Machine learning to support citizen science in urban environmental management.

Machine learning (ML) and citizen science (CS) are increasingly prevalent and rapidly evolving approaches to studying and managing environmental challenges. Municipal and other governance actors can benefit from technology advances in ML and public engagement benefits of CS but must also address validity and other quality assurance concerns in their application to particular management contexts. In this article, we take up the pervasive challenge of urban litter to demonstrate how ML can support CS by providing quality assurance in the regulatory context of California's stormwater program. We gave quantitative CS-collected data to five ML models to compare their predictions of a qualitative, site-specific, multiclass "Litter Index" score, an important regulatory metric typically only assessed by trained experts. XGBoost had the best outcome, with scores of 0.98 for accuracy, precision, recall and F-1. These strong results show that ML can provide a reliable complement to CS assessments and increase quality assurance in a regulatory context. To date, ML and CS have each contributed to litter management in novel ways and we find that their integration can provide important synergies with additional applications in other environmental management domains.

Enrichment of aging yeast cells and budding polarity assay in Saccharomyces cerevisiae.

Replicative lifespan, a measure of the number of times that a yeast cell can divide before senescence, is one model for aging. Here, we provide a protocol for enrichment of yeast as a function of replicative age using a miniature chemostat aging device (mCAD). This protocol allows for isolation of quantities of cells that are sufficient for biochemical or genomic analysis. We also describe an approach to assess bud site selection, a marker for cell polarity, during the aging process. For complete details on the use and execution of this protocol, please refer to Yang et al. (2022).

Identification of a modulator of the actin cytoskeleton, mitochondria, nutrient metabolism and lifespan in yeast.

In yeast, actin cables are F-actin bundles that are essential for cell division through their function as tracks for cargo movement from mother to daughter cell. Actin cables also affect yeast lifespan by promoting transport and inheritance of higher-functioning mitochondria to daughter cells. Here, we report that actin cable stability declines with age. Our genome-wide screen for genes that affect actin cable stability identified the open reading frame YKL075C. Deletion of YKL075C results in increases in actin cable stability and abundance, mitochondrial fitness, and replicative lifespan. Transcriptome analysis revealed a role for YKL075C in regulating branched-chain amino acid (BCAA) metabolism. Consistent with this, modulation of BCAA metabolism or decreasing leucine levels promotes actin cable stability and function in mitochondrial quality control. Our studies support a role for actin stability in yeast lifespan, and demonstrate that this process is controlled by BCAA and a previously uncharacterized ORF YKL075C, which we refer to as actin, aging and nutrient modulator protein 1 (AAN1).

Touch and Go: Membrane Contact Sites Between Lipid Droplets and Other Organelles.

Lipid droplets (LDs) have emerged not just as storage sites for lipids but as central regulators of metabolism and organelle quality control. These critical functions are achieved, in part, at membrane contact sites (MCS) between LDs and other organelles. MCS are sites of transfer of cellular constituents to or from LDs for energy mobilization in response to nutrient limitations, as well as LD biogenesis, expansion and autophagy. Here, we describe recent findings on the mechanisms underlying the formation and function of MCS between LDs and mitochondria, ER and lysosomes/vacuoles and the role of the cytoskeleton in promoting LD MCS through its function in LD movement and distribution in response to environmental cues.

A role for cell polarity in lifespan and mitochondrial quality control in the budding yeast Saccharomyces cerevisiae.

Babies are born young, largely independent of the age of their mothers. Mother-daughter age asymmetry in yeast is achieved, in part, by inheritance of higher-functioning mitochondria by buds and retention of some high-functioning mitochondria in mother cells. The mitochondrial F box protein, Mfb1p, tethers mitochondria at both poles in a cell cycle-regulated manner: it localizes to and anchors mitochondria at the mother cell tip throughout the cell cycle and at the bud tip before cytokinesis. Here, we report that cell polarity and polarized localization of Mfb1p decline with age in Saccharomyces cerevisiae. Moreover, deletion of genes (BUD1, BUD2, and BUD5) that mediate symmetry breaking during establishment of cell polarity and asymmetric yeast cell division cause depolarized Mfb1p localization and defects in mitochondrial distribution and quality control. Our results support a role for the polarity machinery in lifespan through modulating Mfb1 function in asymmetric inheritance of mitochondria during yeast cell division.

Imaging the Actin Cytoskeleton in Live Budding Yeast Cells.

Although budding yeast, Saccharomyces cerevisiae, is widely used as a model organism in biological research, studying cell biology in yeast was hindered due to its small size, rounded morphology, and cell wall. However, with improved techniques, researchers can acquire high-resolution images and carry out rapid multidimensional analysis of a yeast cell. As a result, imaging in yeast has emerged as an important tool to study cytoskeletal organization, function, and dynamics. This chapter describes techniques and approaches for visualizing the actin cytoskeleton in live yeast cells.

Imaging the Actin Cytoskeleton in Fixed Budding Yeast Cells.

Budding yeast, Saccharomyces cerevisiae, is an appealing model organism to study the organization and function of the actin cytoskeleton. With the advent of techniques to perform high-resolution, multidimensional analysis of the yeast cell, imaging of yeast has emerged as an important tool for research on the cytoskeleton. This chapter describes techniques and approaches for visualizing the actin cytoskeleton in fixed yeast cells with wide-field and super-resolution fluorescence microscopy.

Live-Cell Imaging of Mitochondrial Redox State in Yeast Cells.

The redox state of mitochondria is one indicator of the functional state of the organelles. Mitochondria are also the primary endogenous source of reactive oxygen species (ROS). Therefore, the redox state of the organelles also reflects their function in ROS production. Here, we provide step-by-step protocols for live-cell imaging and quantification of mitochondrial redox state using the genetically encoded fluorescent biosensor, mitochondria-targeted redox sensing GFP (mito-roGFP), and mitochondrial ROS using the membrane-permeant small molecule dihydroethidium (DHE) in budding yeast cells. For complete details on the use and execution of this protocol, please refer to Liao et al. (2020c).

NCP activates chloroplast transcription by controlling phytochrome-dependent dual nuclear and plastidial switches.

Phytochromes initiate chloroplast biogenesis by activating genes encoding the photosynthetic apparatus, including photosynthesis-associated plastid-encoded genes (PhAPGs). PhAPGs are transcribed by a bacterial-type RNA polymerase (PEP), but how phytochromes in the nucleus activate chloroplast gene expression remains enigmatic. We report here a forward genetic screen in Arabidopsis that identified NUCLEAR CONTROL OF PEP ACTIVITY (NCP) as a necessary component of phytochrome signaling for PhAPG activation. NCP is dual-targeted to plastids and the nucleus. While nuclear NCP mediates the degradation of two repressors of chloroplast biogenesis, PIF1 and PIF3, NCP in plastids promotes the assembly of the PEP complex for PhAPG transcription. NCP and its paralog RCB are non-catalytic thioredoxin-like proteins that diverged in seed plants to adopt nonredundant functions in phytochrome signaling. These results support a model in which phytochromes control PhAPG expression through light-dependent double nuclear and plastidial switches that are linked by evolutionarily conserved and dual-localized regulatory proteins.

Light-regulated gene repositioning in Arabidopsis.

Plant genomes are extremely sensitive to, and can be developmentally reprogrammed by environmental light cues. Here using rolling-circle amplification of gene-specific circularizable oligonucleotides coupled with fluorescence in situ hybridization, we demonstrate that light triggers a rapid repositioning of the Arabidopsis light-inducible chlorophyll a/b-binding proteins (CAB) locus from the nuclear interior to the nuclear periphery during its transcriptional activation. CAB repositioning is mediated by the red/far-red photoreceptors phytochromes (PHYs) and is inhibited by repressors of PHY signalling, including COP1, DET1 and PIFs. CAB repositioning appears to be a separate regulatory step occurring before its full transcriptional activation. Moreover, the light-inducible loci RBCS, PC and GUN5 undergo similar repositioning behaviour during their transcriptional activation. Our study supports a light-dependent gene regulatory mechanism in which PHYs activate light-inducible loci by relocating them to the nuclear periphery; it also provides evidence for the biological importance of gene positioning in the plant kingdom.