DOI: 10.5061/dryad.cz8w9gj91
title: Gigantic animal cells suggest organellar scaling mechanisms across a 50-fold range in cell volume

description: Across the tree of life, cell size varies by orders of magnitude, and
 organelles scale to maintain cell function. Depending on their shape,
 organelles can scale by increasing volume, length, or number. Scaling may
 also reflect demands placed on organelles by increased cell size. The
 8,653 species of amphibians exhibit diverse cell sizes, providing a
 powerful system to investigate organellar scaling. Using transmission
 electron microscopy and stereology, we analyzed three frog and salamander
 species whose enterocyte cell volumes range from 228 to 10,593 μm3. We
 show that the nucleus increases in radius while the mitochondria increase
 in total network length; the endoplasmic reticulum and Golgi apparatus,
 with their complex shapes, are intermediate. Notably, all four organelles
 increase in volume proportionate to cell volume. This pattern suggests
 that protein concentrations are the same across amphibian species that
 differ 50-fold in cell size, and that organellar building blocks are
 incorporated into more or larger organelles following the same “rules”
 across cell sizes, despite variation in metabolic and transport demands.
 This conclusion contradicts results from experimental cell size increases,
 which produce severe proteome dilution. We hypothesize that salamanders
 have evolved the biosynthetic capacity to maintain a functional proteome
 despite a huge cell volume. 
description: <strong>Tissue Sampling,
 Fixation, Staining, and Imaging</strong> 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 <em>Silurana tropicalis
 </em>(genome size = 1.2 Gb), the northern gray-cheeked salamander
 <em>Plethodon montanus </em>(genome size = 35 Gb), and the
 western waterdog <em>Necturus beyeri</em> (genome size ~100 Gb
 based on congeners that range from 80.5-120.6 Gb). <em>Silurana
 tropicalis</em> were obtained from a lab-reared colony following
 standard husbandry conditions and <em>Necturus beyeri</em>
 were obtained commercially. <em>Plethodon
 montanus</em>
 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 (<em>P, montanus, N.
 beyeri</em>) and University of Wyoming (<em>S.
 tropicalis</em>) 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).  <em>Silurana
 tropicalis</em> and <em>P. montanus</em> tissues were
 imaged at 9,300x direct magnification. <em>Necturus
 beyeri</em> 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. <strong>Stereological
 Approach</strong> 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). <strong>Organelle Volume Fraction
 Estimation</strong> 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 <strong>Organelle and Cell Surface
 Area Estimation</strong> 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). <strong>Organelle and Cell Surface
 Area to Volume Ratio Estimation</strong> 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.
 <strong>Organelle and Cell
 Absolute Volume Estimation</strong> 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. <strong>Among-Species Differences in
 Organelle Volume and Surface Area</strong> 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).
description: The excel files are measurements of organellar volume
 (μm<sup>3</sup>), surface area
 (μm<sup>2</sup>/μm<sup>3</sup>) and the ratio of
 the 2 different measurements. All measurements were made in the stereology
 plugin of IMODS. All relevant data has been converted into data frames
 using .csv files for ease of processing. CSV files marked as V_DF, SA_DF,
 and Ratio_DF are used to calculate statistics using the the
 ANOVA_R_Code.txt, while the complimentary CSV files marked with *Graph are
 used to generate graphs for the relevant data using Graph_R_Code.txt. The
 columns of the CSV are each denoted in the file but for the sake of
 further clarity each preffix of the CSV files coincides with what is being
 measured V_DF is organellar volume (μm<sup>3</sup>), SA_DF is
 organellar surface area
 (μm<sup>2</sup>/μm<sup>3</sup>) and Ratio_DF is
 the ratio of the 2 different measurements. The nucleator excel file is the
 volume measurements of random nuclei (μm<sup>3</sup>) using
 the nucleator plugin provided by Visiopharm VIS stereology software. The 2
 text documents are the general R code version 2023.03.0+386 for both the
 visualization of the graphs and the underlying ANOVA statistics that were
 run in the paper. All following packages were installed in Rstudio at the
 following versions: ggplot2 version 3.4.2, readr version 2.1.4, dplyr
 version 2.3.2, tidyverse version 2.0.0, scales version 1.2.1, ggpubr
 version 0.6.0, RColorBrewer version 1.1-3, ggtext version 0.1.2, lme4
 version 1.1-33, emmeans version 1.8.5, car version 3.1-2, and agricolae
 version 1.3-6. Lastly, the 6 mrc packets are the images that were used for
 all organelles, the packets marked specifically for golgi were used to
 measure the Golgi apparatus the other organelles were measured in the
 unspecified packets. Each image has a reference scale (μm/pixel) included
 in an information box at the bottom.