Completion Of Cytokinesis Research Articles

The Dystrophin-Dystroglycan complex ensures cytokinesis efficiency in Drosophila epithelia.

Cytokinesis physically separates daughter cells at the end of cell division. This step is particularly challenging for epithelial cells, which are connected to their neighbors and to the extracellular matrix by transmembrane protein complexes. To systematically evaluate the impact of the cell adhesion machinery on epithelial cytokinesis efficiency, we performed an RNAi-based modifier screen in the Drosophila follicular epithelium. Strikingly, this unveiled adhesion molecules and transmembrane receptors that facilitate cytokinesis completion. Among these is Dystroglycan, which connects the extracellular matrix to the cytoskeleton via Dystrophin. Live imaging revealed that Dystrophin and Dystroglycan become enriched in the ingressing membrane, below the cytokinetic ring, during and after ring constriction. Using multiple alleles, including Dystrophin isoform-specific mutants, we show that Dystrophin/Dystroglycan localization is linked with unanticipated roles in regulating cytokinetic ring contraction and in preventing membrane regression during the abscission period. Altogether, we provide evidence that, rather than opposing cytokinesis completion, the machinery involved in cell-cell and cell-matrix interactions has also evolved functions to ensure cytokinesis efficiency in epithelial tissues.

Extracellular vesicles, including large translating vesicles called midbody remnants, are released during the cell cycle.

Extracellular vesicles (EVs) play crucial roles in cell-cell communication, but the biogenesis of large EVs has remained elusive. Here, we show that the biogenesis of large EVs (>800nm-2um) occurs predominantly through the completion of successful cytokinesis, and the majority of large extracellular vesicles are midbody remnants (MBRs) with translation activity, and the unique marker MKLP1. Blocking the cell cycle or cytokinesis, genetically or chemically, significantly decreases MBRs and large (800nm-2um), medium (500-800nm), and small (<300nm) EVs, suggesting that proliferative cells can also generate all sizes of EVs. The canonical EV markers including CD9, CD63, CD81 localize to the spindle midzone, midbody and MBRs, suggesting that these markers are not specific for detecting EVs exclusively. Importantly, all commonly used EV isolation methods isolate MBRs, confounding previous EV research. Lastly, isolated MBRs maintain translation activity regardless of the isolation method. We propose a model for the biogenesis of extracellular vesicles throughout the cell cycle and suggest that some large EVs are primarily generated from mitotic cells. The discovery of MBRs as a unique class of large, translating EVs has implications for using them as cancer diagnostic markers and for engineering them for therapeutic cargo delivery during mitosis.

Central Role of the Actomyosin Ring in Coordinating Cytokinesis Steps in Budding Yeast.

Eukaryotic cells must accurately transfer their genetic material and cellular components to their daughter cells. Initially, cells duplicate their chromosomes and subsequently segregate them toward the poles. The actomyosin ring, a crucial molecular machinery normally located in the middle of the cells and underneath the plasma membrane, then physically divides the cytoplasm and all components into two daughter cells, each ready to start a new cell cycle. This process, known as cytokinesis, is conserved throughout evolution. Defects in cytokinesis can lead to the generation of genetically unstable tetraploid cells, potentially initiating uncontrolled proliferation and cancer. This review focuses on the molecular mechanisms by which budding yeast cells build the actomyosin ring and the preceding steps involved in forming a scaffolding structure that supports the challenging structural changes throughout cytokinesis. Additionally, we describe how cells coordinate actomyosin ring contraction, plasma membrane ingression, and extracellular matrix deposition to successfully complete cytokinesis. Furthermore, the review discusses the regulatory roles of Cyclin-Dependent Kinase (Cdk1) and the Mitotic Exit Network (MEN) in ensuring the precise timing and execution of cytokinesis. Understanding these processes in yeast provides insights into the fundamental aspects of cell division and its implications for human health.

De novo TANGLED1 recruitment from the phragmoplast to aberrant cell plate fusion sites in maize.

Division plane positioning is crucial for proper growth and development in many organisms. In plants, the division plane is established before mitosis, by accumulation of a cytoskeletal structure called the preprophase band (PPB). The PPB is thought to be essential for recruitment of division site-localized proteins, which remain at the division site after the PPB disassembles. Here, we show that the division site-localized protein TANGLED1 (TAN1) is recruited independently of the PPB to the cell cortex by the plant cytokinetic machinery, the phragmoplast, from experiments using both the PPB-defective mutant discordia1 (dcd1) and chemical treatments that disrupt the phragmoplast in maize. TAN1 recruitment to de novo sites on the cortex is partially dependent on intact actin filaments and the myosin XI motor protein OPAQUE1 (O1). These data imply a yet unknown role for TAN1 and possibly other division site-localized proteins during the last stages of cell division when the phragmoplast touches the cell cortex to complete cytokinesis.

Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin

BackgroundToxoplasma gondii is an apicomplexan intracellular obligate parasite and the etiological agent of toxoplasmosis in humans, domestic animals and wildlife, causing miscarriages and negatively impacting offspring. During its intracellular development, it relies on nutrients from the host cell, controlling several pathways and the cytoskeleton. T. gondii has been proven to control the host cell cycle, mitosis and cytokinesis, depending on the time of infection and the origin of the host cell. However, no data from parallel infection studies have been collected. Given that T. gondii can infect virtually any nucleated cell, including those of humans and animals, understanding the mechanism by which it infects or develops inside the host cell is essential for disease prevention. Therefore, we aimed here to reveal whether this modulation is dependent on a specific cell type or host cell species.MethodsWe used only primary cells from humans and bovines at a maximum of four passages to ensure that all cells were counted with appropriate cell cycle checkpoint control. The cell cycle progression was analysed using fluorescence-activated cell sorting (FACS)-based DNA quantification, and its regulation was followed by the quantification of cyclin B1 (mitosis checkpoint protein). The results demonstrated that all studied host cells except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Additionally, we used an immunofluorescence assay to track mitosis and cytokinesis in uninfected and T. gondii-infected cells.ResultsThe results demonstrated that all studied host cell except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Our findings showed that the analysed cells developed chromosome segregation problems and failed to complete cytokinesis. Also, the number of centrosomes per mitotic pole was increased after infection in all cell types. Therefore, our data suggest that T. gondii modulates the host cell cycle, chromosome segregation and cytokinesis during infection or development regardless of the host cell origin or type.Graphical

The role of midbody-associated mRNAs in regulating abscission.

Midbodies function during telophase to regulate the abscission step of cytokinesis. Until recently, it was thought that abscission-regulating proteins, such as ESCRT-III complex subunits, accumulate at the MB by directly or indirectly binding to the MB resident protein, CEP55. However, recent studies have shown that depletion of CEP55 does not fully block ESCRT-III targeting the MB. Here, we show that MBs contain mRNAs and that these MB-associated mRNAs can be locally translated, resulting in the accumulation of abscission-regulating proteins. We demonstrate that localized MB-associated translation of CHMP4B is required for its targeting to the abscission site and that 3' UTR-dependent CHMP4B mRNA targeting to the MB is required for successful completion of cytokinesis. Finally, we identify regulatory cis-elements within RNAs that are necessary and sufficient for mRNA trafficking to the MB. We propose a novel method of regulating cytokinesis and abscission by MB-associated targeting and localized translation of selective mRNAs.

The microtubule quartet protein SNAP1 in Trypanosoma brucei facilitates flagellum and cell division plane positioning by promoting basal body segregation

The unicellular protozoan Trypanosoma brucei has a single flagellum that is involved in cell motility, cell morphogenesis, and cell division. Inheritance of the newly assembled flagellum during the cell cycle requires its correct positioning, which depends on the faithful duplication or segregation of multiple flagellum-associated cytoskeletal structures, including the basal body, the flagellum attachment zone, and the hook complex. Along the flagellum attachment zone sites a set of four microtubules termed the microtubule quartet (MtQ), whose molecular function remains enigmatic. We recently reported that the MtQ-localized protein NHL1 interacts with the microtubule-binding protein TbSpef1 and regulates flagellum inheritance by promoting basal body rotation and segregation. Here, we identified a TbSpef1- and NHL1-associated protein named SNAP1, which co-localizes with NHL1 and TbSpef1 at the proximal portion of the MtQ, depends on TbSpef1 for localization and is required for NHL1 localization to the MtQ. Knockdown of SNAP1 impairs the rotation and segregation of the basal body, the elongation of the flagellum attachment zone filament, and the positioning of the newly assembled flagellum, thereby causing mis-placement of the cell division plane, a halt in cleavage furrow ingression, and an inhibition of cytokinesis completion. Together, these findings uncover a coordinating role of SNAP1 with TbSpef1 and NHL1 in facilitating flagellum positioning and cell division plane placement for the completion of cytokinesis.

The mechanism for polar localization of the type IVa pilus machine in Myxococcus xanthus.

Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation, and virulence. Different bacteria have adapted different piliation patterns. To address how these patterns are established, we focused on the bipolar localization of the T4aP machine in the model organism Myxococcus xanthus by studying the localization of the PilQ secretin, the first component of this machine that assembles at the poles. Based on experiments using a combination of fluorescence microscopy, biochemistry, and computational structural analysis, we propose that PilQ, and specifically its AMIN domains, binds septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates PilQ multimerization in the outer membrane. We also propose that the presence and absence of AMIN domains in T4aP secretins contribute to the different piliation patterns across bacteria.

An acytokinetic cell division creates PIP2-enriched membrane asymmetries leading to slit diaphragm assembly in Drosophila nephrocytes.

Vertebrate podocytes and Drosophila nephrocytes display slit diaphragms, specialised cell junctions that are essential for the execution of the basic excretory function of ultrafiltration. To elucidate the mechanisms of slit diaphragm assembly we have studied their formation in Drosophila embryonic garland nephrocytes. These cells of mesenchymal origin lack overt apical-basal polarity. We find that their initial membrane symmetry is broken by an acytokinetic cell division that generates PIP2-enriched domains at their equator. The PIP2-enriched equatorial cortex becomes a favourable domain for hosting slit diaphragm proteins and the assembly of the first slit diaphragms. Indeed, when this division is either prevented or forced to complete cytokinesis, the formation of diaphragms is delayed to larval stages. Furthermore, although apical polarity determinants also accumulate at the equatorial cortex, they do not appear to participate in the recruitment of slit diaphragm proteins. The mechanisms we describe allow the acquisition of functional nephrocytes in embryos, which may confer on them a biological advantage similar to the formation of the first vertebrate kidney, the pronephros.

Rucaparib inhibits lung adenocarcinoma cell proliferation and migration via the SHCBP1/CDK1 pathway.

Src homolog and collagen homolog binding protein 1 (SHCBP1) binds to the SH2 domain of SHC-transforming protein 1 (SHC1) and is involved in midbody organization and cytokinesis completion. SHCBP1 has been reported to be a cancer driver gene, promoting cancer progression. However, the functional role and underlying mechanism of SHCBP1 in regulating lung adenocarcinoma (LUAD) cell proliferation and migration are incompletely understood. Here, we discovered that SHCBP1 is overexpressed in LUAD tissues and is associated with a poor prognosis. SHCBP1 knockdown inhibited LUAD cell proliferation and migration by arresting the cell cycle and preventing epithelial-mesenchymal transition (EMT) via decreasing cyclin-dependent kinase 1 (CDK1) expression. Mechanistically, CDK1 overexpression reversed SHCBP1 knockdown-induced inhibition of proliferation and migration, confirming CDK1 as a key downstream target of SHCBP1. In addition, we proposed that rucaparib may be a small-molecule inhibitor of SHCBP1 and validated both in vitro and in vivo that rucaparib inhibits cell proliferation and migration via suppression of the SHCBP1/CDK1 pathway in LUAD. Our study elucidates a newly identified role of SHCBP1 in promoting cell proliferation and migration in LUAD, and suggests rucaparib as a potential inhibitor for LUAD treatment.

The abscission checkpoint senses chromatin bridges through Top2α recruitment to DNA knots.

In response to chromatin bridges, the abscission checkpoint delays completion of cytokinesis to prevent chromosome breakage or tetraploidization. Here, we show that spontaneous or replication stress-induced chromatin bridges exhibit "knots" of catenated and overtwisted DNA next to the midbody. Topoisomerase IIα (Top2α) forms abortive Top2-DNA cleavage complexes (Top2ccs) on DNA knots; furthermore, impaired Top2α-DNA cleavage activity correlates with chromatin bridge breakage in cytokinesis. Proteasomal degradation of Top2ccs is required for Rad17 localization to Top2-generated double-strand DNA ends on DNA knots; in turn, Rad17 promotes local recruitment of the MRN complex and downstream ATM-Chk2-INCENP signaling to delay abscission and prevent chromatin breakage. In contrast, dicentric chromosomes that do not exhibit knotted DNA fail to activate the abscission checkpoint in human cells. These findings are the first to describe a mechanism by which the abscission checkpoint detects chromatin bridges, through generation of abortive Top2ccs on DNA knots, to preserve genome integrity.

Phosphorylation controls spatial and temporalactivities of motor-PRC1 complexes to complete mitosis.

During mitosis, spindle architecture alters as chromosomes segregate into daughter cells. The microtubule crosslinker protein regulator of cytokinesis 1 (PRC1) is essential for spindle stability, chromosome segregation and completion of cytokinesis, but how it recruits motors to the central spindle to coordinate the segregation of chromosomes is unknown. Here, we combine structural and cell biology approaches to show that the human CENP-E motor, which is essential for chromosome capture and alignment by microtubules, binds to PRC1 through a conserved hydrophobic motif. This binding mechanism is also used by Kinesin-4 Kif4A:PRC1. Using in vitro reconstitution, we demonstrate that CENP-E slides antiparallel PRC1-crosslinked microtubules. We find that the regulation of CENP-E -PRC1 interaction is spatially and temporally coupled with relocalization to overlapping microtubules in anaphase. Finally, we demonstrate that the PRC1-microtubule motor interaction is essential in anaphase to control chromosome partitioning, retain central spindle integrity and ensure cytokinesis. Taken together our findings reveal the molecular basis for the cell cycle regulation of motor-PRC1 complexes to couple chromosome segregation and cytokinesis.

FAZ assembly in bloodstream form Trypanosoma brucei requires kinesin KIN-E.

Trypanosoma brucei, the causative agent of African sleeping sickness, uses its flagellum for movement, cell division, and signaling. The flagellum is anchored to the cell body membrane via the flagellum attachment zone (FAZ), a complex of proteins, filaments, and microtubules that spans two membranes with elements on both flagellum and cell body sides. How FAZ components are carried into place to form this complex is poorly understood. Here, we show that the trypanosome-specific kinesin KIN-E is required for building the FAZ in bloodstream-form parasites. KIN-E is localized along the flagellum with a concentration at its distal tip. Depletion of KIN-E by RNAi rapidly inhibits flagellum attachment and leads to cell death. A detailed analysis reveals that KIN-E depletion phenotypes include failure in cytokinesis completion, kinetoplast DNA missegregation, and transport vesicle accumulation. Together with previously published results in procyclic form parasites, these data suggest KIN-E plays a critical role in FAZ assembly in T. brucei.

Subcellular positioning during cell division and cell plate formation in maize.

During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles. Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells. The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects in Arabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins. Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize.

AtMAC stabilizes the phragmoplast by crosslinking microtubules and actin filaments during cytokinesis.

The phragmoplast, a structure crucial for the completion of cytokinesis in plant cells, is composed of antiparallel microtubules (MTs) and actin filaments (AFs). However, how the parallel structure of phragmoplast MTs and AFs is maintained, especially during centrifugal phragmoplast expansion, remains elusive. Here, we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein (AtMAC). When AtMAC was deleted, the phragmoplast showed disintegrity during centrifugal expansion, and the resulting phragmoplast fragmentation led to incomplete cell plates. Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis. Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro, and the truncated AtMAC protein, N-CC1, was the key domain controlling the ability of AtMAC. Further analysis showed that N-CC1(51-154) is the key domain for binding MTs, and N-CC1(51-125) for binding AFs. In conclusion, AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion, which is required for complete cytokinesis.

Anillin and the microtubule bundler PRC1 maintain myosin in the contractile ring to ensure completion of cytokinesis.

Cytokinesis is the last step of cell division, when one cell physically divides into two cells. Cytokinesis is driven by an equatorial contractile ring and signals from antiparallel microtubule bundles (the central spindle) that form between the two masses of segregating chromosomes. Bundling of central spindle microtubules is essential for cytokinesis in cultured cells. Using a temperature-sensitive mutant of SPD-1, the homolog of the microtubule bundler PRC1, we demonstrate that SPD-1 is required for robust cytokinesis in the Caenorhabditis elegans early embryo. SPD-1 inhibition results in broadening of the contractile ring, creating an elongated intercellular bridge between sister cells at the last stages of ring constriction that fails to seal. Moreover, depleting anillin/ANI-1 in SPD-1-inhibited cells results in myosin loss from the contractile ring during the second half of furrow ingression, which in turn results in furrow regression and cytokinesis failure. Our results thus reveal a mechanism involving the joint action of anillin and PRC1, which operates during the later stages of furrow ingression to ensure continued functioning of the contractile ring until cytokinesis is complete.

Evolutionarily conserved midbody remodeling precedes ring canal formation during gametogenesis.

How canonical cytokinesis is altered during germ cell division to produce stable intercellular bridges, called "ring canals," is poorly understood. Here, using time-lapse imaging in Drosophila, we observe that ring canal formation occurs through extensive remodeling of the germ cell midbody, a structure classically associated with its function in recruiting abscission-regulating proteins in complete cytokinesis. Germ cell midbody cores reorganize and join the midbody ring rather than being discarded, and this transition is accompanied by changes in centralspindlin dynamics. The midbody-to-ring canal transformation is conserved in the Drosophila male and female germlines and during mouse and Hydra spermatogenesis. In Drosophila, ring canal formation depends on Citron kinase function to stabilize the midbody, similar to its role during somatic cell cytokinesis. Our results provide important insights into the broader functions of incomplete cytokinesis events across biological systems, such as those observed during development and disease states.

Cytokinesis in Trypanosoma brucei relies on an orphan kinesin that dynamically crosslinks microtubules

Many single-celled eukaryotes have complex cell morphologies defined by microtubules arranged into higher-order structures. The auger-like shape of the parasitic protist Trypanosoma brucei (T.brucei) is mediated by a parallel array of microtubules that underlies the plasma membrane. The subpellicular array must be partitioned and segregated using a microtubule-based mechanism during cell division. We previously identified an orphan kinesin, KLIF, that localizes to the ingressing cleavage furrow and is essential for the completion of cytokinesis. We have characterized the biophysical properties of a truncated KLIF construct invitro to gain mechanistic insight into the function of this novel kinesin. We find that KLIF is a non-processive dimeric kinesin that dynamically crosslinks microtubules. Microtubules crosslinked by KLIF in an antiparallel orientation are translocated relative to one another, while microtubules crosslinked parallel to one another remain static, resulting in the formation of organized parallel bundles. In addition, we find that KLIF stabilizes the alignment of microtubule plus ends. These features provide a mechanistic understanding for how KLIF functions to form a new pole of aligned microtubule plus ends that defines the shape of the new cell posterior, which is an essential requirement for the completion of cytokinesis in T.brucei.

The centralspindlin complex regulates cytokinesis and morphogenesis in the C. elegans spermatheca.

Organ morphogenesis needs orchestration of a series of cellular events, including cell division, cell shape change, cell rearrangement and cell death. Cytokinesis, the final step of cell division, is involved in the control of organ size, shape and function. Mechanistically, it is unclear how the molecules involved in cytokinesis regulate organ size and shape. Here, we demonstrate that the centralspindlin complex coordinates cell division and epithelial morphogenesis by regulating cytokinesis. Loss of the centralspindlin components CYK-4 and ZEN-4 disrupts cell division, resulting in altered cell arrangement and malformation of the Caenorhabditis elegans spermatheca. Further investigation revealed that most spermathecal cells undergo nuclear division without completion of cytokinesis. Germline mutant-based analyses suggest that CYK-4 regulates cytokinesis of spermathecal cells in a GTPase activator activity-independent manner. Spermathecal morphology defects can be enhanced by double knockdown of rho-1 and cyk-4, and partially suppressed by double knockdown of cdc-42 and cyk-4. Thus, the centralspindlin components CYK-4 and ZEN-4, together with RHO-1 and CDC-42, are central players of a signaling network that guides spermathecal morphogenesis by enabling completion of cytokinesis.

Phosphoproteomic analysis identifies supervillin as an ERK3 substrate regulating cytokinesis and cell ploidy.

Extracellular signal-regulated kinase 3 (ERK3) is a poorly characterized member of the mitogen-activated protein (MAP) kinase family. Functional analysis of the ERK3 signaling pathway has been hampered by a lack of knowledge about the substrates and downstream effectors of the kinase. Here, we used large-scale quantitative phosphoproteomics and targeted gene silencing to identify direct ERK3 substrates and gain insight into its cellular functions. Detailed validation of one candidate substrate identified the gelsolin/villin family member supervillin (SVIL) as a bona fide ERK3 substrate. We show that ERK3 phosphorylates SVIL on Ser245 to regulate myosin II activation and cytokinesis completion in dividing cells. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild type SVIL but not by the non-phosphorylatable S245A mutant. Our results unveil a new function of the atypical MAP kinase ERK3 in cell division and the regulation of cell ploidy.