Biology subjects

Licknack, T.

Publications and source records attributed to Licknack, T..

4 recordsLinked to original sources

bioRxiv2025

Subcellular proteomics of Paradiplonema papillatum reveals digestive capacity of the cell membrane and the plasticity of peroxisomes across euglenozoans

Diplonemids are among the most diverse and abundant protists in the deep ocean, have extremely complex and ancient cellular systems, and exhibit unique metabolic capacities. Despite this, we know very little about this major group of eukaryotes. To establish a model organism for comprehensive investigation, we performed subcellular proteomics on Paradiplonema papillatum and localized 4,870 proteins to 22 cellular compartments. We additionally confirmed the predicted location of several proteins by epitope tagging and fluorescence microscopy. To probe the metabolic capacities of P. papillatum, we explored the proteins predicted to the cell membrane compartment in our subcellular proteomics dataset. Our data revealed an accumulation of many carbohydrate active enzymes (CAZymes). Our predictions suggest that these CAZymes are exposed to extracellular space, supporting proposals that diplonemids may specialize in breaking down carbohydrates in plant and algal cell walls. Further exploration of carbohydrate metabolism revealed an evolutionary divergence in the function of glycosomes (modified peroxisomes) in diplonemids versus kinetoplastids. Our subcellular proteome provides a resource for future investigations into the unique cell biology of diplonemids.

cell biology↗

bioRxiv2025

Subcellular proteomics of Paramecium tetraurelia reveals mosaic localization of glycolysis and gluconeogenesis

Ciliates are unicellular heterotrophic eukaryotes, most of which consume other microbes as prey. They exhibit nuclear dimorphism which requires reconstruction of a transcriptionally active macronucleus from the germline micronucleus after sexual recombination. This complex genomic structure has prevented the development of highly tractable genetic models leaving much of ciliate cell biology unexplored. To complicate matters further, some ciliates tend to accumulate many gene duplicates either singly or via whole genome duplications. Thus, extensive insight into the cell biology of ciliates requires the use of high-throughput tools like subcellular proteomics. Here, we use a subcellular proteomics workflow to classify over 9,000 proteins to 16 subcellular compartments in Paramecium tetraurelia. From these data, we identify a small but robust subcellular cluster containing canonical mitochondrial outer membrane proteins as well as some ER proteins, putatively at membrane contact sites. Within this cluster, we identified the important glycolytic enzyme phosphofructokinase, which contained a transmembrane domain. Further investigation revealed that several latter-acting glycolytic enzymes were localized to the mitochondrial cluster. The location of phosphoenol pyruvate carboxykinase and pyruvate carboxylase in the mitochondria but pyruvate kinase in the cytosol suggests that ciliates prefer gluconeogenesis over glycolysis. The localization of these enzymes was confirmed in a preliminary subcellular proteome of Tetrahymena thermophila. In sum, our findings suggest that mitochondrial localization of glycolytic/gluconeogenic enzymes is widespread across ciliates and that several may preferentially undergo gluconeogenesis over glycolysis using amino acids as a primary carbon source in both catabolic and anabolic metabolism. HighlightsSubcellular proteomics of Paramecium tetraurelia revealed that glycolytic and gluconeogenic enzymes are mosaically distributed between the cytosol, mitochondrial matrix, and mitochondrial outer membrane. A distinct mitochondrial outer membrane compartment was identified with 105 classified proteins, including core mitochondrial biogenesis proteins and a putative Tom70-like protein. Phosphofructokinase, a key glycolytic enzyme, was found embedded in the mitochondrial outer membrane. Localization of biochemical pathways suggest ciliates favor gluconeogenesis over glycolysis. In total, over 9000 Paramecium proteins were identified using subcellular proteomics and classified into 16 different cellular compartments.

cell biology↗

bioRxiv2025

Translation Accuracy in E. coli

We present a method that quantifies nearly all pairwise amino acid substitutions, revealing the rates and spectra of mistranslation per amino acid and per codon. While determining error rates is illuminating, understanding why certain mistranslation events are enriched requires further exploration, particularly in relation to the translation-accuracy hypothesis. We found that codons favored in highly expressed genes are not predictive of translational fidelity and are not selected for translational accuracy. We evaluated our method using historically characterized ribosomal variants of Escherichia coli reported to differ in translation fidelity. However, we found no significant differences among their overall translation-error rates, estimated here to be [~]2 per 1,000 amino acids. Instead they each exhibited unique mistranslation profiles, which likely led to prior overestimates of the translation error-rate in the putative error-prone variant. Together, these results underscore the significance of a direct, proteome-wide measure of translation fidelity.

evolutionary biology↗

bioRxiv2018

Further Investigation of Mitochondrial Biogenesis and Gene Expression of Key Regulators in Ascites- Susceptible and Ascites-Resistant Broiler Research Lines

We have extended our previous survey of the association of mitochondrial prevalence in particular tissues with ascites susceptibility in broilers. We previously reported that in breast muscle of 22 week old susceptible line male birds had significantly higher mtDNA copy number relative to nuclear copy number (mtDNA/nucDNA), compared to resistant line male birds. The higher copy number correlated with higher expression of PPARGC1A mRNA gene. Ascites is a significant metabolic disease associated with fast-growing meat-type chickens (broilers) and is a terminal result of pulmonary hypertension syndrome. We now report the mtDNA/nucDNA ratio in lung, liver, heart, thigh, and breast of both genders at 3, and 20 weeks old. At 3 weeks the mtDNA/nucDNA ratio is significantly higher in lung, breast, and thigh for susceptible line males compared to the resistant line males. Conversely, we see the opposite for lung and breast in females. At 20 weeks of age the differences between males from the two lines is lost for lung, and thigh. Although there is a significant reduction in the mtDNA/nucDNA ratio of breast from 3 weeks to 20 weeks in the susceptible line males, the susceptible males remain higher than resistant line males for this specific tissue. We assessed relative expression of five genes known to regulate mitochondrial biogenesis for lung, thigh and breast muscle from males and females of both lines with no consistent pattern to explain the marked gender and line differences for these tissues. Our results indicate clear sex differences in mitochondrial biogenesis establishing a strong association between the mtDNA quantity in a tissue-specific manner and correlated with ascites-phenotype. We propose that mtDNA/nucDNA levels could serve as a potential predictive marker in breeding programs to reduce ascites.

developmental biology↗