LINE-1: an emerging initiator of cGAS-STING signalling and inflammation that is dysregulated in disease.

The cGAS-STING (cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)) axis integrates DNA damage and cellular stress with type I interferon (IFN) signalling to facilitate transcriptional changes underlying inflammatory stress responses. The cGAS-STING pathway responds to cytosolic DNA in the form of double-stranded DNA, micronuclei, and long interspersed nuclear element 1 (L1) retroelements. L1 retroelements are a class of self-propagating non-long terminal repeat transposons that have remained highly active in mammalian genomes. L1 retroelements are emerging as important inducers of cGAS-STING and IFN signalling, which are often dysregulated in several diseases, including cancer. A key repressor of cGAS-STING and L1 activity is the exonuclease three prime repair exonuclease 1 (TREX1), and loss of TREX1 promotes the accumulation of L1. In addition, L1 dysregulation is a common theme among diseases with chronic induction of type I IFN signalling through cGAS-STING, such as Aicardi-Goutières syndrome, Fanconi anemia, and dermatomyositis. Although TREX1 is highly conserved in tetrapod species, other suppressor proteins exist that inhibit L1 retrotransposition. These suppressor genes when mutated are often associated with diseases characterized by unchecked inflammation that is associated with high cGAS-STING activity and elevated levels of L1 expression. In this review, we discuss these interconnected pathways of L1 suppression and their role in the regulation of cGAS-STING and inflammation in disease.

From biosynthesis and beyond-Loss or overexpression of the cytokinin synthesis gene, iptA, alters cytokinesis and mitochondrial and amino acid metabolism in Dictyostelium discoideum.

Cytokinins (CKs) are a class of growth-promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA-IPTs). In this study, the social amoeba, Dictyostelium discoideum, was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate-IPT gene, iptA. The life cycle of D. discoideum is unique and possesses both single- and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum. To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA-deficiency results in cytokinesis defects, and both iptA-deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development.

Suppressors of cGAS-STING are downregulated during fin-limb regeneration and aging in aquatic vertebrates.

During the early stages of limb and fin regeneration in aquatic vertebrates (i.e., fishes and amphibians), blastema undergo transcriptional rewiring of innate immune signaling pathways to promote immune cell recruitment. In mammals, a fundamental component of innate immune signaling is the cytosolic DNA sensing pathway, cGAS-STING. However, to what extent the cGAS-STING pathway influences regeneration in aquatic anamniotes is unknown. In jawed vertebrates, negative regulation of cGAS-STING activity is accomplished by suppressors of cytosolic DNA such as Trex1, Pml, and PML-like exon 9 (Plex9) exonucleases. Here, we examine the expression of these suppressors of cGAS-STING, as well as inflammatory genes and cGAS activity during caudal fin and limb regeneration using the spotted gar (Lepisosteus oculatus) and axolotl (Ambystoma mexicanum) model species, and during age-related senescence in zebrafish (Danio rerio). In the regenerative blastema of wounded gar and axolotl, we observe increased inflammatory gene expression, including interferon genes and interleukins 6 and 8. We also observed a decrease in axolotl Trex1 and gar pml expression during the early phases of wound healing which correlates with a dramatic increase in cGAS activity. In contrast, the plex9.1 gene does not change in expression during wound healing in gar. However, we observed decreased expression of plex9.1 in the senescing cardiac tissue of aged zebrafish, where 2'3'-cGAMP levels are elevated. Finally, we demonstrate a similar pattern of Trex1, pml, and plex9.1 gene regulation across species in response to exogenous 2'3'-cGAMP. Thus, during the early stages of limb-fin regeneration, Pml, Trex1, and Plex9.1 exonucleases are downregulated, presumably to allow an evolutionarily ancient cGAS-STING activity to promote inflammation and the recruitment of immune cells.

PML and PML-like exonucleases restrict retrotransposons in jawed vertebrates.

We have uncovered a role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the mammalian PML protein forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish function as cytoplasmic DEDDh exonucleases. In contrast, PML proteins from amniote species localized both to the cytoplasm and formed nuclear bodies. We also identified the PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases. Plex9 proteins resemble TREX1 but are unique from the TREX family and share homology to gar PML. We also characterized the molecular evolution of TREX1 and the first non-mammalian TREX1 homologs in axolotl. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus by enhancing ORF1p degradation through the ubiquitin-proteasome system. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its new role in the assembly of nuclear bodies.

A Dietary Antioxidant Formulation Ameliorates DNA Damage Caused by γ-Irradiation in Normal Human Bronchial Epithelial Cells In Vitro.

Antioxidants can be used as radioprotectants to reduce DNA damage due to exposure to radiation that could result in malignancies, including lung cancer. Mortality rates are consistently higher in lung cancer, which is usually diagnosed at later stages of cancer development and progression. In this preliminary study, we examined the potential of an antioxidant formulation (AOX2) to reduce DNA damage using a cell model of human normal bronchial epithelial cells (BEAS-2B). Cells were exposed to γ-irradiation or smoke-related hydrocarbon 4[(acetoxymethyl)nitrosamino]-1 (3-pyridyl) 1-butanone (NNKOAc) to induce DNA damage. We monitored intracellular reactive oxygen species (ROS) levels and evidence of genotoxic damage including DNA fragmentation ELISA, γ-H2AX immunofluorescence, and comet assays. Pre-incubation of the cells with AOX2 before exposure to γ-irradiation and NNKOAc significantly reduced DNA damage. The dietary antioxidant preparation AOX2 significantly reduced the induction of the tumor suppressor protein p53 and DNA damage-associated γ-H2AX phosphorylation by radiation and the NNKOAc treatment. Thus, AOX2 has the potential to act as a chemoprotectant by lowering ROS levels and DNA damage caused by exposure to radiation or chemical carcinogens.

The Cellular and Developmental Roles of Cullins, Neddylation, and the COP9 Signalosome in Dictyostelium discoideum.

Cullins (CULs) are a core component of cullin-RING E3 ubiquitin ligases (CRLs), which regulate the degradation, function, and subcellular trafficking of proteins. CULs are post-translationally regulated through neddylation, a process that conjugates the ubiquitin-like modifier protein neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target cullins, as well as non-cullin proteins. Counteracting neddylation is the deneddylase, COP9 signalosome (CSN), which removes NEDD8 from target proteins. Recent comparative genomics studies revealed that CRLs and the CSN are highly conserved in Amoebozoa. A well-studied representative of Amoebozoa, the social amoeba Dictyostelium discoideum, has been used for close to 100 years as a model organism for studying conserved cellular and developmental processes owing to its unique life cycle comprised of unicellular and multicellular phases. The organism is also recognized as an exceptional model system for studying cellular processes impacted by human diseases, including but not limited to, cancer and neurodegeneration. Recent work shows that the neddylation inhibitor, MLN4924 (Pevonedistat), inhibits growth and multicellular development in D. discoideum, which supports previous work that revealed the cullin interactome in D. discoideum and the roles of cullins and the CSN in regulating cellular and developmental processes during the D. discoideum life cycle. Here, we review the roles of cullins, neddylation, and the CSN in D. discoideum to guide future work on using this biomedical model system to further explore the evolutionarily conserved functions of cullins and neddylation.

Tinker, Tailor, Tumour Suppressor: The Many Functions of PRP4K.

Pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is an essential kinase first identified in the fission yeast Schizosaccharomyces pombe that is evolutionarily conserved from amoebae to animals. During spliceosomal assembly, PRP4K interacts with and phosphorylates PRPF6 and PRPF31 to facilitate the formation of the spliceosome B complex. However, over the past decade additional evidence has emerged that PRP4K has many diverse cellular roles beyond splicing that contribute to tumour suppression and chemotherapeutic responses in mammals. For example, PRP4K appears to play roles in regulating transcription and the spindle assembly checkpoint (SAC), a key pathway in maintaining chromosomes stability and the response of cancer cells to taxane-based chemotherapy. In addition, PRP4K has been revealed to be a haploinsufficient tumour suppressor that promotes aggressive cancer phenotypes when partially depleted. PRP4K is regulated by both the HER2 and estrogen receptor, and its partial loss increases resistance to the taxanes in multiple malignancies including cervical, breast and ovarian cancer. Moreover, ovarian and triple negative breast cancer patients harboring tumours with low PRP4K expression exhibit worse overall survival. The depletion of PRP4K also enhances both Yap and epidermal growth factor receptor (EGFR) signaling, the latter promoting anoikis resistance in breast and ovarian cancer. Finally, PRP4K is negatively regulated during epithelial-to-mesenchymal transition (EMT), a process that promotes increased cell motility, drug resistance and cancer metastasis. Thus, as we discuss in this review, PRP4K likely plays evolutionarily conserved roles not only in splicing but in a number of cellular pathways that together contribute to tumour suppression.

Running 'LAPS' Around nLD: Nuclear Lipid Droplet Form and Function.

The nucleus harbours numerous protein subdomains and condensates that regulate chromatin organization, gene expression and genomic stress. A novel nuclear subdomain that is formed following exposure of cells to excess fatty acids is the nuclear lipid droplet (nLD), which is composed of a neutral lipid core surrounded by a phospholipid monolayer and associated regulatory and lipid biosynthetic enzymes. While structurally resembling cytoplasmic LDs, nLDs are formed by distinct but poorly understood mechanisms that involve the emergence of lipid droplets from the lumen of the nucleoplasmic reticulum and de novo lipid synthesis. Luminal lipid droplets that emerge into the nucleoplasm do so at regions of the inner nuclear membrane that become enriched in promyelocytic leukemia (PML) protein. The resulting nLDs that retain PML on their surface are termed lipid-associated PML structures (LAPS), and are distinct from canonical PML nuclear bodies (NB) as they lack key proteins and modifications associated with these NBs. PML is a key regulator of nuclear signaling events and PML NBs are sites of gene regulation and post-translational modification of transcription factors. Therefore, the subfraction of nLDs that form LAPS could regulate lipid stress responses through their recruitment and retention of the PML protein. Both nLDs and LAPS have lipid biosynthetic enzymes on their surface suggesting they are active sites for nuclear phospholipid and triacylglycerol synthesis as well as global lipid regulation. In this review we have summarized the current understanding of nLD and LAPS biogenesis in different cell types, their structure and composition relative to other PML-associated cellular structures, and their role in coordinating a nuclear response to cellular overload of fatty acids.

SARS-CoV-2 and wastewater: What does it mean for non-human primates?

In most of our lifetimes, we have not faced a global pandemic such as the novel coronavirus disease 2019. The world has changed as a result. However, it is not only humans who are affected by a pandemic of this scale. Our closest relatives, the non-human primates (NHPs) who encounter researchers, sanctuary/zoo employees, and tourists, are also potentially at risk of contracting the virus from humans due to similar genetic susceptibility. "Anthropozoonosis"-the transmission of diseases from humans to other species-has occurred historically, resulting in infection of NHPs with human pathogens that have led to disastrous outbreaks. Recent studies have assessed the susceptibility of NHPs and predict that catarrhine primates and some lemurs are potentially highly susceptible to infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. There is accumulating evidence that a new factor to consider with the spread of the virus is fecal-oral transmission. The virus has been detected in the watersheds of countries with underdeveloped infrastructure where raw sewage enters the environment directly without processing. This may expose NHPs, and other animals, to SARS-CoV-2 through wastewater contact. Here, we address these concerns and discuss recent evidence. Overall, we suggest that the risk of transmission of SARS-CoV-2 via wastewater is low. Nonetheless, tracking of viral RNA in wastewater does provide a unique testing approach to help protect NHPs at zoos and wildlife sanctuaries. A One Health approach going forward is perhaps the best way to protect these animals from a novel virus, the same way that we would protect ourselves.

Haploinsufficient tumor suppressor PRP4K is negatively regulated during epithelial-to-mesenchymal transition.

The pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is an essential gene. However, reduced PRP4K expression is associated with aggressive breast and ovarian cancer phenotypes including taxane therapy resistance, increased cell migration and invasion in vitro, and cancer metastasis in mice. These results are consistent with PRP4K being a haploinsufficient tumor suppressor. Increased cell migration and invasion is associated with epithelial-to-mesenchymal transition (EMT), but how reduced PRP4K levels affect normal epithelial cell migration or EMT has not been studied. Depletion of PRP4K by small hairpin RNA (shRNA) in non-transformed mammary epithelial cell lines (MCF10A, HMLE) reduced or had no effect on 2D migration in the scratch assay but resulted in greater invasive potential in 3D transwell assays. Depletion of PRP4K in mesenchymal triple-negative breast cancer cells (MDA-MB-231) resulted in both enhanced 2D migration and 3D invasion, with 3D invasion correlated with higher fibronectin levels in both MDA-MB-231 and MCF10A cells and without changes in E-cadherin. Induction of EMT in MCF10A cells, by treatment with WNT-5a and TGF-ß1, or depletion of eukaryotic translation initiation factor 3e (eIF3e) by shRNA, resulted in significantly reduced PRP4K expression. Mechanistically, induction of EMT by WNT-5a/TGF-ß1 reduced PRP4K transcript levels, whereas eIF3e depletion led to reduced PRP4K translation. Finally, reduced PRP4K levels after eIF3e depletion correlated with increased YAP activity and nuclear localization, both of which are reversed by overexpression of exogenous PRP4K. Thus, PRP4K is a haploinsufficient tumor suppressor negatively regulated by EMT, that when depleted in normal mammary cells can increase cell invasion without inducing full EMT.

Adding Some "Splice" to Stress Eating: Autophagy, ESCRT and Alternative Splicing Orchestrate the Cellular Stress Response.

Autophagy is a widely studied self-renewal pathway that is essential for degrading damaged cellular organelles or recycling biomolecules to maintain cellular homeostasis, particularly under cellular stress. This pathway initiates with formation of an autophagosome, which is a double-membrane structure that envelopes cytosolic components and fuses with a lysosome to facilitate degradation of the contents. The endosomal sorting complexes required for transport (ESCRT) proteins play an integral role in controlling autophagosome fusion events and disruption to this machinery leads to autophagosome accumulation. Given the central role of autophagy in maintaining cellular health, it is unsurprising that dysfunction of this process is associated with many human maladies including cancer and neurodegenerative diseases. The cell can also rapidly respond to cellular stress through alternative pre-mRNA splicing that enables adaptive changes to the cell's proteome in response to stress. Thus, alternative pre-mRNA splicing of genes that are involved in autophagy adds another layer of complexity to the cell's stress response. Consequently, the dysregulation of alternative splicing of genes associated with autophagy and ESCRT may also precipitate disease states by either reducing the ability of the cell to respond to stress or triggering a maladaptive response that is pathogenic. In this review, we summarize the diverse roles of the ESCRT machinery and alternative splicing in regulating autophagy and how their dysfunction can have implications for human disease.

Aberrant Autophagy Impacts Growth and Multicellular Development in a Dictyostelium Knockout Model of CLN5 Disease.

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. While the precise role of CLN5 in NCL pathogenesis is not known, recent work revealed that the protein has glycoside hydrolase activity. Previous work on the Dictyostelium discoideum homolog of human CLN5, Cln5, revealed its secretion during the early stages of development and its role in regulating cell adhesion and cAMP-mediated chemotaxis. Here, we used Dictyostelium to examine the effect of cln5-deficiency on various growth and developmental processes during the life cycle. During growth, cln5 - cells displayed reduced cell proliferation, cytokinesis, viability, and folic acid-mediated chemotaxis. In addition, the growth of cln5 - cells was severely impaired in nutrient-limiting media. Based on these findings, we assessed autophagic flux in growth-phase cells and observed that loss of cln5 increased the number of autophagosomes suggesting that the basal level of autophagy was increased in cln5 - cells. Similarly, loss of cln5 increased the amounts of ubiquitin-positive proteins. During the early stages of multicellular development, the aggregation of cln5 - cells was delayed and loss of the autophagy genes, atg1 and atg9, reduced the extracellular amount of Cln5. We also observed an increased amount of intracellular Cln5 in cells lacking the Dictyostelium homolog of the human glycoside hydrolase, hexosaminidase A (HEXA), further supporting the glycoside hydrolase activity of Cln5. This observation was also supported by our finding that CLN5 and HEXA expression are highly correlated in human tissues. Following mound formation, cln5 - development was precocious and loss of cln5 affected spore morphology, germination, and viability. When cln5 - cells were developed in the presence of the autophagy inhibitor ammonium chloride, the formation of multicellular structures was impaired, and the size of cln5 - slugs was reduced relative to WT slugs. These results, coupled with the aberrant autophagic flux observed in cln5 - cells during growth, support a role for Cln5 in autophagy during the Dictyostelium life cycle. In total, this study highlights the multifaceted role of Cln5 in Dictyostelium and provides insight into the pathological mechanisms that may underlie CLN5 disease.

Inhibiting Neddylation with MLN4924 Suppresses Growth and Delays Multicellular Development in Dictyostelium discoideum.

Neddylation is a post-translational modification that is essential for a variety of cellular processes and is linked to many human diseases including cancer, neurodegeneration, and autoimmune disorders. Neddylation involves the conjugation of the ubiquitin-like modifier neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target proteins, and has been studied extensively in various eukaryotes including fungi, plants, and metazoans. Here, we examine the biological processes influenced by neddylation in the social amoeba, Dictyostelium discoideum, using a well-established inhibitor of neddylation, MLN4924 (pevonedistat). NEDD8, and the target of MLN4924 inhibition, NEDD8-activating enzyme E1 (NAE1), are highly conserved in D. discoideum (Nedd8 and Nae1, respectively). Treatment of D. discoideum cells with MLN4924 increased the amount of free Nedd8, suggesting that MLN4924 inhibited neddylation. During growth, MLN4924 suppressed cell proliferation and folic acid-mediated chemotaxis. During multicellular development, MLN4924 inhibited cyclic adenosine monophosphate (cAMP)-mediated chemotaxis, delayed aggregation, and suppressed fruiting body formation. Together, these findings indicate that neddylation plays an important role in regulating cellular and developmental events during the D. discoideum life cycle and that this organism can be used as a model system to better understand the essential roles of neddylation in eukaryotes, and consequently, its involvement in human disease.

The converging roles of Batten disease proteins in neurodegeneration and cancer.

Epidemiological studies have reported an inverse correlation between cancer and neurodegenerative disorders, and increasing evidence shows that similar genes and pathways are dysregulated in both diseases but in a contrasting manner. Given the genetic convergence of the neuronal ceroid lipofuscinoses (NCLs), a family of rare neurodegenerative disorders commonly known as Batten disease, and other neurodegenerative diseases, we sought to explore the relationship between cancer and the NCLs. In this review, we survey data from The Cancer Genome Atlas and available literature on the roles of NCL genes in different oncogenic processes to reveal links between all the NCL genes and cancer-related processes. We also discuss the potential contributions of NCL genes to cancer immunology. Based on our findings, we propose that further research on the relationship between cancer and the NCLs may help shed light on the roles of NCL genes in both diseases and possibly guide therapy development.

Cancer and the breakdown of multicellularity: What Dictyostelium discoideum, a social amoeba, can teach us.

Ancient pathways promoting unicellularity and multicellularity are associated with cancer, the former being pro-oncogenic and the latter acting to suppress oncogenesis. However, there are only a limited number of non-vertebrate models for studying these pathways. Here, we review Dictyostelium discoideum and describe how it can be used to understand these gene networks. D. discoideum has a unicellular and multicellular life cycle, making it possible to study orthologs of cancer-associated genes in both phases. During development, differentiated amoebae form a fruiting body composed of a mass of spores that are supported atop a stalk. A portion of the cells sacrifice themselves to become non-reproductive stalk cells. Cheating disrupts the principles of multicellularity, as cheater cells alter their cell fate to preferentially become spores. Importantly, D. discoideum has gene networks and several strategies for maintaining multicellularity. Therefore, D. discoideum can help us better understand how conserved genes and pathways involved in multicellularity also influence cancer development, potentially identifying new therapeutic avenues.

Pandemic danger to the deep: The risk of marine mammals contracting SARS-CoV-2 from wastewater.

We are in unprecedented times with the ongoing COVID-19 pandemic. The pandemic has impacted public health, the economy and our society on a global scale. In addition, the impacts of COVID-19 permeate into our environment and wildlife as well. Here, we discuss the essential role of wastewater treatment and management during these times. A consequence of poor wastewater management is the discharge of untreated wastewater carrying infectious SARS-CoV-2 into natural water systems that are home to marine mammals. Here, we predict the susceptibility of marine mammal species using a modelling approach. We identified that many species of whale, dolphin and seal, as well as otters, are predicted to be highly susceptible to infection by the SARS-CoV-2 virus. In addition, geo-mapping highlights how current wastewater management in Alaska may lead to susceptible marine mammal populations being exposed to the virus. Localities such as Cold Bay, Naknek, Dillingham and Palmer may require additional treatment of their wastewater to prevent virus spillover through sewage. Since over half of these susceptibility species are already at risk worldwide, the release of the virus via untreated wastewater could have devastating consequences for their already declining populations. For these reasons, we discuss approaches that can be taken by the public, policymakers and wastewater treatment facilities to reduce the risk of virus spillover in our natural water systems. Thus, we indicate the potential for reverse zoonotic transmission of COVID-19 and its impact on marine wildlife; impacts that can be mitigated with appropriate action to prevent further damage to these vulnerable populations.

Lions, tigers and kittens too: ACE2 and susceptibility to COVID-19.

SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2) has been reported to infect domesticated animals in a species-specific manner, where cats were susceptible but not dogs. Using the recently published crystal structure of the SARS-CoV-2 spike protein complexed with the human host cell receptor angiotensin converting enzyme 2 (ACE2), we characterized the structure and evolution of ACE2 in several of these species and identify a single interacting amino acid residue conserved between human and Felidae ACE2 but not in Canidae that correlates with virus susceptibility. Using computational analyses we describe how this site likely affects ACE2 targeting by the virus. Thus, we highlight how evolution-based approaches can be used to form hypotheses and study animal transmission of such viruses in the future.

Mfsd8 localizes to endocytic compartments and influences the secretion of Cln5 and cathepsin D in Dictyostelium.

The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases that affect people of all ages and ethnicities, yet many of the associated genes/proteins are not well characterized. Mutations in MFSD8 (major facilitator superfamily domain-containing 8) cause an infantile form of NCL referred to as CLN7 disease. In this study, we revealed the localization and binding partners of an ortholog of human MFSD8 (Mfsd8) in the social amoeba Dictyostelium discoideum. Putative lysosomal targeting motifs are conserved in Dictyostelium Mfsd8, as are several residues mutated in CLN7 disease patients. Mfsd8 tagged with GFP localizes to endocytic compartments, which includes acidic intracellular vesicles and late endosomes. We pulled-down GFP-Mfsd8 and used mass spectrometry to reveal the Mfsd8 interactome during Dictyostelium growth and starvation. Among the identified hits were the Dictyostelium ortholog of human cathepsin D (CtsD), as well as proteins linked to the functions of the CLN3 (Cln3) and CLN5 (Cln5) orthologs in Dictyostelium. To study the function of Mfsd8, we validated a publically available mfsd8- cell line (GWDI Project) and then used this knockout cell line to show that Mfsd8 influences the secretion of Cln5 and CtsD. This information is then integrated into an emerging model describing the molecular networking of NCL proteins in Dictyostelium. In total, this study identifies Dictyostelium as a new model system for studying CLN7 disease.

Calmodulin-mediated events during the life cycle of the amoebozoan Dictyostelium discoideum.

This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin-binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin-dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin-dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin-dependent transdifferentiation to re-establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin-dependent germination of spores. Specific calmodulin-binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.

PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination.

Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.