Circadian rhythm shows potential for mRNA efficiency and self-organized division of labor in multinucleate cells

Multinucleate cells occur in every biosphere and across the kingdoms of life, including in the human body as muscle cells and bone-forming cells. Data from filamentous fungi suggest that, even when bathed in a common cytoplasm, nuclei are capable of autonomous behaviors, including division. How does this potential for autonomy affect the organization of cellular processes between nuclei? Here we analyze a simplified model of circadian rhythm, a form of cellular oscillator, in a mathematical model of the filamentous fungus Neurospora crassa. Our results highlight a potential role played by mRNA-protein phase separation to keep mRNAs close to the nuclei from which they originate, while allowing proteins to diffuse freely between nuclei. Our modeling shows that syncytism allows for extreme mRNA efficiency—we demonstrate assembly of a robust oscillator with a transcription rate a thousand-fold less than in comparable uninucleate cells. We also show self-organized division of the labor of mRNA production, with one nucleus in a two-nucleus syncytium producing at least twice as many mRNAs as the other in 30% of cycles. This division can occur spontaneously, but division of labor can also be controlled by regulating the amount of cytoplasmic volume available to each nucleus. Taken together, our results show the intriguing richness and potential for emergent organization among nuclei in multinucleate cells. They also highlight the role of previously studied mechanisms of cellular organization, including nuclear space control and localization of mRNAs through RNA-protein phase separation, in regulating nuclear coordination.

Multinucleate parietal cells and cytoplasmic inclusion bodies in the gastric epithelium of callitrichids

Inclusion bodies (IBs) and multinucleate cells can be associated with viral infections; however, IBs and multinucleate cells have been described in normal tissue and with non-viral disease processes in multiple species. We examined fundic stomach from 50 callitrichids histologically for bi- and multinucleate parietal cells and cytoplasmic IBs in gastric epithelial cells. Callitrichids represented included 6 genera: Saguinus (4 spp.), Leontopithecus (1 sp.), Mico (3 spp.), Cebuella (1 sp.), Callithrix (1 sp.), Callimico (1 sp.), and 13 unspecified marmosets. Gastric epithelial IBs were present in 46 of 47 (98%) of the callitrichids from which the stomach was sufficiently well preserved to identify IBs. Cytoplasmic IBs were identified in gastric surface pit epithelial cells (43 of 44, 98%), mucous neck cells (43 of 44, 98%), parietal cells (43 of 44, 98%), and chief cells (43 of 44, 98%). The IBs were eosinophilic, ovoid, round, elongate, or variably indented, sometimes slightly refractile, and 1–6 × 1–13 µm. IBs were sometimes perinuclear and molded around the nucleus. Electron microscopy of the gastric epithelium of one marmoset indicated that IBs were composed of intermediate filaments. The IBs did not stain with immunohistochemical markers for cytokeratin AE1/AE3 or vimentin. Binucleate parietal cells were found in 49 of 50 (98%) callitrichids, and multinucleate parietal cells were observed in 40 of 49 (82%) callitrichids. Gastric epithelial cytoplasmic IBs and bi- and multinucleate parietal cells are likely a normal finding in callitrichids, and, to our knowledge, have not been reported previously.

Drosophila larval epidermal cells only exhibit epidermal aging when they persist to the adult stage

Holometabolous insects undergo a complete transformation of the body plan from the larval to the adult stage. In Drosophila, this transformation includes replacement of larval epidermal cells (LECs) by adult epidermal cells (AECs). AECs in Drosophila undergo a rapid and stereotyped aging program where they lose both cell membranes and nuclei. Whether LECs are capable of undergoing aging in a manner similar to AECs remains unknown. Here, we address this question in two ways. First, we looked for hallmarks of epidermal aging in larvae that have a greatly extended third instar and/or carry mutations that would cause premature epidermal aging at the adult stage. Such larvae, irrespective of genotype, did not show any of the signs of epidermal aging observed in the adult. Second, we developed a procedure to effect a heterochronic persistence of LECs into the adult epidermal sheet. Lineage tracing verified that presumptive LECs in the adult epidermis are not derived from imaginal epidermal histoblasts. LECs embedded within the adult epidermal sheet undergo clear signs of epidermal aging; they form multinucleate cells with each other and with the surrounding AECs. The incidence of adult cells with mixed AEC nuclei (small) and persistent LEC nuclei (large) increased with age. Our data reveals that epidermal aging in holometabolous Drosophila is a stage-specific phenomenon and that the capacity of LECs to respond to aging signals does exist.

Circadian rhythm shows potential for mRNA efficiency and self-organized division of labor in multinucleate cells

Multinucleate cells occur in every biosphere and across the kingdoms of life, including in the human body as muscle cells and bone-forming cells. Data from filamentous fungi suggest that, even when bathed in a common cytoplasm, nuclei are capable of autonomous behaviors, including division. How does this potential for autonomy affect the organization of cellular processes between nuclei? Here we analyze a simplified model of circadian rhythm, a form of cellular oscillator, in a mathematical model of the filamentous fungus Neurospora crassa. Our results highlight the role played by mRNA-protein phase separation to keep mRNAs close to the nuclei from which they originate, while allowing proteins to diffuse freely between nuclei. Our modeling shows that syncytism allows for extreme mRNA efficiency — we demonstrate assembly of a robust oscillator with transcription levels 104-fold less than in comparable uninucleate cells. We also show self-organized division of the labor of mRNA production, with one nucleus in a two-nucleus syncytium producing at least twice as many mRNAs as the other in 30% of cycles. This division can occur spontaneously, but division of labor can also be controlled by regulating the amount of cytoplasmic volume available to each nucleus. Taken together, our results show the intriguing richness and potential for emergent organization among nuclei in multinucleate cells. They also highlight the role of previously studied mechanisms of cellular organization, including nuclear space control and localization of mRNAs through RNA-protein phase separation, in regulating nuclear coordination.Author summaryCircadian rhythms are among the most researched cellular processes, but limited work has been done on how these rhythms are coordinated between nuclei in multinucleate cells. In this work, we analyze a mathematical model for circadian oscillations in a multinucleate cell, motivated by frequency mRNA and protein data from the filamentous fungus Neurospora crassa. Our results illuminate the importance of mRNA-protein phase separation, in which mRNAs are kept close to the nucleus in which they were transcribed, while proteins can diffuse freely across the cell. We demonstrate that this phase separation allows for a robust oscillator to be assembled with very low mRNA counts. We also investigate how the labor of transcribing mRNAs is divided between nuclei, both when nuclei are evenly spaced across the cell and when they are not. Division of this labor can be regulated by controlling the amount of cytoplasmic volume available to each nucleus. Our results show that there is potential for emergent organization and extreme mRNA efficiency in multinucleate cells.

Unilateral Cleavage Furrows in Multinucleate Cells

Multinucleate cells can be produced in Dictyostelium by electric pulse-induced fusion. In these cells, unilateral cleavage furrows are formed at spaces between areas that are controlled by aster microtubules. A peculiarity of unilateral cleavage furrows is their propensity to join laterally with other furrows into rings to form constrictions. This means cytokinesis is biphasic in multinucleate cells, the final abscission of daughter cells being independent of the initial direction of furrow progression. Myosin-II and the actin filament cross-linking protein cortexillin accumulate in unilateral furrows, as they do in the normal cleavage furrows of mononucleate cells. In a myosin-II-null background, multinucleate or mononucleate cells were produced by cultivation either in suspension or on an adhesive substrate. Myosin-II is not essential for cytokinesis either in mononucleate or in multinucleate cells but stabilizes and confines the position of the cleavage furrows. In fused wild-type cells, unilateral furrows ingress with an average velocity of 1.7 µm × min−1, with no appreciable decrease of velocity in the course of ingression. In multinucleate myosin-II-null cells, some of the furrows stop growing, thus leaving space for the extensive broadening of the few remaining furrows.

Unilateral Cleavage Furrows in Multinucleate Cells

Multinucleate cells can be produced in Dictyostelium by electric-pulse induced fusion. In these cells unilateral cleavage furrows are formed at spaces between areas that are controlled by aster microtubules. A peculiarity of unilateral cleavage furrows is their propensity to join laterally with other furrows into rings to form constrictions. This means, cytokinesis is biphasic in multinucleate cells, the final abscission of daughter cells being independent of the initial direction of furrow progression. Myosin-II and the actin-filament cross-linking protein cortexillin accumulate in the unilateral furrows, as they do in the normal cleavage furrows of mononucleate cells. Myosin-II is not essential for cytokinesis, but stabilizes and confines the position of the cleavage furrows.

Drosophila larval epidermal cells only exhibit epidermal aging when they persist to the adult stage

Holometabolous insects undergo a complete transformation of the body plan from the larval to the adult stage. In Drosophila, this transformation includes replacement of larval epidermal cells (LECs) by adult epidermal cells (AECs). AECs in Drosophila undergo a rapid and stereotyped aging program where they lose both cell membranes and nuclei. Whether LEC’s are capable of undergoing aging in a manner similar to AECs remains unknown. Here, we address this question in two ways. First, we looked for hallmarks of epidermal aging in larvae that have a greatly extended third instar and/or carry mutations that would cause premature epidermal aging at the adult stage. Such larvae, irrespective of genotype, did not show any of the signs of epidermal aging observed in the adult. Second, we developed a procedure to effect a heterochronic persistence of LECs into the adult epidermal sheet. LECs embedded within the adult epidermal sheet undergo clear signs of epidermal aging; they form multinucleate cells with each other and with the surrounding AECs on the same schedule as the AECs themselves. Our data reveals that epidermal aging in holometabolous Drosophila is a stage-specific phenomenon and that the capacity of LECs to respond to aging signals does exist.Summary StatementWe show that Drosophila larval epidermal cells do not age at the larval stage. They do, however, exhibit signs of aging if they persist into the adult.

Codanin-1 Mutations Engineered in Human Erythroid Cells Allow Investigation of Mechanisms Underlying Congenital Dyserythropoietic Anemia

The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during a time of hemoglobin accumulation, rapid cell division, and nuclear condensation. Disruption of this process is associated with myelodysplastic syndromes and congenital anemias. Congenital Dyserythropoietic Anemia type I (CDA-I), is an autosomal recessive disease that presents with severe macrocytic anemia early in infancy or early childhood. Patients with CDA-I have erythroid hyperplasia in the bone marrow. The erythroblasts in CDA-I are frequently binucleate, have chromatin bridging, and defective chromatin condensation (Renella 2011). CDA-I is most commonly caused by mutations in the protein codanin 1 (CDAN1). The function of CDAN1 is poorly understood, but it is thought to regulate histone incorporation into nascent DNA during cellular replication (Ask 2012). The study of CDA-1 has been limited by lack of in vitro models that recapitulate key features of the disease, and to date, the majority of studies on CDAN1 function have been done in non-erythroid cells. To model CDA-I we introduced a point mutation (PM) commonly observed in CDA-1 patients (R1042W) into HUDEP2 cells. In addition, we generated two HUDEP2 cell lines with heterozygous deletion of CDAN1 (del-R1042). All CDAN1 mutant cell lines had decreased viability in during both expansion and terminal maturation compared to control lines. Chromatin bridges and multinucleate cells were observed in all three mutant CDAN1 lines but were most prominent in the PM line. Intriguingly, global gene analysis demonstrated that all mutated lines had significantly elevated gamma-globin expression compared to controls, consistent with reports of elevated fetal globin expression in CDA-1 patients. Interestingly, the PM cell line had faster cell divisions than the del-R1042 or control lines, characterized by decreased doubling time and verified by quantifying dilution of a fluorescent dye. In contrast, the del-R1042 lines had slower cell doubling times than both the controls and the PM line. The PM line also had an increased median intensity of BrdU compared to controls and del-R1042 lines, suggesting an accelerated S-phase. KI67 staining of the PM line showed an increase percentage of mitotic cells. These data are consistent with the finding that multinucleate cells and chromatin bridges are more common in the PM line. Furthermore, electron microscopy suggests the PM cell line may have defects in heterochromatin formation. Together, these data imply a specific functional role for residue R1042, and suggest that within the context of R1042W, loss of the arginine residue and replacement thereof with a tryptophan may have different mechanistic consequences. Collectively, our preliminary data suggests that CDAN1 is important in the regulation of DNA replication and organization in maturing erythroblasts. Specific mutations may confer a change of function which results in dysplastic erythroid cells that are sensitive to dysregulated cell cycle mechanics due to the high rate of cell division. We hypothesize R1042W substitution may accelerate cell division at the expense of appropriate checkpoints and result in dysplastic erythroid cells and mimic the clinical presentation of increased multinuclearity, decreased cell viability, and increased gamma globin expression. Most importantly, generation of models with specific patient mutations will provide further mechanistic insight into CDA-I pathology. Disclosures No relevant conflicts of interest to declare.