Spatial transcriptomic and single-nucleus analysis reveals heterogeneity in a gigantic single-celled syncytium

Abstract

In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleate syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters and if nuclei within the cell diversify into heterogeneous states. Here, we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleus cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life.

A special envelope separates extra-chromosomal from mammalian chromosomal DNA in the cytoplasm

“…Remarkably, syncytia, which are cells with multiple stable nuclei, exist. All these nuclei are roughly of similar size as seen in the slime mold Physarum polycephalum and human osteoclasts (Gerber et al, 2022; Kopesky et al, 2014). In contrast micronuclei in mammalian cells are at least 5 times smaller than their corresponding nuclei (Kneissig et al, 2019).…”

Molecular compartmentalization in a syncytium: restricted mobility of proteins within the sea urchin skeletogenic mesenchyme

“…Our findings are relevant to the functional and molecular compartmentalization of other syncytia. Heterogeneous patterns of gene expression have been documented in several other syncytia, including vertebrate muscle (Bursztajn et al, 1989) and syncytiotrophoblast (Fogarty et al, 2011), slime mold pseudoplasmodia (Gerber et al, 2022), and multinucleate fungi (Dundon et al, 2016), although the molecular mechanisms that establish and maintain these domains are not well understood. In the best-studied case, the early, syncytial development of Drosophila embryos, anterior-posterior compartments of gene expression are generated through the limited diffusibility of several transcription factors, including Bicoid (Huang and Saunders, 2020).…”

“…Syncytia can also arise under pathogenic conditions, when host cell fusion is triggered by viral and bacterial pathogens (Leroy et al, 2020; Lin et al, 2021; Bzdyl et al, 2022). In some well-studied cases, biochemical and cellular activities such as gene expression programs and patterns of nuclear division, are regionalized with syncytia (Bursztajn et al, 1989; Fogarty et al, 2011; Roberts and Gladfelter, 2013; Dundon et al, 2016; Gerber et al, 2022). Such studies reveal that distinct molecular and functional compartments can exist within a single, large, multinucleated cell.…”

Microtubules in Microorganisms: How Tubulin Isotypes Contribute to Diverse Cytoskeletal Functions

“…Expression of the α-tubulin isotypes is differentially regulated in various life stages of Physarum . Despite having only 1 MT organelle, the anastral mitotic spindle, in the coenocytic plasmodia stage, more tubulin isotypes were found to be definitively expressed in this stage [ ALTA , ALTB(N) , ALTB(E) , BETB , BETC ] than the amoeba-like myxamoeba stage ( ALTA , BETA , BETB ) ( Burland et al, 1983 ; Roobol et al, 1984 ; Gerber et al, 2022 ). Although the α-tubulin population contains two isoelectric variants, one was found to originate via acetylation of AltA ( Green and Dove, 1984 ; Blindt et al, 1986 ; Diggins and Dove, 1987 ; Sasse et al, 1987 ), leaving only one major α-isotype expressed in the myxamoeba phase ( Singhofer-Wowra and Little, 1987 ).…”

“…Of the three β-tubulin isotypes, BETA and BETC are exclusively expressed and incorporated into MT structures of the myxamoeba and plasmodia stages, respectively ( Burland et al, 1988 ; Diggins-Gilicinski et al, 1989 ; Gerber et al, 2022 ). On the other hand, BETB is constitutively expressed during both the myxamoeba and plasmodia phases ( Werenskiold et al, 1988 ) and incorporated into all MT structures ( Paul et al, 1989 ).…”