Open Biology

Centrosome dysfunction is linked to developmental disorders affecting brain and body size, including microcephaly and primordial dwarfism. However, the cellular mechanisms underlying these rare conditions remain poorly understood. In this study, we investigate a rare variant of the centrosome-associated protein Pericentrin, which was discovered in a single family with Majewski/microcephalic osteodysplastic primordial dwarfism type II (MOPD II). Unlike the majority of pathogenic PCNT variants that cause severe protein truncation, the p.Lys3154del variant ({Delta}K3154) involves a single amino acid deletion in the proteins only conserved functional domain, providing a unique opportunity to explore PCNT function in MOPD II. To model PCNT{Delta}K3154, we examined the effects of Drosophila Pericentrin-like protein (PLP) carrying an orthologous deletion (Plp{Delta}R). Our results show that plp{Delta}R animals exhibit smaller tissues that recapitulate MOPD II phenotypes. Behavioral assays revealed defects in climbing and mechanosensation, suggesting impaired sensory cilia function. We also found that Plp{Delta}R cells exhibit accelerated mitosis, increased apoptosis, and reduced pericentriolar material recruitment. In silico structural modeling, yeast two-hybrid, and co-immunoprecipitation experiments show that Plp{Delta}R produces a protein that disrupts PLP dimerization and PLP interaction with Asterless, another centrosome protein. Overall, modeling the human MOPD II patient variant PCNT{Delta}K3154 in Drosophila reveals how a single amino acid deletion affects biological processes from the molecular level to the organismal level. Our work offers new insights into the defective cellular mechanisms underlying MOPD II in patients with the PCNT{Delta}K3154 variant, potentially linking the etiology of the disease in these individuals to the loss of a single protein-protein interaction.

The high efficiency of genome editing presents a challenge when modifying genes associated with viability, welfare, or fertility issues, as implementation of the technology frequently results in mosaic animals with bi-allelic mutations. Combining deactivated Cas9 (dCas9) with Cas9 has been proposed as a strategy to protect one of the two target alleles from editing. We piloted this strategy with 11 genes that are reported as homozygous lethal or associated with welfare issues. We showed that the viability of founders was significantly increased when using 80:20 or 90:10 dCas9:Cas9 ratios, whereas the 70:30 ratio did not yield an equivalent protective effect. The associated overall production rate of mutated founder per manipulated embryo was significantly higher for the 80:20 ratio. Concomitantly, an increased proportion of dCas9 was associated with a significant increase in retention of unedited target alleles but, importantly, did not hinder germline transmission. In addition, editing genes in a paralog cluster with a combination of dCas9 and Cas9 reduced unwanted off-target editing, illustrating a further potential applicability of this approach. This study defines the optimal ratio between dCas9 and Cas9 for strategies aimed at achieving mono-allelic mutations within mosaic founders and proposes a means to reduce the incidence of off-target effects in experiments with limited gRNA options.

Microproteins are proteins of <100 amino acids. They represent a major, and until recently, overlooked fraction of the human proteome. However, it has now been demonstrated that many of these proteins play key roles in cellular physiology. Our group reported the expression of a microprotein expressed from an ioORF within the 53BP1 CDS arising as a result of delayed translational reinitiation mediated by a small uORF within the 5 TL. We named this microprotein SEP53BP1. We have sought to expand these studies with the ultimate aim of establishing a function for this microprotein. Although this remains elusive, we report findings providing new insights into the elements regulating its translation and demonstrate that the SEP53BP1 sequence serves as a Golgi targeting tag. Lastly, despite the fact that subunits of the proteasome feature prominently on interactome studies we were unable to demonstrate an impact of microprotein over-expression on the activities of both the proteasome and immunoproteasome.

The biological conservation between fungi and mammals due to a common ancestor has made development of selective antifungal drugs a difficult challenge. Further complicating this situation is the selection of antifungal drug-resistant organisms during drug treatment. The pathogenic yeast Nakaseomyces glabratus (called here Candida glabrata) presents an especially challenging organism due to its tendency to frequently lose susceptibility to the major antifungal drug class the azoles. Additionally, C. glabrata develops resistance to echinocandin drugs, a second, more recently described antifungal agent at 10 times the rate of other organisms. Previous work has established that the sterol responsive transcriptional regulator Upc2A is a key determinant of azole susceptibility in C. glabrata and plays a role in echinocandin resistance. We used a biochemical approach to identify proteins that co-purified with Upc2A and identified the Ypk2 AGC kinase as an interacting protein. Strains lacking YPK2 exhibited increased susceptibility to fluconazole and the echinocandin caspofungin. A ypk2{Delta} strain failed to normally induce transcription of several ERG genes but exhibited normal induction of the CDR1 ATP-binding cassette transporter gene. Isogenic ypk2{Delta} strains were also highly susceptible to the three major classes of antifungal drugs, indicating that this kinase behaves as a multidrug susceptibility factor. RNA-seq analyses indicated that the transcriptional response to exposure is different for each drug and each response is differentially altered upon loss of Ypk2. Our data indicate that Ypk2 plays an important role in coordinating gene expression that impacts susceptibility to all major antifungal drug classes.

Silk glands have been found in two groups of amphipods: the Corophiida and the Ampeliscidae. The silk glands in Ampeliscidae, however, have yet to be examined in detail. Here we report, for the first time, the morphology and distribution of pereopodal glands in the Ampeliscidae, in non-thread producing Synopiidae, and in the Paragammaropsidae. In the Ampeliscidae we found two gland types distributed throughout all pereopods which have the ability to create threads. Pereopods three and four have additional silk extrusion morphology at the tip of the dactylus in which silk is transformed into semi-cylindrical threads used for building domiciles. Synopiid outgroup species have one of the gland types but lack silk extrusion morphology. Using ancestral state reconstruction analysis, we find that glands in the Synopiidae are likely ancestral and hypothesize that silk glands in Ampeliscidae are derived from these ancestral glands. Silk-spinning pereopods in the Paragammaropsidae had similarities with both Corophiida and Ampeliscidae but had distinctions. Ampeliscidae silk-spinning systems bear surprising resemblance to the Corophiida which presents one to reconsider the taxonomic placement of Ampeliscidae and the origins of silk-spinning in amphipods. This is the first comprehensive study on the glandular systems of Ampeliscidae, Synopiidae, and Paragammaropsidae using advanced microscopy, providing pertinent morphological data to the study of arthropod silk gland evolution and complex traits.

Astrocytic homeostatic programs, many of which are regulated by the circadian clock, are disrupted early in neurodegenerative disease. The core clock transcription factor (TF) BMAL1 is required for normal astrocyte function, but its role during disease remains unclear. This is partly because methods for identifying cell type-specific TF binding sites are limited. Here, we developed MACS-Calling Cards (MACS-CC), a strategy for mapping astrocyte-specific TF occupancy in vivo. We used MACS-CC to define BMAL1 binding in the Cln3{Delta}ex7/8 mouse model of CLN3 disease, a fatal neurodegenerative disorder marked by early astrocyte dysfunction and circadian disruption. BMAL1 binding was extensively redistributed in Cln3{Delta}ex7/8 astrocytes: wild-type-specific binding sites enriched near glial differentiation genes, whereas Cln3{Delta}ex7/8-specific sites lacked functional enrichment. Consistent with these changes, Cln3{Delta}ex7/8 astrocytes decreased expression of mature astrocyte markers. To define mechanisms underlying BMAL1 retargeting, we tested whether altered chromatin accessibility explained the changes in BMAL1 binding. Although chromatin accessibility was broadly remodeled, differential accessibility did not predict BMAL1 redistribution. Instead, motif analysis suggested that loss of cooperative TF interactions drives BMAL1 retargeting. These findings demonstrate that MACS-CC enables astrocyte-specific TF occupancy mapping and reveals mechanisms behind early rewiring of circadian regulatory programs within a model of a neurodegenerative disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/721783v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1ada239org.highwire.dtl.DTLVardef@7564a3org.highwire.dtl.DTLVardef@122222forg.highwire.dtl.DTLVardef@1f2729c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Arthropods exhibit an exceptional diversity of life histories, where developmental modes involve moulting stage progressions with changes ranging from the bare minimal to the dramatically transformative. While this variability drives many research questions aiming to understand evolutionary and developmental underpinnings of life history differences, it can complicate comparative analyses across taxa. However, this can be approached by applying a framework that defines metamorphosis as a post-embryonic stage progression characterised by substantial changes in morphology and adaptive landscape. Employing this framework with a phylogenomic dataset spanning 26 orders and encompassing four independently arising metamorphic lineages, we explore gene repertoire evolutionary dynamics potentially associated with metamorphosis in Pancrustacea. The approach contrasts gene family evolutionary dynamics inferred to have occurred in the last common ancestors of the metamorphic Insecta, Copepoda, Eucarida, and Thecostraca, with those of their sister lineages, as well as of descendent and ancestral nodes. The results reveal that the metamorphosis ancestors are characterised by an elevated number of gene family births and expansions. Expanded gene families share a set of commonly enriched biological processes across all metamorphosis ancestors, suggesting functional convergence by independent evolution of distinct gene families involved in embryonic and post-embryonic development and nervous system differentiation. Evolutionary modelling further highlights a subset of these families exhibiting signatures of adaptive, lineage-specific gene family size increases associated with metamorphic development. These families include genes implicated in neural and sensory development, segmentation, and moulting. These findings support a model of the evolution of pancrustacean metamorphosis where distinct gene families from a common functional toolkit expand and are co-opted into facilitating transitions to multi-phasic life cycles. This reframes the role of moulting in arthropod diversification to be recognised as an important reservoir of genetic change that can potentiate truly remarkable life history transitions.

O-fucosylation plays an essential role in controlling protein folding, secretion and protein-protein interactions within the extracellular space. Recently, we identified a new form of protein O-fucosylation occurring on the N-terminal Elastin Microfibril Interaction (EMI) domain of several secreted proteins, mediated by two previously uncharacterized protein O-fucosyltransferases, POFUT3 (FUT10) and POFUT4 (FUT11). As all POFUT enzymes (POFUT1-4) are highly specific for the three-dimensional (3D) structure of their substrate protein domains, we postulated that structural homologues of these domains in other proteins may also be O-fucosylated. Here, we employed iterative protein structural homology searches as a novel strategy for identifying EMI-like domains that may serve as potential substrates for POFUT3/4. We discovered that microfibrillar-associated protein 2 and 5 (MFAP2/MFAP5) contain EMI-like domains and are O-fucosylated at high stoichiometry in human tissues. Unexpectedly, we showed that only POFUT3 is both necessary and sufficient for MFAP2/MFAP5 O-fucosylation, despite POFUT4 also having strong protein-protein interactions with MFAP2/MFAP5. Finally, we determined that O-fucosylation of MFAP2/MFAP5 is required for their efficient secretion, similar to other EMI domain-containing proteins. Together, these data demonstrate the power of sensitive structural homology analysis in identifying new enzyme-substrate relationships and protein-protein interactions.

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium that causes scrub typhus, a potentially life-threatening disease. To systematically identify host factors regulating early stages of infection, we performed a microscopy-based genome-wide siRNA screen in HeLa cells. This approach identified 2,989 genes grouped into 55 functional networks that modulate bacterial entry and intracellular translocation. In addition to confirming previously described pathways, including endocytosis and microtubule-dependent trafficking, the screen revealed an association between Ot infection and host cell cycle regulation. We found that Ot preferentially infects and/or replicates in host cells in the S and G2 phases, where intracellular bacterial accumulation is increased relative to G1. Early infection was associated with a shift in host cell cycle distribution, consistent with a delay in progression through S and G2 phases. Longitudinal analysis further showed that these cell cycle states support enhanced bacterial expansion. In parallel, infected cells exhibited reduced proliferation compared to uninfected cells, suggesting that Ot infection alters host cell division dynamics. Together, these findings support a model in which host cell cycle state influences susceptibility to Ot infection and intracellular growth. This work provides a systems-level map of host pathways involved in early infection and identifies cell cycle regulation as an important component of host-pathogen interactions in scrub typhus. Author SummaryScrub typhus is a potentially life-threatening disease caused by the bacterium Orientia tsutsugamushi, which can only survive and replicate inside human cells. Although some host factors involved in infection have been identified, many remain unknown. In this study, we used a large-scale screening approach to systematically identify human genes that influence the bacteriums ability to enter and move within host cells. Our analysis uncovered multiple pathways required for infection, including a role for the host cell cycle. We found that O. tsutsugamushi preferentially accumulates in cells during specific stages of the cell cycle, particularly when cells are preparing to divide. At the same time, infection slows host cell division, suggesting that the bacterium alters the cellular environment to support its own growth. These findings provide new insight into how O. tsutsugamushi interacts with human cells and identify potential host processes that could be targeted to limit infection.

Octopamine is involved in a variety of different physiological and behavioral mecha-nisms in Drosophila melanogaster. Throughout the life cycle of the fruit fly, from the larva to the adult, octopaminergic neurons in both the central and the peripheral nerv-ous system target a multitude of neurons and even non-neuronal tissues, making it challenging to analyze individual mechanisms of octopamine function. One approach to deconstructing this complex system is to examine the postsynaptic components of signal transmission. In Drosophila, octopamine interacts with six distinct G-protein-coupled receptors. For some of these receptors, expression maps and functional im-plications have been described. In contrast, other receptors have been neglected, partly due to the lack of suitable genetic tools. Here, for the first time, we compiled a complete set of mutant lines of all known octopamine receptors, all generated using the same genetic tool, the recently established Trojan Exon system. It integrates the Gal4/UAS binary expression strategy while simultaneously impairing receptor func-tion. This enabled us to generate a comprehensive anatomical map of receptor ex-pression in the larva and, at the same time, analyze the function of individual octopa-mine receptors during larval development, chemosensory perception and locomotion. All octopamine receptors (Oamb, Oct2R, Oct{beta}1R, Oct{beta}2R, Oct{beta}3R, and Oct-TyrR) showed extensive signal in the central nervous system. The same was found for the peripheral nervous system, with the exception of Oct{beta}2R, which showed pronounced expression in the somatic muscles. We also observed a previously undescribed role of Oct{beta}1R, Oct{beta}3R, and Oct-TyrR in larval hatching and in the survival of larvae and pupae. Molecular evaluation of the Trojan Exon octopamine lines supports our analy-sis. In addition, we combined the experimental results with gene expression data from the different development stages of Drosophila melanogaster and from different tis-sues and cell populations throughout the body. Overall, we compiled, analyzed and validated a complete set of octopamine lines which, together with gene expression analysis, provides a basis for further functional studies on the larval octopaminergic system.

Acropora spp. are the dominant reef-builders of the Indo-Pacific but are also amongst the most stress-sensitive corals. For these reasons, Acropora spp. have become the most studied of corals, two species (A. digitifera and A. millepora) often essentially serving as the basis for understanding molecular responses and processes across the sub-order Refertina and corals in general. The early development of these species has been well-characterised in terms of morphology and gene expression but as yet we have a limited understanding of how transcription is regulated during development. In "higher" animals (bilaterians) microRNAs (miRNAs) are critical regulators of gene expression but until now their involvement in coral development has not been investigated. Building on the existing developmental data for Acropora spp., we catalogued microRNAs (miRNAs) expressed during the early development of Acropora digitifera and profiled their expression in 21 stages from unfertilised eggs to 24h after treatment with a natural settlement cue (CCA chips). 157 miRNAs were recognised, many of which ([~]60%) were novel. These fell into three distinct groups, corresponding to three distinct developmental phases: (1) those present in eggs through to gastrulation (2) a larvally expressed group and (3) those expressed following settlement induction. Exposure of competent larvae to a natural settlement inducer resulted in major changes in the miRNA profile within 10 minutes, indicating that miRNAs may be particularly important in mediating the larva/polyp transition but are also likely to play important regulatory roles throughout early coral development in addition to possible roles in disease resistance.

Eukaryotic cells control their size by coordinating growth and division. Fission yeast divide at a reproducible cell size due to regulated activation of the cyclin-dependent kinase Cdk1. The nuclear concentration of mitotic cyclin Cdc13 increases in a time-dependent manner to promote Cdk1 activation as cells grow. Here, we show that interphase Cdc13 is stable against degradation and nuclear export, but is diluted by cell growth. Low glucose reduced cell growth rate but not time-dependent accumulation of Cdc13. Uncoupling the rates of cell growth and Cdc13 accumulation resulted in higher concentrations of nuclear Cdc13 despite reduced cell size. This change coincided with reduced activating phosphorylation of Cdk1-T167 and occurred dynamically during abrupt changes in glucose concentration. Mathematical modeling and experiments showed that cells maintain size homeostasis under these conditions. In contrast to low glucose, poor nitrogen reduced both cell growth rate and Cdc13 accumulation rate. Therefore, Cdc13 accumulation is independent of cell growth rate but can be altered by nutrient-specific mechanisms.

KaiC, a clock protein in cyanobacteria, cycles between dephosphorylated and phosphorylated states in a 24-hour period in the presence of KaiA and KaiB. We identified the 322nd residue of KaiC as a third example of period-tuning sites. 322nd-site-directed saturation mutagenesis resulted in a variety of KaiC mutants exhibiting either shortened or lengthened cycles. The tunable range of the periods was from approximately 11 to 78 h without significantly compromising temperature compensation. We conducted biochemical analyses of the 322nd variants and examined their predicted structural models. In contrast to another known period-tuning site, where the period decreases sharply as the side-chain volume increases due to mutations, the cycle lengths correlate only modestly with bulkiness at the 322nd residues. The 322nd residue is located in a C-terminal domain of KaiC and influences ATPase cycles in both the C-terminal domain and an N-terminal domain through its interaction with a flexible loop connecting the two domains. The structural models predict that placing less bulky but polar side chains, such as serine and threonine, at the 322nd position leads to the formation of a hydrogen-bonding network between that site and the loop. This reduces the mobility of the loop, resulting in the longer cycles due to decreases in the ATPase activity of the N-terminal domain. Conversely, placing bulky residues such as phenylalanine at the 322nd position appears to alter the loop structure, shortening the periods by enhancing the ATP activities of both the domains. The third period-tuning mechanism is distinct from other known mechanisms. Significance StatementA Kai-protein clock system serves as a model for studying how long circadian rhythms are achieved. We identified the 322nd residue of KaiC as a third example of period-tuning sites that allow tuning of the period in either long- and short-period directions. The third period-tuning mechanism differs from the two previously known types in several respects. Previous studies have suggested that the ATPase activity in an N-terminal domain of KaiC is the primary regulator of the period. On the other hand, the 322nd residues of KaiC can affect the period by activating the ATPase cycle in its C-terminal domain. Our findings will stimulate future studies on the period-tuning mechanism mediated by the ATPase activity in the C-terminal domain of KaiC.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

The brain is one of the most complex animal organs but the development of the many different neuron types remains enigmatic. A set of brain-specific transcription factors is known to be involved in brain patterning but their specific contributions remain to be elucidated in most cases, including foxQ2II. This transcription factor is known to be conserved in anterior neuroectodermal patterning of most animals while it has been lost from vertebrates. However, the contribution of foxQ2II-positive neurons to the adult brain has remained enigmatic. Here, we use an enhancer trap, immunostainings and our newly established beetle brainbow system to categorize Tc-foxQ2II-positive neurons into nine clusters with different projection patterns. All clusters contain neurons with the fast activating neurotransmitters acetylcholine and glutamate while no Tc-foxQ2II positive neuron is GABA-ergic or serotonin-positive. Interestingly, we found that many dopaminergic neurons were Tc-foxQ2II positive and we homologize them with dopaminergic neurons of the PPL2c, PPM1 and PPL1 cluster described in the Drosophila brain. Our results show that Tc-foxQ2II marks subsets of fast-acting interneurons contributing to the higher order brain centers mushroom bodies and central complex. Taken together, our work expands the known functional range of foxQ2 genes from sensory and neurosecretory cell specification to interneurons involved in the function of higher order brain centers.

Calpains constitute an ancient, extensive family of calcium-dependent cysteine proteases found in some bacteria and most eukaryotes. They are involved in a wide variety of developmental and cellular processes and are implicated in major human diseases, yet it remains to be seen if they have a common core function explaining their widespread and varied presence across taxa. Beyond their core CysPc catalytic domain, calpains contain diverse domain combinations and can be either cytosolic or membrane bound. Here we hypothesize a general role for both cytosolic and transmembrane calpains in cellular cytokinesis through positional anchoring and organization of microtubules (MTs). We propose that during plant cell division, the singular transmembrane calpain DEK1 localizes and organizes the array of cortical MTs from the microtubule organizing center (MTOC) to establish the location of the preprophase band and/or the site of cell plate formation according to the positional activation of DEK1 proteins in the nuclear membrane. Similarly, during cell division in animals, their calpains may be involved in setting the point of membrane invagination via their association with membrane-bound proteins. This proposition adds to the current picture of animal MTOC/centrosome function and suggests how a calcium peak during the initial cytokinetic furrowing might be transmitted. We discuss this novel mechanistic model for calpain activity in the context of data from the animal and plant literature, as well as of our novel discovery here of calpain sequences in both brown and red algal genomes. Finally, we speculate that the ancestral role of calpains in early eukaryotes, before the split into the major eukaryotic supergroups, may have been to facilitate the formation and function of MT arrays in flagella and cilia. From this origin, calpains may have developed new functions in eukaryote cell division processes by anchoring centrosomes/MTOC to set the cell division orientations that are especially important for complex multicellularity.

During early development of the placenta, a subset of murine trophectoderm stem cells (TSCs) undergo endoreplication, an unusual form of cell division cycle that decouples DNA synthesis from cytokinesis, resulting in physiological polyploidy. Oscillations in CDK2 activity are essential for the orderly progression of the cell cycle to ensure replicated DNA is accurately partitioned into two daughter cells. However, it remains underexplored how the dynamics of CDK2 activity regulate endoreplication in the context of TSCs differentiation. To address this question, we leveraged the variability in cell fate decisions in an established in vitro system of TSCs differentiation that relies on removal of a growth factor, FGF4, to induce endoreplication. Using quantitative single-cell live confocal microscopy of a precise CDK2 biosensor, DHB-Venus, we identified at least three different outcomes upon FG4 removal: self-renewal, endoreplication, and migration. Our quantitative analyses showed high levels of Cdk2 activity in self-renewing cells whereas intermediate DHB-Venus turnover is linked to increased nuclear and cell size, indicating a shift to endoreplication. Importantly, we also characterize a third class of differentiating TSCs with migratory characteristics that correlate with low levels Cdk2 activity without a change in nuclear size. In sum, our results demonstrated a correlation between different fate outcomes and specific thresholds of CDK2 activity. Our findings show that TSCs can distinguish between different outcomes through modulating the central kinase of the cell cycle, CDK2, positioning it as a key regulator of early trophoblast differentiation. Summary StatementThis study investigates the oscillatory behavior of CDK2 activity during murine trophectoderm differentiation and its potential role in guiding cell fate decisions.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

Actin superfamily members are critical for the biology of eukaryotes and archaea. Actin-related proteins (Arps) are a subgroup within the actin superfamily and play essential roles in trafficking, replication and motility. The genome of the malaria parasite Plasmodium contains a set of Arps unique to apicomplexans, termed actin-like proteins (Alps). However, the importance and specific roles of many of these Alps in Plasmodium progression are not yet understood. Here, we determined the functional contribution of Plasmodium berghei Alp3 and Alp5a (recently relabelled as Arp3) by generation of knock-out (KO) lines and their subsequent characterisation across different life cycle stages. Deletion of either Alp did not affect blood stage growth, gametogenesis and ookinete gliding motility. However, deletion of Alp5a lead to smaller and fewer oocysts as well as severely impaired sporozoite formation. The Alp3KO line had highly reduced oocyst loads compared to wild-type parasites. This striking decrease was due to impaired ookinete penetration of the mosquito midgut epithelium. Our study shows that both Alp3 and Alp5a are indispensable for Plasmodium transmission at different steps of initial mosquito infection, provides insights into the role of specific unique members of the actin superfamily during parasite progression and the requirements for efficient midgut penetration.

The origin of eukaryotes is a key event in the evolution of cellular life hypothesized to involve a symbiotic integration between a member of the Asgard archaea and the Alphaproteobacteria. Recent work has provided evidence for additional genetic input from other prokaryotes to the eukaryotic proteome yet the extent and sources of these contributions remain debated. Here we aimed to further resolve the prokaryotic origins of eukaryotic genes to inform our understanding of eukaryogenesis. Specifically, we developed a phylogenetic framework to investigate the origins of eukaryotic gene families associated with metabolism and informational processing for comparison. We found that informational processing genes were predominantly derived by archaea whereas eukaryotic metabolism is highly chimeric in its origin. In contrast to previous studies, we report a substantial number of archaeal origins of diverse metabolic enzymes including key metabolic regulators. This highlights an overlooked participation of archaeal metabolism and pinpoints potential metabolic integrations during eukaryogenesis. Apart from the alphaproteobacterial contributions to the eukaryotic metabolism, we found an additional dominant phylogenetic signal of genes potentially derived from Myxococcota, especially for gene families associated with lipid metabolism. By systematically analysing the origins of eukaryotic metabolism, this research offers novel insights into the origin of eukaryotic membranes and refine our current models for the origin of the eukaryotic cell.