Gigantic animal cells suggest organellar scaling mechanisms across a 50-fold range in cell volume

Tissue Sampling, Fixation, Staining, and Imaging Intestinal tissue was chosen for analyses as it is made up of only four cell types, and 80 percent of the total cell population is enterocytes, resulting in a relatively homogenous population of cells (De Santa Barbara et al. 2003). Three species of amphibians were chosen that span much of the range of amphibian genome and cell sizes: the western clawed frog Silurana tropicalis (genome size = 1.2 Gb), the northern gray-cheeked salamander Plethodon montanus (genome size = 35 Gb), and the western waterdog Necturus beyeri (genome size ~100 Gb based on congeners that range from 80.5-120.6 Gb). Silurana tropicalis were obtained from a lab-reared colony following standard husbandry conditions and Necturus beyeri were obtained commercially. Plethodon montanus were field collected between May and August of 2018 in Avery County, North Carolina under the wildlife collection license # 18-SC01250 issued by the North Carolina Wildlife Resources Commission. One individual was sampled per species, and all specimens were euthanized in MS222. Work was carried out in accordance with Colorado State University (P, montanus, N. beyeri) and University of Wyoming (S. tropicalis) IACUC protocols (17-7189A and 20200714DL00443-01, respectively). Intestinal tissue from each individual was dissected and immersion fixed in 2.5% glutaraldehyde/2% formaldehyde. The tissues then underwent secondary fixation and staining in 1% OsO4 in a 0.1 M cacodylate buffered solution followed by embedding in PELCO Eponate 12 epoxy (Cushing et al. 2014). Thin sections (60-80 nm) of resin-embedded samples were cut using a Leica UCT ultramicrotome, collected onto Formvar-coated TEM slot grids, and poststained with 2% aqueous uranyl acetate followed by Reynold’s lead citrate. Sample preparation, fixation, and mounting were done at Colorado State University. The samples were then sectioned, stained, and imaged at the University of Colorado, Boulder Electron Microscopy Services Core Facility. Sections were imaged using a Tecnai T12 Spirit transmission electron microscope, operating at 100 kV, with an AMT CCD (digital camera). Silurana tropicalis and P. montanus tissues were imaged at 9,300x direct magnification. Necturus beyeri tissue was imaged at 6,800x direct magnification because the larger cell sizes could not be captured at the higher magnification. All images were evaluated for quality, ensuring intact tissues undamaged by the fixation process. Stereological Approach Stereology uses 2-dimensional image sampling protocols that allow the estimation of surface area and volume of 3-dimensional shapes through unbiased sampling using grids (Howard and Reed 2004; Russ and Dehoff 2012). Grids are superimposed randomly onto preselected, non-overlapping TEM images. Depending on the type of probes used in the grid and how the user measures the probes’ interactions with the images, the volume, surface area, length, and number of 3-dimensional structures can be estimated from the 2-D TEM images. For the individual representing each species, 60 TEM images were randomly selected, each image was overlayed with a grid of probes using the IMOD image processing program (Kremer et al. 1996). Volume fraction of organelles per cell (point probes), as well as surface area of organelles per unit of cell volume (line probes), were measured for nucleus, mitochondria, ER, and Golgi apparatus using the 3dmod stereology plugin in IMOD (Noske 2010). Organelle Volume Fraction Estimation For measuring volume fractions of the organelle, each of the 60 images for each individual was fitted with a 7 x 7 grid using crosshair probes (points) for a total of 2,940 probes per species. Next, each crosshair was visually identified and manually assigned to one of the following categories: nucleus, mitochondria, ER, Golgi apparatus, or cytoplasm (which included cytosol and other non-focal organelles). If a crosshair fell on a part of the image that included damaged cellular material or extracellular material, it was categorized as “not in bounds” and was removed from the data set. The center of the crosshair was used to define the object category and only one class was permitted per point (Howard and Reed 2004; Russ and Dehoff 2012). A denser grid was used for the Golgi apparatus due to the rarity of the organelle; the same 60 images per individual were overlaid with 18 x 18 grids for a total of 19,440 crosshair probes Organelle and Cell Surface Area Estimation For organelle and cell surface area per unit of cell volume estimates (surface area density), the same 60 images per individual were used as for volume estimates. The nucleus, mitochondria, ER, and plasma membrane were measured simultaneously using alternating cycloid probes in a 2 x 2 grid per image for a total of 240 probes. The Golgi apparatus was again measured using a denser grid due to the organelle’s scarcity, using a 6 x 6 grid of alternating cycloids on each image for a total of 2,160 cycloids per species. The cycloids were visually identified and manually assigned as being “in bounds” and “not in bounds” as in the volume estimation. From there, cycloids were marked with intercepts each time a cycloid interacted with the boundary of an organelle or cell; if a cycloid went entirely through an organelle, then an intercept was marked both entering and leaving the structure. Each intercept was categorized as one of the four groups of organelles from the volume assessment or as the cell boundary; cytoplasm was not used in surface area estimation. Cycloids that were fully inside an organelle and did not contact the outside of the organelle were not marked with intercepts. The surface area estimates were then calculated from these results with the equation 2*Intercepts/Total Length of all “In Bound” Cycloids (Howard and Reed 2004; Russ and Dehoff 2012). Organelle and Cell Surface Area to Volume Ratio Estimation Organelle and whole cell SA:V ratios for each species were calculated by dividing the estimates for surface area per unit cell volume (above) by the organellar volume fraction estimates (above) for each grid. Because organellar structures are so variable in shape (e.g. the nucleus and mitochondria exhibiting spherical vs. tubular network shapes) (McCarron et al. 2013; Malerba and Marshall 2021), their SA:V ratios scale in different ways with increases in organellar size (Chan and Marshall 2010; Marshall 2020). Thus, SA:V estimates across species act as a proxy for organelle shape. Organelle and Cell Absolute Volume Estimation Nuclear volume was estimated using the Nucleator probe (Gundersen et al. 1988) in the Visiopharm VIS stereology software (version 2017.7). The Nucleator randomly assigns two perpendicular rays that radiate outward from a fixed point in the nucleus, defined to be the nucleolus, and uses these rays and their intersection points with the nuclear membrane to estimate mean nuclear volume. 100 nuclei were analyzed from each species. Then, using the proportional estimates of nuclear volume obtained from IMOD (Kremer et al. 1996), cell volume and surface area, and all other organelle volumes and surface areas, were extrapolated from nuclear volume. Among-Species Differences in Organelle Volume and Surface Area We tested for associations between cell size (i.e. species) and 1) organelle volume fraction, 2) organelle surface area per unit of cell volume, and 3) organelle surface area to organelle volume ratio using ANOVA, followed by a pairwise Tukey post-hoc method of comparison. We carried out all analyses in R Studio (Martin 2021; R Core Team 2021) using R packages emmeans (Lenth 2021), car (Fox and Weisberg 2011) and lme4 (Bates et al. 2015).