RESUMO
Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with lipids and proteins synthesized in the ER. However, little is known about the structure of these ER-NE junctions. Here, we systematically study the ultrastructure of ER-NE junctions in cryo-fixed mammalian cells staged in anaphase, telophase, and interphase by correlating live cell imaging with three-dimensional electron microscopy. Our results show that ER-NE junctions in interphase cells have a pronounced hourglass shape with a constricted neck of 7-20 nm width. This morphology is significantly distinct from that of junctions within the ER network, and their morphology emerges as early as telophase. The highly constricted ER-NE junctions are seen in several mammalian cell types, but not in budding yeast. We speculate that the unique and highly constricted ER-NE junctions are regulated via novel mechanisms that contribute to ER-to-NE lipid and protein traffic in higher eukaryotes.
Assuntos
Retículo Endoplasmático , Mitose , Membrana Nuclear , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Humanos , Animais , Núcleo Celular/metabolismo , Células HeLa , Interfase , TelófaseRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Citocinese , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HeLa , RNA Mensageiro/genética , TelófaseRESUMO
Cell mechanics is a factor that determines cell growth, migration, proliferation, or differentiation, as well as trafficking inside the cytoplasm and organization of organelles. Knowledge about cell mechanics is critical to gaining insight into these biological processes. Here, we used atomic force microscopy to examine the elasticity, an important parameter of cell mechanics, of non-adherent Jurkat leukemic T-cells in both interphase and mitotic phases. We found that the elasticity of an individual cell does not significantly change at interphase. When a cell starts to divide, its elasticity increases in the transition from metaphase to telophase during normal division while the cell is stiffened right after it enters mitosis during abnormal division. At the end of the division, the cell elasticity gradually returned to the value of the mother cell. These changes may originate from the changes in cell surface tension during modulating actomyosin at the cleavage furrow, redistributing cell organelles, and constricting the contractile ring to sever mother cell to form daughters. The difference in elasticity patterns suggests that there is a discrepancy in the redistribution of the cell organelles during normal and abnormal division.
Assuntos
Mitose , Linfócitos T , Ciclo Celular , Telófase , InterfaseRESUMO
Cell divisions are accurately positioned to generate cells of the correct size and shape. In plant cells, the new cell wall is built in the middle of the cell by vesicles trafficked along an antiparallel microtubule and a microfilament array called the phragmoplast. The phragmoplast expands toward a specific location at the cell cortex called the division site, but how it accurately reaches the division site is unclear. We observed microtubule arrays that accumulate at the cell cortex during the telophase transition in maize (Zea mays) leaf epidermal cells. Before the phragmoplast reaches the cell cortex, these cortical-telophase microtubules transiently interact with the division site. Increased microtubule plus end capture and pausing occur when microtubules contact the division site-localized protein TANGLED1 or other closely associated proteins. Microtubule capture and pausing align the cortical microtubules perpendicular to the division site during telophase. Once the phragmoplast reaches the cell cortex, cortical-telophase microtubules are incorporated into the phragmoplast primarily by parallel bundling. The addition of microtubules into the phragmoplast promotes fine-tuning of the positioning at the division site. Our hypothesis is that division site-localized proteins such as TANGLED1 organize cortical microtubules during telophase to mediate phragmoplast positioning at the final division plane.
Assuntos
Arabidopsis , Zea mays , Zea mays/genética , Citocinese , Telófase , Microtúbulos/metabolismo , MitoseRESUMO
Several studies have suggested a role for the LEM-domain protein emerin and the DNA binding factor BAF in nuclear envelope reformation after mitosis, but the exact molecular mechanisms are not understood. Using HeLa cells deficient for emerin or both emerin and lamin A, we show that emerin deficiency induces abnormal aggregation of lamin A at the nuclear periphery in telophase. As a result, nuclear membrane expansion is impaired and BAF accumulates at the core region, the middle part of telophase nuclei. Aggregates do not form when lamin A carries the mutation R435C in the immunoglobulin fold known to prevent interaction of lamin A with BAF suggesting that aggregation is caused by a stabilized association of lamin A with BAF bound to chromosomal DNA. Reintroduction of emerin in the cells prevents formation of lamin A clusters and BAF accumulation at the core region. Therefore emerin is required for the expansion of the nuclear membrane at the core region to enclose the nucleus and for the rapid reformation of the nuclear lamina based on lamin A/C in telophase. Finally, we show that LEM-domain and lumenal domain are required for the targeting of emerin to exert its function at the core region.
Assuntos
Lamina Tipo A , Membrana Nuclear , Humanos , Membrana Nuclear/metabolismo , Lamina Tipo A/metabolismo , Lâmina Nuclear/metabolismo , Telófase , Células HeLa , Proteínas de Ligação a DNA/metabolismo , Mitose , Cromossomos/metabolismoRESUMO
The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate, moving through the four phases of the cell cycle: G1, S, G2, and M. Mitosis is the most dynamic period of the cell cycle, involving a major reorganization of virtually all cell components. Mitosis is further divided into prophase, prometaphase, metaphase, anaphase, and telophase, which can be easily distinguished from one another by protein markers and/or comparing their chromosome morphology under fluorescence microscope. The progression of the cell cycle through these mitotic subphases is tightly regulated by complicated molecular mechanisms. Synchronization of cells to the mitotic subphases is important for understanding these molecular mechanisms. Here, we describe a protocol to synchronize Hela cells to prometaphase, metaphase, and anaphase/telophase. In this protocol, Hela cells are first synchronized to the early S phase by a double thymidine block. Following the release of the block, the cells are treated with nocodazole, MG132, and blebbistatin to arrest them at prometaphase, metaphase, and anaphase/telophase, respectively. Successful synchronization is assessed using Western blot and fluorescence microscopy.
Assuntos
Anáfase , Mitose , Células HeLa , Humanos , Metáfase , TelófaseRESUMO
DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)âlike membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.
Assuntos
Núcleo Celular/fisiologia , DNA/genética , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Plasmídeos/genética , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/genética , Mutação , Proteínas Nucleares/genética , Análise de Célula Única , Telófase , TransfecçãoRESUMO
Dephosphorylation of lamin A, which triggers nuclear lamina reconstitution, is crucial for the completion of mitosis. However, the specific phosphatase and regulatory mechanism that allow timely lamin A dephosphorylation remain unclear. Here, we report that RepoMan (also known as CDCA2), a regulatory subunit of protein phosphatase 1γ (PP1γ) is transiently modified with SUMO-2 at K762 during late telophase. SUMOylation of RepoMan markedly enhanced its binding affinity with lamin A. Moreover, SUMOylated RepoMan contributes to lamin A recruitment to telophase chromosomes and dephosphorylation of the mitotic lamin A phosphorylation. Expression of a SUMO-2 mutant that has a defective interaction with the SUMO-interacting motif (SIM) resulted in failure of the lamin A and RepoMan association, along with abrogation of lamin A dephosphorylation and subsequent nuclear lamina formation. These findings strongly suggest that RepoMan recruits lamin A through SUMO-SIM interaction. Thus, transient SUMOylation of RepoMan plays an important role in the spatiotemporal regulation of lamin A dephosphorylation and the subsequent nuclear lamina formation at the end of mitosis.
Assuntos
Lamina Tipo A , Sumoilação , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Mitose , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , TelófaseRESUMO
Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope (NE) during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III assembly at the NE occurs transiently during mitotic (M) exit and is initiated when CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, interacts with the Inner Nuclear Membrane (INM) protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during M-exit. Firstly, we show that CHMP7 plays an important role in the dissolution of LEM2 clusters that form at the NE during M-exit. Secondly, we show that CDK1 phosphorylates CHMP7 upon M-entry at Ser3 and Ser441 and that this phosphorylation reduces CHMP7's interaction with LEM2, limiting its assembly during M-phase. We show that spatiotemporal differences in the dephosphorylation of CHMP7 license its assembly at the NE during telophase, but restrict its assembly on the ER at this time. Without CDK1 phosphorylation, CHMP7 undergoes inappropriate assembly in the peripheral ER during M-exit, capturing LEM2 and downstream ESCRT-III components. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration.
Assuntos
Proteína Quinase CDC2/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membrana Nuclear/metabolismo , Proteína Quinase CDC2/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos , Proteínas de Membrana , Microtúbulos/metabolismo , Mitose , Proteínas Nucleares , TelófaseRESUMO
Several lines of evidence suggest the existence in the eukaryotic cells of a tight, yet largely unexplored, connection between DNA replication and sister chromatid cohesion. Tethering of newly duplicated chromatids is mediated by cohesin, an evolutionarily conserved hetero-tetrameric protein complex that has a ring-like structure and is believed to encircle DNA. Cohesin is loaded onto chromatin in telophase/G1 and converted into a cohesive state during the subsequent S phase, a process known as cohesion establishment. Many studies have revealed that down-regulation of a number of DNA replication factors gives rise to chromosomal cohesion defects, suggesting that they play critical roles in cohesion establishment. Conversely, loss of cohesin subunits (and/or regulators) has been found to alter DNA replication fork dynamics. A critical step of the cohesion establishment process consists in cohesin acetylation, a modification accomplished by dedicated acetyltransferases that operate at the replication forks. Defects in cohesion establishment give rise to chromosome mis-segregation and aneuploidy, phenotypes frequently observed in pre-cancerous and cancerous cells. Herein, we will review our present knowledge of the molecular mechanisms underlying the functional link between DNA replication and cohesion establishment, a phenomenon that is unique to the eukaryotic organisms.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Replicação do DNA/fisiologia , Fase G1/fisiologia , Telófase/fisiologia , Animais , Humanos , CoesinasRESUMO
Tungsten oxide nanoparticles or nanopowder (WO3NPs) is commonly used in various industries and also in biomedical applications such as additives, pigments, and biomedical sensors. Non-judicious excessive use of these nanoparticles (NPs) could be a serious human health concern. Therefore, the current study aimed to explore the cytotoxic and genotoxic assessment of WO3NPs through Allium cepa anaphase-telophase and comet assays. Nanoparticles were characterized through the scanning and transmission electron microscopy (TEM), zetasizer, and energy-dispersive X-ray spectroscopy. The mean size and the average diameter of WO3NPs were determined as 21.57 ± 2.48 nm and 349.42 ± 80.65 nm using TEM and a Zetasizer measurement system, respectively. Five concentrations (12.5 mg/L, 25 mg/L, 50 mg/L, 75 mg/L, and 100 mg/L) of WO3NPs were employed on the Allium cepa (A. cepa) roots for 4 h. Significant (p ≤ 0.05) decrease in mitotic index (MI) was shown by WO3NPs at all concentrations. The increase of chromosomal aberrations (CAs) was also observed in a concentration-dependent manner due to the WO3NPs exposure. There was a significant increase (p ≤ 0.05) in DNA damage at all concentrations of WO3NPs on the A. cepa cells. It was concluded that WO3NPs had cytotoxic and genotoxic effects on A. cepa meristematic cells. Moreover, further cytogenetic effects of WO3NPs should be investigated at the molecular level to assess its safety margin.
Assuntos
Nanopartículas , Cebolas/genética , Óxidos/toxicidade , Tungstênio/toxicidade , Aberrações Cromossômicas , Ensaio Cometa , Dano ao DNA , Nanopartículas/toxicidade , Cebolas/efeitos dos fármacos , Raízes de Plantas , TelófaseRESUMO
The eukaryotic nucleus remodels extensively during mitosis. Upon mitotic entry, the nuclear envelope breaks down and chromosomes condense into rod-shaped bodies, which are captured by the spindle apparatus and segregated during anaphase. Through telophase, chromosomes decondense and the nuclear envelope reassembles, leading to a functional interphase nucleus. While the molecular processes occurring in early mitosis are intensively investigated, our knowledge about molecular mechanisms of nuclear reassembly is rather limited. Using cell free and cellular assays, we identify the histone variant H2A.Z and its chaperone VPS72/YL1 as important factors for reassembly of a functional nucleus after mitosis. Live-cell imaging shows that siRNA-mediated downregulation of VPS72 extends the telophase in HeLa cells. In vitro, depletion of VPS72 or H2A.Z results in malformed and nonfunctional nuclei. VPS72 is part of two chromatin-remodeling complexes, SRCAP and EP400. Dissecting the mechanism of nuclear reformation using cell-free assays, we, however, show that VPS72 functions outside of the SRCAP and EP400 remodeling complexes to deposit H2A.Z, which in turn is crucial for formation of a functional nucleus.
Assuntos
Núcleo Celular/metabolismo , Histonas/metabolismo , Mitose , Proteínas Repressoras/metabolismo , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Sequência Conservada , Regulação para Baixo , Células HeLa , Humanos , Domínios Proteicos , Proteínas Repressoras/química , Telófase , XenopusRESUMO
The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent-or independent-on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Aurora Quinase A/metabolismo , Linhagem Celular , Cromatina/metabolismo , Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Humanos , Lamina Tipo B/metabolismo , Metáfase , Ligação Proteica , TelófaseRESUMO
In this study pillar[5]arene (P5) and a quinoline-functionalized pillar[5]arene (P5-6Q) which is used for detecting radioactive element, gas adsorption and toxic ions were synthesized. These materials were characterized by Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared (FTIR), elemental analysis, melting point, Mass Spectroscopy, Scanning Electron Microscopy (SEM) and Zeta Potential. The cytotoxic and genotoxic potential of P5 and P5-6Q at distinct concentrations of 12.5, 25, 50, and 100 µg/mL were also investigated by Allium ana-telophase and comet assays on Allium cepa roots and Drosophila melanogaster haemocytes. P5 and P5-6Q showed dose dependent cytotoxic effect by decreasing mitotic index (MI) and genotoxic effect by increasing chromosomal aberrations (CAs such as disturbed anaphase-telophase, polyploidy, stickiness, chromosome laggards and bridges) and DNA damage at the exposed concentrations. These changes in P5-6Q were lower than P5. Further research is necessary to clarify the cytotoxic and genotoxic action mechanisms of P5 and P5-6Q at molecular levels.
Assuntos
Calixarenos/toxicidade , Dano ao DNA , Drosophila melanogaster/efeitos dos fármacos , Cebolas/efeitos dos fármacos , Anáfase/efeitos dos fármacos , Animais , Calixarenos/química , Aberrações Cromossômicas , Ensaio Cometa , Citotoxinas/química , Citotoxinas/toxicidade , Drosophila melanogaster/genética , Hemócitos/efeitos dos fármacos , Índice Mitótico , Cebolas/genética , Raízes de Plantas/efeitos dos fármacos , Quinolinas/síntese química , Quinolinas/química , Quinolinas/toxicidade , Telófase/efeitos dos fármacosRESUMO
Purpose: To examine the phenomena governing the quantitative relationships between acentric fragments and micronuclei and understand which formulas are useful for curve-fitting of experimental data of micronuclei.Materials and methods: A stochastic model, including the phenomena of inclusion, coalescence and culling out, was developed and applied to experimental data.Results: Probabilities for inclusion/exclusion of acentric fragments into daughter nuclei and for coalescence of many fragments into a single micronucleus were found to be not cell type-specific. The biological basis for this result is explained with the lack of DNA damage checkpoints between metaphase (when acentric fragments are scored) and telophase (when micronuclei are formed). The phenomenon of "culling out" cells with high numbers of acentric fragments is also described, along with its proposed biological mechanism.Conclusions: Apart from complex formulas that describe these phenomena, we discuss which simple formulas can best approximate them and when is the case to use them for curve fitting of micronuclei data.
Assuntos
Núcleo Celular/metabolismo , Aberrações Cromossômicas/efeitos da radiação , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Testes para Micronúcleos , Apoptose , Dano ao DNA , Relação Dose-Resposta à Radiação , Células Epiteliais/efeitos da radiação , Fibroblastos/efeitos da radiação , Humanos , Cinética , Modelos Lineares , Metáfase , Probabilidade , TelófaseRESUMO
During organogenesis, precise control of spindle orientation balances proliferation and differentiation. In the developing murine epidermis, planar and perpendicular divisions yield symmetric and asymmetric fate outcomes, respectively. Classically, division axis specification involves centrosome migration and spindle rotation, events occurring early in mitosis. Here, we identify a novel orientation mechanism which corrects erroneous anaphase orientations during telophase. The directionality of reorientation correlates with the maintenance or loss of basal contact by the apical daughter. While the scaffolding protein LGN is known to determine initial spindle positioning, we show that LGN also functions during telophase to reorient oblique divisions toward perpendicular. The fidelity of telophase correction also relies on the tension-sensitive adherens junction proteins vinculin, α-E-catenin, and afadin. Failure of this corrective mechanism impacts tissue architecture, as persistent oblique divisions induce precocious, sustained differentiation. The division orientation plasticity provided by telophase correction may enable progenitors to adapt to local tissue needs.
Assuntos
Células Epidérmicas/citologia , Células Epiteliais/citologia , Telófase/fisiologia , Actomiosina/fisiologia , Anáfase , Animais , Autorrenovação Celular , Forma Celular , Citoesqueleto/ultraestrutura , Epiderme/embriologia , Feminino , Genes Reporter , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Conformação Proteica , Interferência de RNA , Fuso Acromático/ultraestrutura , Vinculina/genética , Vinculina/fisiologia , alfa Catenina/genética , alfa Catenina/fisiologiaRESUMO
Chromosome folding is modulated as cells progress through the cell cycle. During mitosis, condensins fold chromosomes into helical loop arrays. In interphase, the cohesin complex generates loops and topologically associating domains (TADs), while a separate process of compartmentalization drives segregation of active and inactive chromatin. We used synchronized cell cultures to determine how the mitotic chromosome conformation transforms into the interphase state. Using high-throughput chromosome conformation capture (Hi-C) analysis, chromatin binding assays and immunofluorescence, we show that, by telophase, condensin-mediated loops are lost and a transient folding intermediate is formed that is devoid of most loops. By cytokinesis, cohesin-mediated CTCF-CTCF loops and the positions of TADs emerge. Compartment boundaries are also established early, but long-range compartmentalization is a slow process and proceeds for hours after cells enter G1. Our results reveal the kinetics and order of events by which the interphase chromosome state is formed and identify telophase as a critical transition between condensin- and cohesin-driven chromosome folding.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Complexos Multiproteicos/genética , Telófase , Adenosina Trifosfatases/metabolismo , Compartimento Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Mapeamento Cromossômico , Citocinese/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Células HeLa , Humanos , Interfase , Complexos Multiproteicos/metabolismo , Fase S , CoesinasRESUMO
In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis.