunits in Cytoskeletal Septin Filaments in Cells Measured by Fluorescence Microscopy.

We resolved the organization of subunits in cytoskeletal polymers in cells by light microscopy. Septin GTPases form linear complexes of about 32 nm length that polymerize into filaments. We visualized both termini of septin complexes by single molecule microscopy in vitro. Complexes appeared as 32 nm spaced localization pairs, and filaments appeared as stretches of equidistant localizations. Cellular septins were resolved as localization pairs and thin stretches of equidistant localizations.

Localization microscopy in yeast.

Conventional light and fluorescence microscopy techniques have offered tremendous insight into cellular processes and structures. Their resolution is however intrinsically limited by diffraction. Superresolution techniques achieve an order of magnitude higher resolution. Among these, localization microscopy relies on the position determination of single emitters with nanometer accuracy, which allows the subsequent reconstruction of an image of the target structure. In this chapter, we provide general guidelines for localization microscopy with a focus on Saccharomyces cerevisiae. Its different cellular architecture complicates efforts to directly transfer protocols established in mammalian cells to yeast. We compare different methodologies to label structures of interest and provide protocols for the respective sample preparation, which are not limited to yeast. Using these guidelines, nanoscopic subcellular structures in yeast can be investigated by localization microscopy, which perfectly complements live-cell fluorescence and electron microscopy.

A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.

We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.

Indoor air pollution from unprocessed solid fuels in developing countries.

Approximately half of the world's population relies on biomass (primarily wood and agricultural residues) or coal fuels (collectively termed solid fuels) for heating, lighting, and cooking. The incomplete combustion of such materials releases byproducts with well-known adverse health effects, hence increasing the risk of many diseases and death. Among these conditions are acute respiratory infections, chronic obstructive pulmonary disease, heart disease, stroke, lung cancer, cataracts and blindness, tuberculosis, asthma, and adverse pregnancy outcomes. The International Agency for Research on Cancer has classified the indoor combustion of coal emissions as Group 1, a known carcinogen to humans. Indoor air pollution exposure is greatest in individuals who live in rural developing countries. Interventions have been limited and show only mixed results. To reduce the morbidity and mortality from indoor air pollution, countermeasures have to be developed that are practical, efficient, sustainable, and economical with involvement from the government, the commercial sector, and individuals. This review focuses on the contribution of solid fuels to indoor air pollution.

The influence of myosin converter and relay domains on cross-bridge kinetics of Drosophila indirect flight muscle.

We are investigating the influence of the converter and relay domains on elementary rate constants of the actomyosin cross-bridge cycle. The converter and relay domains vary between Drosophila myosin heavy chain isoforms due to alternative mRNA splicing. Previously, we found that separate insertions of embryonic myosin isoform (EMB) versions of these domains into the indirect flight muscle (IFM) myosin isoform (IFI) both decreased Drosophila IFM power and slowed muscle kinetics. To determine cross-bridge mechanisms behind the changes, we employed sinusoidal analysis while varying phosphate and MgATP concentrations in skinned Drosophila IFM fibers. Based on a six-state cross-bridge model, the EMB converter decreased myosin rate constants associated with actin attachment and work production, k(4), but increased rates related to cross-bridge detachment and work absorption, k(2). In contrast, the EMB relay domain had little influence on kinetics, because only k(4) decreased. The main alteration was mechanical, in that work production amplitude decreased. That both domains decreased k(4) supports the hypothesis that these domains are critical to lever-arm-mediated force generation. Neither domain significantly influenced MgATP affinity. Our modeling suggests the converter domain is responsible for the difference in rate-limiting cross-bridge steps between EMB and IFI myosin--i.e., a myosin isomerization associated with MgADP release for EMB and Pi release for IFI.