Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.

Glycerol suppresses glucose consumption in trypanosomes through metabolic contest.

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.

The biochemistry and physiology of long-chain dicarboxylic acid metabolism.

Mitochondrial ß-oxidation is the most prominent pathway for fatty acid oxidation but alternative oxidative metabolism exists. Fatty acid ω-oxidation is one of these pathways and forms dicarboxylic acids as products. These dicarboxylic acids are metabolized through peroxisomal ß-oxidation representing an alternative pathway, which could potentially limit the toxic effects of fatty acid accumulation. Although dicarboxylic acid metabolism is highly active in liver and kidney, its role in physiology has not been explored in depth. In this review, we summarize the biochemical mechanism of the formation and degradation of dicarboxylic acids through ω- and ß-oxidation, respectively. We will discuss the role of dicarboxylic acids in different (patho)physiological states with a particular focus on the role of the intermediates and products generated through peroxisomal ß-oxidation. This review is expected to increase the understanding of dicarboxylic acid metabolism and spark future research.

Peroxisomal ß-oxidation stimulates cholesterol biosynthesis in the liver in diabetic mice.

Although diabetes normally causes an elevation of cholesterol biosynthesis and induces hypercholesterolemia in animals and human, the mechanism linking diabetes to the dysregulation of cholesterol biosynthesis in the liver is not fully understood. As liver peroxisomal ß-oxidation is induced in the diabetic state and peroxisomal oxidation of fatty acids generates free acetate, we hypothesized that peroxisomal ß-oxidation might play a role in liver cholesterol biosynthesis in diabetes. Here, we used erucic acid, a specific substrate for peroxisomal ß-oxidation, and 10,12-tricosadiynoic acid, a specific inhibitor for peroxisomal ß-oxidation, to specifically induce and suppress peroxisomal ß-oxidation. Our results suggested that induction of peroxisomal ß-oxidation increased liver cholesterol biosynthesis in streptozotocin-induced diabetic mice. We found that excessive oxidation of fatty acids by peroxisomes generated considerable free acetate in the liver, which was used as a precursor for cholesterol biosynthesis. In addition, we show that specific inhibition of peroxisomal ß-oxidation decreased cholesterol biosynthesis by reducing acetate formation in the liver in diabetic mice, demonstrating a crosstalk between peroxisomal ß-oxidation and cholesterol biosynthesis. Based on these results, we propose that induction of peroxisomal ß-oxidation serves as a mechanism for a fatty acid-induced upregulation in cholesterol biosynthesis and also plays a role in diabetes-induced hypercholesterolemia.

Asymmetric horseshoe-like assembly of peroxisomal yeast oxalyl-CoA synthetase.

Oxalyl-CoA synthetase from Saccharomyces cerevisiae is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in a homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target.

Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase.

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process.

Biogenesis and metabolic homeostasis of trypanosomatid glycosomes: New insights and new questions.

Kinetoplastea and Diplonemea possess peroxisome-related organelles that, uniquely, contain most of the enzymes of the glycolytic pathway and are hence called glycosomes. Enzymes of several other core metabolic pathways have also been located in glycosomes, in addition to some characteristic peroxisomal systems such as pathways of lipid metabolism. A considerable amount of research has been performed on glycosomes of trypanosomes since their discovery four decades ago. Not only the role of the glycosomal enzyme systems in the overall cell metabolism appeared to be unique, but also the organelles display remarkable features regarding their biogenesis and structural properties. These features are similar to those of the well-studied peroxisomes of mammalian and plant cells and yeasts yet exhibit also differences reflecting the large evolutionary distance between these protists and the representatives of other major eukaryotic lineages. Despite all research performed, many questions remain about various properties and the biological roles of glycosomes and peroxisomes. Here, we review the current knowledge about glycosomes, often comparing it with information about peroxisomes. Furthermore, we highlight particularly many questions that remain about the biogenesis, and the heterogeneity in structure and content of these enigmatic organelles, and the properties of their boundary membrane.

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

Glycosome heterogeneity in kinetoplastids.

Kinetoplastid parasites have essential organelles called glycosomes that are analogous to peroxisomes present in other eukaryotes. While many of the processes that regulate glycosomes are conserved, there are several unique aspects of their biology that are divergent from other systems and may be leveraged as therapeutic targets for the treatment of kinetoplastid diseases. Glycosomes are heterogeneous organelles that likely exist as sub-populations with different protein composition and function in a given cell, between individual cells, and between species. However, the limitations posed by the small size of these organelles makes the study of this heterogeneity difficult. Recent advances in the analysis of small vesicles by flow-cytometry provide an opportunity to overcome these limitations. In this review, we describe studies that document the diverse nature of glycosomes and propose an approach to using flow cytometry and organelle sorting to study the diverse composition and function of these organelles. Because the cellular machinery that regulates glycosome protein import and biogenesis is likely to contribute, at least in part, to glycosome heterogeneity we highlight some ways in which the glycosome protein import machinery differs from that of peroxisomes in other eukaryotes.

Effect of lysine acetylation on the regulation of Trypanosoma brucei glycosomal aldolase activity.

Post-translational modifications provide suitable mechanisms for cellular adaptation to environmental changes. Lysine acetylation is one of these modifications and occurs with the addition of an acetyl group to Nε-amino chain of this residue, eliminating its positive charge. Recently, we found distinct acetylation profiles of procyclic and bloodstream forms of Trypanosoma brucei, the agent of African Trypanosomiasis. Interestingly, glycolytic enzymes were more acetylated in the procyclic, which develops in insects and uses oxidative phosphorylation to obtain energy, compared with the bloodstream form, whose main source of energy is glycolysis. Here, we investigated whether acetylation regulates the T. brucei fructose 1,6-bisphosphate aldolase. We found that aldolase activity was reduced in procyclic parasites cultivated in the absence of glucose and partial recovered by in vitro deacetylation. Similarly, acetylation of protein extracts from procyclics cultivated in glucose-rich medium, caused a reduction in the aldolase activity. In addition, aldolase acetylation levels were higher in procyclics cultivated in the absence of glucose compared with those cultivated in the presence of glucose. To further confirm the role of acetylation, lysine residues near the catalytic site were substituted by glutamine in recombinant T. brucei aldolase. These replacements, especially K157, inhibited enzymatic activity, changed the electrostatic surface potential, decrease substrate binding and modify the catalytic pocket structure of the enzyme, as predicted by in silico analysis. Taken together, these data confirm the role of acetylation in regulating the activity of an enzyme from the glycolytic pathway of T. brucei, expanding the factors responsible for regulating important pathways in this parasite.

Hysteresis of pyruvate phosphate dikinase from Trypanosoma cruzi.

Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.

First report of Trypanosoma dionisii (Trypanosomatidae) identified in Australia.

Trypanosomes are blood-borne parasites that can infect a variety of different vertebrates, including animals and humans. This study aims to broaden scientific knowledge about the presence and biodiversity of trypanosomes in Australian bats. Molecular and morphological analysis was performed on 86 blood samples collected from seven different species of microbats in Western Australia. Phylogenetic analysis on 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) sequences identified Trypanosoma dionisii in five different Australian native species of microbats; Chalinolobus gouldii, Chalinolobus morio, Nyctophilus geoffroyi, Nyctophilus major and Scotorepens balstoni. In addition, two novels, genetically distinct T. dionisii genotypes were detected and named T. dionisii genotype Aus 1 and T. dionisii genotype Aus 2. Genotype Aus 2 was the most prevalent and infected 20.9% (18/86) of bats in the present study, while genotype Aus 1 was less prevalent and was identified in 5.8% (5/86) of Australian bats. Morphological analysis was conducted on trypomastigotes identified in blood films, with morphological parameters consistent with trypanosome species in the subgenus Schizotrypanum. This is the first report of T. dionisii in Australia and in Australian native bats, which further contributes to the global distribution of this cosmopolitan bat trypanosome.

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites.

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in 'sub G0/G1' phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.

The dynamic life of the glycogen granule.

Glycogen, the primary storage form of glucose, is a rapid and accessible form of energy that can be supplied to tissues on demand. Each glycogen granule, or "glycosome," is considered an independent metabolic unit composed of a highly branched polysaccharide and various proteins involved in its metabolism. In this Minireview, we review the literature to follow the dynamic life of a glycogen granule in a multicompartmentalized system, i.e. the cell, and how and where glycogen granules appear and the factors governing its degradation. A better understanding of the importance of cellular compartmentalization as a regulator of glycogen metabolism is needed to unravel its role in brain energetics.

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids.

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Cytochemical Localization of Polyphenol Oxidase Activity in K2-Bodies of Saprolegnia ferax Secondary Zoospores.

Zoospores of the oomycete Saprolegnia ferax release adhesive material from K-bodies at the onset of attachment to substrates. To understand more fully how K-bodies function in adhesion, enzyme activity was investigated cytochemically in secondary zoospores. Presence of catalase, a marker enzyme for microbodies, was explored in the diaminobenzidine (DAB) reaction. Although pH 9.2 DAB-staining characteristic of catalase activity was detected in the granular matrix regions of K-bodies, reaction controls indicated that the reaction was due to oxidative enzyme activity other than catalase. Because polyphenol oxidase (PPO) is another metal-containing enzyme capable of oxidizing DAB, activity of this enzyme was tested with a more specific substrate, dihydroxyphenylalanine (DOPA). In the DOPA procedure, reaction product was exclusively localized within K-bodies, indicating the presence of PPO. Results with three methods of reaction controls (elimination of substrate, addition of a PPO enzyme inhibitor, and heat-inactivation of enzymes) all supported the presence of PPO in K-bodies. This study highlights potential roles for K-body PPO in stabilization of adhesion bodies by: cross-linking matrix phenolic proteins or glycoproteins as K-bodies discharge adhesives onto substrates, or polymerizing phenolics protective against microbial attacks of the adhesion pad.

The hydrophobic region of the Leishmania peroxin 14: requirements for association with a glycosome mimetic membrane.

Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.

Proteomic Profiling of Leishmania donovani Promastigote Subcellular Organelles.

To facilitate a greater understanding of the biological processes in the medically important Leishmania donovani parasite, a combination of differential and density-gradient ultracentrifugation techniques were used to achieve a comprehensive subcellular fractionation of the promastigote stage. An in-depth label-free proteomic LC-MS/MS analysis of the density gradients resulted in the identification of ∼50% of the Leishmania proteome (3883 proteins detected), which included ∼645 integral membrane proteins and 1737 uncharacterized proteins. Clustering and subcellular localization of proteins was based on a subset of training Leishmania proteins with known subcellular localizations that had been determined using biochemical, confocal microscopy, or immunoelectron microscopy approaches. This subcellular map will be a valuable resource that will help dissect the cell biology and metabolic processes associated with specific organelles of Leishmania and related kinetoplastids.

pH regulation in glycosomes of procyclic form Trypanosoma brucei.

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+ exchangers are required for glycosomal pH regulation.

Fossil melanosomes or bacteria? A wealth of findings favours melanosomes: Melanin fossilises relatively readily, bacteria rarely, hence the need for clarification in the debate over the identity of microbodies in fossil animal specimens.

The discovery of fossil melanosomes has resulted in a wealth of research over the last 7 years, notably the reconstruction of colour in dinosaurs and fossil mammals. In spite of these discoveries some authors persist in arguing that the observed microbodies could represent preserved bacteria. They contend that bacteria fossilise easily and everywhere, which means that one can never be certain that a microbody is a melanosome without an extraordinary burden of evidence. However, this critique mischaracterises the morphological and structural evidence for interpreting microbodies as fossil melanosomes, and hence the basis for using them in reconstructing prehistoric colours. The claims for bacterial omnipresence in the fossil record are themselves not supported, thus tipping the scales strongly towards melanosomes in the bacteria-versus-melanosome controversy.