In the event of a large-scale incident involving radiological or nuclear exposures, there is a potential for large numbers of individuals to have received doses of radiation sufficient to cause adverse health effects. It is imperative to quickly identify these individuals in order to provide information to the medical community to assist in making decisions about their treatment. The cytokinesis-block micronucleus assay is a well-established method for performing biodosimetry. This assay has previously been adapted to imaging flow cytometry and has been validated as a high-throughput option for providing dose estimates in the range of 0-10 Gy. The goal of this study was to test the ability to further optimize the assay by reducing the time of culture to 48 h from 68 h as well as reducing the volume of blood required for the analysis to 200 μL from 2 mL. These modifications would provide efficiencies in time and ease of processing impacting the ability to manage large numbers of samples and provide dose estimates in a timely manner. Results demonstrated that either the blood volume or the culture time could be reduced while maintaining dose estimates with sufficient accuracy for triage analysis. Reducing both the blood volume and culture time, however, resulted in poor dose estimates. In conclusion, depending on the needs of the scenario, either culture time or the blood volume could be reduced to improve the efficiency of analysis for mass casualty scenarios.

The endosomal sorting complex required for transport (ESCRT) machinery is composed of an articulated architecture of proteins that assemble at multiple cellular sites. The ESCRT machinery is involved in pathways that are pivotal for the physiology of the cell, including vesicle transport, cell division, and membrane repair. The subunits of the ESCRT I complex are mainly responsible for anchoring the machinery to the action site. The ESCRT II subunits function to bridge and recruit the ESCRT III subunits. The latter are responsible for finalizing operations that, independently of the action site, involve the repair and fusion of membrane edges. In this review, we report on the data related to the activity of the ESCRT machinery at two sites: the nuclear membrane and the midbody and the bridge linking cells in the final stages of cytokinesis. In these contexts, the machinery plays a significant role for the protection of genome integrity by contributing to the control of the abscission checkpoint and to nuclear envelope reorganization and correlated resilience. Consistently, several studies show how the dysfunction of the ESCRT machinery causes genome damage and is a codriver of pathologies, such as laminopathies and cancer.

An Arabidopsis sterol mutant, smt2 smt3, defective in sterolmethyltransferase2 (SMT2), exhibits severe growth abnormalities. The loss of C-24 ethyl sterols, maintaining the biosynthesis of C-24 methyl sterols and brassinosteroids, suggests specific roles of C-24 ethyl sterols. We characterized the subcellular localizations of fluorescent protein-fused sterol biosynthetic enzymes, such as SMT2-GFP, and found these enzymes in the endoplasmic reticulum during interphase and identified their movement to the division plane during cytokinesis. The mobilization of endoplasmic reticulum-localized SMT2-GFP was independent of the polarized transport of cytokinetic vesicles to the division plane. In smt2 smt3, SMT2-GFP moved to the abnormal division plane, and unclear cell plate ends were surrounded by hazy structures from SMT2-GFP fluorescent signals and unincorporated cellulose debris. Unusual cortical microtubule organization and impaired cytoskeletal function accompanied the failure to determine the cortical division site and division plane formation. These results indicated that both endoplasmic reticulum membrane remodeling and cytokinetic vesicle transport during cytokinesis were impaired, resulting in the defects of cell wall generation. The cell wall integrity was compromised in the daughter cells, preventing the correct determination of the subsequent cell division site. We discuss the possible roles of C-24 ethyl sterols in the interaction between the cytoskeletal network and the plasma membrane.

Cytokinesis, the last step in cell division, separates daughter cells through mechanical force. This is often through the force produced by an actomyosin contractile ring. In fission yeast cells, the ring helps recruit a mechanosensitive ion channel, Pkd2, to the cleavage furrow, whose activation by membrane tension promotes calcium influx and daughter cell separation. However, it is unclear how the activities of Pkd2 may affect the actomyosin ring. Here, through both microscopic and genetic analyses of a hypomorphic pkd2 mutant, we examined the potential role of this essential gene in assembling the contractile ring. The pkd2-81KD mutation significantly increased the counts of the type II myosin heavy chain Myo2 (+18%), its regulatory light chain Rlc1 (+37%) and actin (+100%) molecules in the ring, compared to the wild type. Consistent with a regulatory role of Pkd2 in the ring assembly, we identified a strong negative genetic interaction between pkd2-81KD and the temperature-sensitive mutant myo2-E1. The pkd2-81KD myo2-E1 cells often failed to assemble a complete contractile ring. We conclude that Pkd2 modulates the recruitment of type II myosin and actin to the contractile ring, suggesting a novel calcium-dependent mechanism regulating the actin cytoskeletal structures during cytokinesis.

A new analysis of cytokinetic furrow ingression in the Caenorhabditis elegans zygote at high spatiotemporal resolution demonstrates that, rather than being a process of steady, spatially uniform constriction, furrow ingression is modulated by complex contractile oscillations that move around the furrow, possibly in the form of propagating waves.

The actin-like FtsA protein is essential for function of the cell division machinery, or divisome, in many bacteria including Escherichia coli. Previous in vitro studies demonstrated that purified wild-type FtsA assembles into closed mini-rings on lipid membranes, but oligomeric variants of FtsA such as FtsAR286W and FtsAG50E can bypass certain divisome defects and form arc and double-stranded (DS) oligomeric states, respectively, which may reflect conversion of an inactive to an active form of FtsA. However, it remains unproven which oligomeric forms of FtsA are responsible for assembling and activating the divisome. Here, we used an in vivo crosslinking assay for FtsA DS filaments to show that they largely depend on proper divisome assembly and are prevalent at later stages of cell division. We also used a previously reported variant that fails to assemble DS filaments, FtsAM96E R153D, to investigate the roles of FtsA oligomeric states in divisome assembly and activation. We show that FtsAM96E R153D cannot form DS filaments in vivo, fails to replace native FtsA, and confers a dominant negative phenotype, underscoring the importance of the DS filament stage for FtsA function. Surprisingly, however, activation of the divisome through the ftsL* or ftsW* superfission alleles suppressed the dominant negative phenotype and rescued the functionality of FtsAM96E R153D. Our results suggest that FtsA DS filaments are needed for divisome activation once it is assembled, but they are not essential for divisome assembly or guiding septum synthesis.IMPORTANCECell division is fundamental for cellular duplication. In simple cells like Escherichia coli bacteria, the actin homolog FtsA is essential for cell division and assembles into a variety of protein filaments at the cytoplasmic membrane. These filaments not only help tether polymers of the tubulin-like FtsZ to the membrane at early stages of cell division but also play crucial roles in recruiting other cell division proteins to a complex called the divisome. Once assembled, the E. coli divisome subsequently activates synthesis of the division septum that splits the cell in two. One recently discovered oligomeric conformation of FtsA is an antiparallel double-stranded filament. Using a combination of in vivo crosslinking and genetics, we provide evidence suggesting that these FtsA double filaments have a crucial role in activating the septum synthesis enzymes.

Septins can function as scaffolds for protein recruitment, membrane-bound diffusion barriers, or membrane curvature sensors. Septins are important for cytokinesis, but their exact roles are still obscure. In fission yeast, four septins (Spn1 to Spn4) accumulate at the rim of the division plane as rings. The octameric exocyst complex, which tethers exocytic vesicles to the plasma membrane, exhibits a similar localization and is essential for plasma membrane deposition during cytokinesis. Without septins, the exocyst spreads across the division plane but absent from the rim during septum formation. These results suggest that septins and the exocyst physically interact for proper localization. Indeed, we predicted six pairs of direct interactions between septin and exocyst subunits by AlphaFold2 ColabFold, most of them are confirmed by co-immunoprecipitation and yeast two-hybrid assays. Exocyst mislocalization results in mistargeting of secretory vesicles and their cargos, which leads to cell-separation delay in septin mutants. Our results indicate that septins guide the targeting of exocyst complex on the plasma membrane for vesicle tethering during cytokinesis through direct physical interactions.

Phenotypic heterogeneity in bacteria can result from stochastic processes or deterministic programs. The deterministic programs often involve the versatile second messenger c-di-GMP, and give rise to daughter cells with different c-di-GMP levels by deploying c-di-GMP metabolizing enzymes asymmetrically during cell division. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum. Here, we identify a deterministic c-di-GMP-dependent program that is hardwired into the cell cycle of Myxococcus xanthus to minimize phenotypic heterogeneity and guarantee the formation of phenotypically similar daughter cells during division. Cells lacking the diguanylate cyclase DmxA have an aberrant motility behaviour. DmxA is recruited to the cell division site and its activity is switched on during cytokinesis, resulting in a transient increase in the c-di-GMP concentration. During cytokinesis, this c-di-GMP burst ensures the symmetric incorporation and allocation of structural motility proteins and motility regulators at the new cell poles of the two daughters, thereby generating phenotypically similar daughters with correct motility behaviours. Thus, our findings suggest a general c-di-GMP-dependent mechanism for minimizing phenotypic heterogeneity, and demonstrate that bacteria can ensure the formation of dissimilar or similar daughter cells by deploying c-di-GMP metabolizing enzymes to distinct subcellular locations.

Invertebrate and vertebrate species have many unusual cellular structures, such as long- or short-lived cell-in-cell structures and coenocytes. Coenocytes (often incorrectly described as syncytia) are multinuclear cells derived, unlike syncytia, not from the fusion of multiple cells but from multiple nuclear divisions without cytokinesis. An example of a somatic coenocyte is the coenocytic blastoderm in Drosophila. An astonishing property of coenocytes is the ability to differentiate the nuclei sharing a common cytoplasm into different subpopulations with different fate trajectories. An example of a germline coenocyte is the oogenic precursor of appendicularian tunicates, which shares many features with the somatic coenocyte of Drosophila. The germline coenocyte (coenocyst) is quite an unexpected structure because in most animals, including Drosophila, Xenopus, and mice, oogenesis proceeds within a group (cyst, nest) of sibling cells (cystocytes) connected by the intercellular bridges (ring canals, RCs) derived from multiple divisions with incomplete cytokinesis of a progenitor cell called the cystoblast. Here, I discuss the differences and similarities between cystocyte-based and coenocyst-based oogenesis, and the resemblance of coenocystic oogenesis to coenocytic somatic blastoderm in Drosophila. I also describe cell-in-cell structures that although not mechanistically, cytologically, or molecularly connected to somatic or germline coenocytes, are both unorthodox and intriguing cytological phenomena rarely covered by scientific literature.

Cytokinetic abscission marks the final stage of cell division, during which the daughter cells physically separate through the generation of new barriers, such as the plasma membrane or cell wall. While the contractile ring plays a central role during cytokinesis in bacteria, fungi and animal cells, the process diverges in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the mother cell by endodyogeny. The mechanism by which the progeny cells acquire their plasma membrane during the disassembly of the mother cell, allowing daughter cells to emerge, remains unknown. Here we identify and characterize five T. gondii proteins, including three protein phosphatase 2A subunits, which exhibit a distinct and dynamic localization pattern during parasite division. Individual downregulation of these proteins prevents the accumulation of plasma membrane at the division plane, preventing the completion of cellular abscission. Remarkably, the absence of cytokinetic abscission does not hinder the completion of subsequent division cycles. The resulting progeny are able to egress from the infected cells but fail to glide and invade, except in cases of conjoined twin parasites.