The role of mitochondrial energetics in the origin and diversification of eukaryotes.

The origin of eukaryotic cell size and complexity is often thought to have required an energy excess supplied by mitochondria. Recent observations show energy demands to scale continuously with cell volume, suggesting that eukaryotes do not have higher energetic capacity. However, respiratory membrane area scales superlinearly with the cell surface area. Furthermore, the consequences of the contrasting genomic architectures between prokaryotes and eukaryotes have not been precisely quantified. Here, we investigated (1) the factors that affect the volumes at which prokaryotes become surface area-constrained, (2) the amount of energy divested to DNA due to contrasting genomic architectures and (3) the costs and benefits of respiring symbionts. Our analyses suggest that prokaryotes are not surface area-constrained at volumes of 100‒103 µm3, the genomic architecture of extant eukaryotes is only slightly advantageous at genomes sizes of 106‒107 base pairs and a larger host cell may have derived a greater advantage (lower cost) from harbouring ATP-producing symbionts. This suggests that eukaryotes first evolved without the need for mitochondria since these ranges hypothetically encompass the last eukaryotic common ancestor and its relatives. Our analyses also show that larger and faster-dividing prokaryotes would have a shortage of respiratory membrane area and divest more energy into DNA. Thus, we argue that although mitochondria may not have been required by the first eukaryotes, eukaryote diversification was ultimately dependent on mitochondria.

Evolutionary bioenergetics of ciliates.

Understanding why various organisms evolve alternative ways of living requires information on both the fitness advantages of phenotypic modifications and the costs of constructing and operating cellular features. Although the former has been the subject of a myriad of ecological studies, almost no attention has been given to how organisms allocate resources to alternative structures and functions. We address these matters by capitalizing on an array of observations on diverse ciliate species and from the emerging field of evolutionary bioenergetics. A relatively robust and general estimator for the total cost of a cell per cell cycle (in units of ATP equivalents) is provided, and this is then used to understand how the magnitudes of various investments scale with cell size. Among other things, we examine the costs associated with the large macronuclear genomes of ciliates, as well as ribosomes, various internal membranes, osmoregulation, cilia, and swimming activities. Although a number of uncertainties remain, the general approach taken may serve as blueprint for expanding this line of work to additional traits and phylogenetic lineages.

Flagellar energy costs across the tree of life.

Flagellar-driven motility grants unicellular organisms the ability to gather more food and avoid predators, but the energetic costs of construction and operation of flagella are considerable. Paths of flagellar evolution depend on the deviations between fitness gains and energy costs. Using structural data available for all three major flagellar types (bacterial, archaeal, and eukaryotic), flagellar construction costs were determined for Escherichia coli, Pyrococcus furiosus, and Chlamydomonas reinhardtii. Estimates of cell volumes, flagella numbers, and flagellum lengths from the literature yield flagellar costs for another ~200 species. The benefits of flagellar investment were analysed in terms of swimming speed, nutrient collection, and growth rate; showing, among other things, that the cost-effectiveness of bacterial and eukaryotic flagella follows a common trend. However, a comparison of whole-cell costs and flagellum costs across the Tree of Life reveals that only cells with larger cell volumes than the typical bacterium could evolve the more expensive eukaryotic flagellum. These findings provide insight into the unsolved evolutionary question of why the three domains of life each carry their own type of flagellum.

Most creatures on Earth are single cell organisms. The tree of life comprises three domains, two of which ­ bacteria and archaea ­ are formed exclusively of creatures that spend their existence as independent cells. Yet even eukaryotes, the domain which include animals and plants, feature single cell species such as yeasts and algae. Regardless of which group they belong to, all single-celled organisms must find food in their environment. For this, many are equipped with flagella, whip-like structures that protrude from the cell and allow it to swim. In fact, archaea, bacteria and eukaryotes have all independently evolved these structures. However, flagella are also expensive for an organism to build, maintain and operate. They are only worth having if the advantages they bring to the cell compensate for their cost; many single-cell species do not carry flagella and obtain their food without having to swim. To explore this trade-off, Schavemaker and Lynch calculated the cost of building and using flagella for about 200 species across the tree of life. The analysis show that the amount of energy spent on flagella varied between 0.1% and 40% of the entire cell budget. This investment is only worthwhile if the cell is above a certain size. Smaller than this, and the organism is better off obtaining its food passively. The results also show that while eukaryotic flagella are much bigger and quite different than their bacterial counterpart, both appendages share the same patterns of cost effectiveness. However only eukaryotic cells, which are on average larger than bacteria, can afford to evolve such sizable and complex structures; making just one would cost more than the entire energy budget of a bacterial cell. Many single-cell species which are critical for the health of the planet are equipped with flagella, such as the microorganisms which recycle matter in the oceans and release carbon dioxide. Understanding the costs and benefits of flagella could explain more about this aspect of the carbon cycle, and therefore global warming.

How Important Is Protein Diffusion in Prokaryotes?

That diffusion is important for the proper functioning of cells is without question. The extent to which the diffusion coefficient is important is explored here for prokaryotic cells. We discuss the principles of diffusion focusing on diffusion-limited reactions, summarize the known values for diffusion coefficients in prokaryotes and in in vitro model systems, and explain a number of cases where diffusion coefficients are either limiting for reaction rates or necessary for the existence of phenomena. We suggest a number of areas that need further study including expanding the range of organism growth temperatures, direct measurements of diffusion limitation, expanding the range of cell sizes, diffusion limitation for membrane proteins, and taking into account cellular context when assessing the possibility of diffusion limitation.

(Membrane) Protein Production in Context.

Great progress has been made in elucidating the structural and mechanistic basis of (membrane) protein production. Here, we attempt to look ahead and indicate four directions in which our understanding of the protein production process can grow: (i) determine how the molecular mechanisms influence higher-level processes, such as the distribution of protein copy number over a population of cells or the cell growth rate; (ii) explore the functional landscape that the molecular mechanisms of protein production exist in, for instance by comparing membrane protein insertion mechanisms; (iii) uncover the life history of proteins - that is, what happens to them between their synthesis and degradation; and (iv) determine, and connect by calculation, the numbers that are associated with (membrane) protein production.

Steric exclusion and protein conformation determine the localization of plasma membrane transporters.

The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

Ribosome surface properties may impose limits on the nature of the cytoplasmic proteome.

Much of the molecular motion in the cytoplasm is diffusive, which possibly limits the tempo of processes. We studied the dependence of protein mobility on protein surface properties and ionic strength. We used surface-modified fluorescent proteins (FPs) and determined their translational diffusion coefficients (D) in the cytoplasm of Escherichia coli, Lactococcus lactis and Haloferax volcanii. We find that in E. coli D depends on the net charge and its distribution over the protein, with positive proteins diffusing up to 100-fold slower than negative ones. This effect is weaker in L. lactis and Hfx. volcanii due to electrostatic screening. The decrease in mobility is probably caused by interaction of positive FPs with ribosomes as shown in in vivo diffusion measurements and confirmed in vitro with purified ribosomes. Ribosome surface properties may thus limit the composition of the cytoplasmic proteome. This finding lays bare a paradox in the functioning of prokaryotic (endo)symbionts.

Impact of osmotic stress on protein diffusion in Lactococcus lactis.

We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram-positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12-transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ∼ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram-positive and Gram-negative bacteria.