The BRCT domains of ECT2 have distinct functions during cytokinesis

Schneid, Sandra; Wolff, Friederike; Buchner, Kristina; Bertram, Nils; Baygün, Seren; Barbosa, Pedro; Mangal, Sriyash; Zanin, Esther

doi:10.1016/j.celrep.2021.108805

Cited by 25 publications

(31 citation statements)

References 69 publications

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“…Ect2 encodes for ECT2 protein, a GEF of the Rho family of small GTPases, that controls cytokinesis during cell cycle, and cell polarity ( 30 ). Structural studies found that exon 4, which codes for part of the BRCT 0 domain of ECT2, contributes to GEF activation and interactions with proteins important for cell spreading ( 31 , 32 ) (Figure 3A ). RNA-seq revealed that exon 4 of Ect2 is more excluded in Rbfox2 -CKO hearts.…”

Section: Resultsmentioning

confidence: 99%

RBFOX2 is required for establishing RNA regulatory networks essential for heart development

Verma

Deshmukh

Thatcher

et al. 2022

Nucleic Acids Research

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Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.

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Section: Resultsmentioning

confidence: 99%

RBFOX2 is required for establishing RNA regulatory networks essential for heart development

Verma

Deshmukh

Thatcher

et al. 2022

Nucleic Acids Research

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“…Centralspindlin is a heterotetrametric complex consisting of a dimer of kinesin-6 family member mitotic kinesin-like protein (Mklp1), and a dimer of Cyk4 (Pavicic-Kaltenbrunner et al, 2007). The centralspindlin helps in recruiting the conserved RhoA guanine nucleotide exchange factor (RhoGEF)-epithelial cell transforming sequence 2 (Ect2) to the spindle midzone (Yuce et al, 2005; Chalamalasetty et al, 2006; Kamijo et al, 2006; Su et al, 2011; Kotynkova et al, 2016; Gomez-Cavazos et al, 2020; Schneid et al, 2021). Ect2 is essential for RhoA activation and thus for cleavage furrow ingression and cytokinesis in animal cells (Miki et al, 1993; Prokopenko et al, 1999; Kimura et al, 2000; Su et al, 2011; reviewed in Basant and Glotzer, 2017; Pollard and O’Shaughnessy, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…Ect2 is essential for RhoA activation and thus for cleavage furrow ingression and cytokinesis in animal cells (Miki et al, 1993; Prokopenko et al, 1999; Kimura et al, 2000; Su et al, 2011; reviewed in Basant and Glotzer, 2017; Pollard and O’Shaughnessy, 2019). Ect2 consists of BRCT (BRCA1-C-terminal) domains at the N-terminus, and RhoGEF, pleckstrin homology (PH), and poly-basic cluster (PBC) domains at the C-terminus (Chalamalasetty et al, 2006; Su et al, 2011; Kotynkova et al, 2016; Schneid et al, 2021; Figure 3A). Through BRCT domains, Ect2 interacts with Cyk4, and the PH and PBC domains are critical for its membrane localization (Burkard et al, 2007; Somers and Saint, 2003; Yuce et al, 2005; Wolfe et al, 2009; Su et al, 2011; Kotynkova et al, 2016; Gomez-Cavazos et al, 2020; Schneid et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…Ect2 consists of BRCT (BRCA1-C-terminal) domains at the N-terminus, and RhoGEF, pleckstrin homology (PH), and poly-basic cluster (PBC) domains at the C-terminus (Chalamalasetty et al, 2006; Su et al, 2011; Kotynkova et al, 2016; Schneid et al, 2021; Figure 3A). Through BRCT domains, Ect2 interacts with Cyk4, and the PH and PBC domains are critical for its membrane localization (Burkard et al, 2007; Somers and Saint, 2003; Yuce et al, 2005; Wolfe et al, 2009; Su et al, 2011; Kotynkova et al, 2016; Gomez-Cavazos et al, 2020; Schneid et al, 2021). The presence of these multiple domains in Ect2 ensures Ect2 localization and RhoA-activation at the equatorial region of the membrane for cleavage furrow formation.…”

Section: Introductionmentioning

confidence: 99%

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Membrane compartmentalization of Ect2/Cyk4/Mklp1 and NuMA/dynein/dynactin is essential for cleavage furrow formation during anaphase

Sana

Rajeevan

Kotak

2022

Preprint

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In animal cells, spindle elongation during anaphase is temporally coupled with cleavage furrow formation. Spindle elongation during anaphase is regulated by NuMA/dynein/dynactin complexes that occupy the polar region of the cell membrane and are excluded from the equatorial membrane. How NuMA/dynein/dynactin are excluded from the equatorial membrane and the biological significance of this exclusion remains unknown. Here, we show that the centralspindlin (Cyk4/Mklp1) and its interacting partner RhoGEF Ect2 are required for NuMA/dynein/dynactin exclusion from the equatorial cell membrane. The Ect2-based (Ect2/Cyk4/Mklp1) and NuMA-based (NuMA/dynein/dynactin) complexes occupy mutually exclusive membrane surfaces during anaphase. The equatorial membrane enrichment of Ect2-based complexes is essential for NuMA/dynein/dynactin exclusion and proper spindle elongation. Conversely, NuMA-based complexes at the polar region of the cell membrane ensure spatially confined localization of Ect2-based complexes and thus RhoA. Overall, our work establishes that membrane compartmentalization of NuMA-based and Ect2-based complexes at the two distinct cell surfaces restricts dynein/dynactin and RhoA for coordinating spindle elongation with cleavage furrow formation.

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“…However, both Ect2 and RhoA show more variability and/or cause cytokinesis phenotypes when over-expressed (Chalamalasetty et al, 2006; Piekny & Glotzer, 2008; Yuce et al, 2005). TCA-fixation-based immunofluorescence microscopy is still used as one of the more reliable methods to visualize the enrichment of RhoA at the equatorial cortex (Koh et al, 2021; Schneid et al, 2021; Yonemura et al, 2004; Yuce et al, 2005), while Ect2 overexpression can lead to cytokinesis failure (Chalamalasetty et al, 2006). In addition, measurements can be confounded by the variability in expression between transfected cells, and endogenous probes enable more quantitative measurements (Husser et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Imaging tools generated by CRISPR/Cas9 tagging reveal cytokinetic diversity in mammalian cells

Husser

Ozugergin

Resta

et al. 2022

Preprint

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Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This process occurs via the ingression of an actomyosin ring that assembles in anaphase and pulls in the overlying plasma membrane as it constricts. Mechanistic studies have uncovered different pathways that regulate the assembly and position of the ring in mammalian cells, but the majority of these studies were done using HeLa cells with overexpressed transgenes, and the relative requirement for these mechanisms among the majority of cell types is not known. Here, we used CRISPR/Cas9 gene editing to endogenously tag cytokinesis proteins, anillin, Ect2 and RhoA, as well as other cellular components, with fluorescent proteins. These tools enabled the visualization of cytokinesis by live imaging to quantitatively study these proteins at endogenous levels. As a proof-of-concept, tagging anillin in multiple mammalian cell lines revealed cytokinetic diversity, which will be useful for studies of how mechanisms controlling cytokinesis vary among cell types. We also successfully tagged multiple cellular components in the same cell line, demonstrating the versatility of these tagging tools.

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The BRCT domains of ECT2 have distinct functions during cytokinesis

Cited by 25 publications

References 69 publications

RBFOX2 is required for establishing RNA regulatory networks essential for heart development

RBFOX2 is required for establishing RNA regulatory networks essential for heart development

Membrane compartmentalization of Ect2/Cyk4/Mklp1 and NuMA/dynein/dynactin is essential for cleavage furrow formation during anaphase

Imaging tools generated by CRISPR/Cas9 tagging reveal cytokinetic diversity in mammalian cells

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