measurements-of-physiological-stress-responses-in-c-elegans

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

surveying-low-cost-methods-to-measure-lifespan-healthspan

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

imaging-quantifying-mitochondrial-morphology-c-elegans-during

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this ...

University of California, Berkeley

University of Southern California

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 Live 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.

Imaging the Actin Cytoskeleton in Fixed Budding Yeast Cells.

The actin cytoskeleton plays a fundamental role in the regulation of multiple cellular pathways, including trafficking and locomotion. The functional integrity of the cytoskeleton is important during aging, as the decline of cytoskeletal integrity contributes to the physiological consequence of aging. Moreover, improving cytoskeletal form and function throughout aging is sufficient to drive life span extension and promote organismal health in multiple model systems. For these reasons, optimized protocols for visualization of the actin cytoskeleton and its downstream consequences on health span and life span are critical for understanding the aging process. In C. elegans, the actin cytoskeleton shows diverse morphologies across tissues, potentially due to the significantly different functions of each cell type. This chapter describes an imaging platform utilizing LifeAct to visualize the actin cytoskeleton in live, whole nematodes throughout the aging process and methods to perform follow-up studies on the life span and health span of these organisms.

Imaging of Actin Cytoskeletal Integrity During Aging in C. elegans.

The dysfunction of mitochondria is associated with the physiological consequences of aging and many age-related diseases. Therefore, critical quality control mechanisms exist to protect mitochondrial functions, including the unfolded protein response of the mitochondria (UPR). However, it is still unclear how UPR is regulated in mammals with mechanistic discrepancies between previous studies. Here, we reasoned that a study of conserved mechanisms could provide a uniquely powerful way to reveal previously uncharacterized components of the mammalian UPR. We performed cross-species comparison of genetic requirements for survival under—and in response to—mitochondrial stress between karyotypically normal human stem cells and the nematode . We identified a role for EPS-8/EPS8 (epidermal growth factor receptor pathway substrate 8), a signaling protein adaptor, in general mitochondrial homeostasis and UPR regulation through integrin-mediated remodeling of the actin cytoskeleton. This study also highlights the use of cross-species comparisons in genetic screens to interrogate cellular pathways.

Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization.

Mitochondrial fitness is critical to organismal health and its impairment is associated with aging and age-related diseases. As such, numerous quality control mechanisms exist to preserve mitochondrial stability, including the unfolded protein response of the mitochondria (UPR). The UPR is a conserved mechanism that drives the transcriptional activation of mitochondrial chaperones, proteases, autophagy (mitophagy), and metabolism to promote restoration of mitochondrial function under stress conditions. UPR has direct ramifications in aging, and its activation is often ascribed to improve health whereas its dysfunction tends to correlate with disease. This review pairs a description of the most recent findings within the field of UPR with a more poorly understood field: mitochondria-derived peptides (MDPs). Similar to UPR, MDPs are microproteins derived from the mitochondria that can impact organismal health and longevity. We then highlight a tantalizing interconnection between UPR and MDPs wherein both mechanisms may be efficiently coordinated to maintain organismal health.

A tale of two pathways: Regulation of proteostasis by UPR and MDPs.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), MTZ, a newly employed ETC complex I inhibitor, exhibited higher cytotoxicity against cancer cell lines compared to certain non-cancer cell lines. Interestingly, 8-OAc demonstrated greater selectivity for cancer cells when compared to both MTZ and Rote, which has promising potential for anticancer therapeutic development. Furthermore, in vivo experiments with these small molecules utilizing a C. elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPR) pathway, that extends lifespan of worms when applied in their adult stage. We also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilizes the transcription factors ATFS-1 and HSF1, which are involved in the UPR and heat shock response (HSR) pathways respectively, 8-OAc only required HSF1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

Article Total : 3	Year
Publication title	2020
Publication title	2022
Publication title	2025

Article	Year
Imaging the Actin Cytoskeleton in Live Budding Yeast Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34542848	2022
Imaging the Actin Cytoskeleton in Fixed Budding Yeast Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34542849	2022
Imaging of Actin Cytoskeletal Integrity During Aging in C. elegans. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34542850	2022
Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization. Science advances\| PubMed ID: 34714674	2021
A tale of two pathways: Regulation of proteostasis by UPR and MDPs. Current opinion in neurobiology\| PubMed ID: 36621224	2023
Investigating impacts of the mycothiazole chemotype as a chemical probe for the study of mitochondrial function and aging. GeroScience\| PubMed ID: 38570396	2024

Ryo Higuchi-Sanabria

Biography

JoVE Journal Publications

Other Publications