Automated human induced pluripotent stem cell culture and sample preparation for 3D live-cell microscopy.

To produce abundant cell culture samples to generate large, standardized image datasets of human induced pluripotent stem (hiPS) cells, we developed an automated workflow on a Hamilton STAR liquid handler system. This was developed specifically for culturing hiPS cell lines expressing fluorescently tagged proteins, which we have used to study the principles by which cells establish and maintain robust dynamic localization of cellular structures. This protocol includes all details for the maintenance, passage and seeding of cells, as well as Matrigel coating of 6-well plastic plates and 96-well optical-grade, glass plates. We also developed an automated image-based hiPS cell colony segmentation and feature extraction pipeline to streamline the process of predicting cell count and selecting wells with consistent morphology for high-resolution three-dimensional (3D) microscopy. The imaging samples produced with this protocol have been used to study the integrated intracellular organization and cell-to-cell variability of hiPS cells to train and develop deep learning-based label-free predictions from transmitted-light microscopy images and to develop deep learning-based generative models of single-cell organization. This protocol requires some experience with robotic equipment. However, we provide details and source code to facilitate implementation by biologists less experienced with robotics. The protocol is completed in less than 10 h with minimal human interaction. Overall, automation of our cell culture procedures increased our imaging samples' standardization, reproducibility, scalability and consistency. It also reduced the need for stringent culturist training and eliminated culturist-to-culturist variability, both of which were previous pain points of our original manual pipeline workflow.

Mitochondrial Fission and Fusion Dynamics Generate Efficient, Robust, and Evenly Distributed Network Topologies in Budding Yeast Cells.

The simplest configuration of mitochondria in a cell is as small separate organellar units. Instead, mitochondria often form a dynamic, intricately connected network. A basic understanding of the topological properties of mitochondrial networks, and their influence on cell function is lacking. We performed an extensive quantitative analysis of mitochondrial network topology, extracting mitochondrial networks in 3D from live-cell microscopic images of budding yeast cells. In the presence of fission and fusion, mitochondrial network structures exhibited certain topological properties similar to other real-world spatial networks. Fission and fusion dynamics were required to efficiently distribute mitochondria throughout the cell and generate highly interconnected networks that can facilitate efficient diffusive search processes. Thus, mitochondrial fission and fusion combine to regulate the underlying topology of mitochondrial networks, which may independently impact cell function.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization.

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1-4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line-generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community.

Shaping the multi-scale architecture of mitochondria.

Mitochondria are complex organelles with a highly regulated architecture across all levels of organization. The architecture of the inner mitochondrial membrane (IMM) provides a crucial platform for many mitochondrial functions while mitochondrial network architecture is crucial for coordinating these activities throughout the cell. This review summarizes the recent findings regarding the most important shaping factors that regulate IMM organization, how IMM architecture supports bioenergetic functions and how IMM morphology adapts to meet other physiological needs of the cell. This review also highlights recent work suggesting that the functional connectivity of mitochondrial networks can be achieved not just by matrix continuity but also by inter-mitochondrial contact sites, which generate conductive continuity within a matrix-discontinuous mitochondrial network.

Exposure to critical thermal maxima increases oxidative stress in hearts of white- but not red-blooded Antarctic notothenioid fishes.

Antarctic icefishes have a significantly lower critical thermal maximum (CT(max)) compared with most red-blooded notothenioid fishes. We hypothesized that the lower thermal tolerance of icefishes compared with red-blooded notothenioids may stem from a greater vulnerability to oxidative stress as temperature increases. Oxidative muscles of icefishes have high volume densities of mitochondria, rich in polyunsaturated fatty acids, which can promote the production of reactive oxygen species (ROS). Moreover, icefishes have lower levels of antioxidants compared with red-blooded species. To test our hypothesis, we measured levels of oxidized proteins and lipids, and transcript levels and maximal activities of antioxidants in heart ventricle and oxidative pectoral adductor muscle of icefishes and red-blooded notothenioids held at 0°C and exposed to their CT(max). Levels of oxidized proteins and lipids increased in heart ventricle of some icefishes but not in red-blooded species in response to warming, and not in pectoral adductor muscle of any species. Thus, increases in oxidative damage in heart ventricles may contribute to the reduced thermal tolerance of icefishes. Despite an increase in oxidative damage in hearts of icefishes, neither transcript levels nor activities of antioxidants increased, nor did they increase in any tissue of any species in response to exposure to CT(max). Rather, transcript levels of the enzyme superoxide dismutase (SOD) decreased in hearts of icefishes and the activity of SOD decreased in hearts of the red-blooded species Gobionotothen gibberifrons. These data suggest that notothenioids may have lost the ability to elevate levels of antioxidants in response to heat stress.

Inter-relationship between mitochondrial function and susceptibility to oxidative stress in red- and white-blooded Antarctic notothenioid fishes.

It is unknown whether Antarctic fishes can defend themselves against oxidative stress induced by elevations in temperature. We hypothesized that Antarctic icefishes, lacking the oxygen-binding protein hemoglobin, might be more vulnerable to temperature-induced oxidative stress compared with red-blooded notothenioids because of differences in their mitochondrial properties. Mitochondria from icefishes have higher densities of phospholipids per mg of mitochondrial protein compared with red-blooded species, and these phospholipids are rich in polyunsaturated fatty acids (PUFA), which can promote the formation of reactive oxygen species (ROS). Additionally, previous studies have shown that multiple tissues in icefishes have lower levels of antioxidants compared with red-blooded species. We quantified several properties of mitochondria, including proton leak, rates of ROS production, membrane composition and susceptibility to lipid peroxidation (LPO), the activity of superoxide dismutase (SOD) and total antioxidant power (TAOP) in mitochondria isolated from hearts of icefishes and red-blooded notothenioids. Mitochondria from icefishes were more tightly coupled than those of red-blooded fishes at both 2°C and 10°C, which increased the production of ROS when the electron transport chain was disrupted. The activity of SOD and TAOP per mg of mitochondrial protein was equivalent between icefishes and red-blooded species, but TAOP normalized to mitochondrial phospholipid content was significantly lower in icefishes compared with red-blooded fishes. Additionally, membrane susceptibility to peroxidation was only detectable in icefishes at 1°C and not in red-blooded species. Together, our results suggest that the high density of mitochondrial phospholipids in hearts of icefishes may make them particularly vulnerable to oxidative stress as temperatures rise.

The unique mitochondrial form and function of Antarctic channichthyid icefishes.

Antarctic icefishes of the family Channichthyidae are the only vertebrate animals that as adults do not express the circulating oxygen-binding protein hemoglobin (Hb). Six of the 16 family members also lack the intracellular oxygen-binding protein myoglobin (Mb) in the ventricle of their hearts and all lack Mb in oxidative skeletal muscle. The loss of Hb has led to substantial remodeling in the cardiovascular system of icefishes to facilitate adequate oxygenation of tissues. One of the more curious adaptations to the loss of Hb and Mb is an increase in mitochondrial density in cardiac myocytes and oxidative skeletal muscle fibers. The proliferation of mitochondria in the aerobic musculature of icefishes does not arise through a canonical pathway of mitochondrial biogenesis. Rather, the biosynthesis of mitochondrial phospholipids is up-regulated independently of the synthesis of proteins and mitochondrial DNA, and newly-synthesized phospholipids are targeted primarily to the outer-mitochondrial membrane. Consequently, icefish mitochondria have a higher lipid-to-protein ratio compared to those from red-blooded species. Elevated levels of nitric oxide in the blood plasma of icefishes, compared to red-blooded notothenioids, may mediate alterations in mitochondrial density and architecture. Modifications in mitochondrial structure minimally impact state III respiration rates but may significantly enhance intracellular diffusion of oxygen. The rate of oxygen diffusion is greater within the hydrocarbon core of membrane lipids compared to the aqueous cytosol and impeded only by proteins within the lipid bilayer. Thus, the proliferation of icefish's mitochondrial membranes provides an optimal conduit for the intracellular diffusion of oxygen and compensates for the loss of Hb and Mb. Currently little is known about how mitochondrial phospholipid synthesis is regulated and integrated into mitochondrial biogenesis. The unique architecture of the oxidative muscle cells of icefishes highlights the need for further studies in this area.