Lethal Giant Disc is a target of Cdk1 and regulates ESCRT-III localization during germline stem cell abscission.

Abscission is the final step of cytokinesis that allows the physical separation of sister cells through the scission of the cellular membrane. This deformation is driven by ESCRT-III proteins, which can bind membranes and form dynamic helices. A crucial step in abscission is the recruitment of ESCRT-III proteins at the right time and place. Alix is one of the best characterized proteins that recruits ESCRT-III proteins from yeast to mammals. However, recent studies in vivo have revealed that pathways acting independently or redundantly with Alix are also required at abscission sites in different cellular contexts. Here, we show that Lgd acts redundantly with Alix to properly localize ESCRT-III to the abscission site in germline stem cells (GSCs) during Drosophila oogenesis. We further demonstrate that Lgd is phosphorylated at multiple sites by the CycB/Cdk1 kinase. We found that these phosphorylation events potentiate the activity of Shrub, a Drosophila ESCRT-III, during abscission of GSCs. Our study reveals that redundancy between Lgd and Alix, and coordination with the cell cycle kinase Cdk1, confers robust and timely abscission of Drosophila germline stem cells.

Mating-induced Ecdysone in the testis disrupts soma-germline contacts and stem cell cytokinesis.

Germline maintenance relies on adult stem cells to continually replenish lost gametes over a lifetime and respond to external cues altering the demands on the tissue. Mating worsens germline homeostasis over time, yet a negative impact on stem cell behavior has not been explored. Using extended live imaging of the Drosophila testis stem cell niche, we find that short periods of mating in young males disrupts cytokinesis in germline stem cells (GSCs). This defect leads to failure of abscission, preventing release of differentiating cells from the niche. We find that GSC abscission failure is caused by increased Ecdysone hormone signaling induced upon mating, which leads to disrupted somatic encystment of the germline. Abscission failure is rescued by isolating males from females, but recurs with resumption of mating. Importantly, reiterative mating also leads to increased GSC loss, requiring increased restoration of stem cells via symmetric renewal and de-differentiation. Together, these results suggest a model whereby acute mating results in hormonal changes that negatively impact GSC cytokinesis but preserves the stem cell population.

The human AAA-ATPase VPS4A isoform and its co-factor VTA1 have a unique function in regulating mammalian cytokinesis abscission.

Mutations in the human AAA-ATPase VPS4 isoform, VPS4A, cause severe neurodevelopmental defects and congenital dyserythropoietic anemia (CDA). VPS4 is a crucial component of the endosomal sorting complex required for transport (ESCRT) system, which drives membrane remodeling in numerous cellular processes, including receptor degradation, cell division, and neural pruning. Notably, while most organisms encode for a single VPS4 gene, human cells have 2 VPS4 paralogs, namely VPS4A and VPS4B, but the functional differences between these paralogs is mostly unknown. Here, we set out to investigate the role of the human VPS4 paralogs in cytokinetic abscission using a series of knockout cell lines. We found that VPS4A and VPS4B hold both overlapping and distinct roles in abscission. VPS4A depletion resulted in a more severe abscission delay than VPS4B and was found to be involved in earlier stages of abscission. Moreover, VPS4A and a monomeric-locked VPS4A mutant bound the abscission checkpoint proteins CHMP4C and ANCHR, while VPS4B did not, indicating a regulatory role for the VPS4A isoform in abscission. Depletion of VTA1, a co-factor of VPS4, disrupted VPS4A-ANCHR interactions and accelerated abscission, suggesting that VTA1 is also involved in the abscission regulation. Our findings reveal a dual role for VPS4A in abscission, one that is canonical and can be compensated by VPS4B, and another that is regulatory and may be delivered by its monomeric form. These observations provide a potential mechanistic explanation for the neurodevelopmental defects and other related disorders reported in VPS4A-mutated patients with a fully functional VPS4B paralog.

Molecular basis for curvature formation in SepF polymerization.

The self-assembly of proteins into curved structures plays an important role in many cellular processes. One good example of this phenomenon is observed in the septum-forming protein (SepF), which forms polymerized structures with uniform curvatures. SepF is essential for regulating the thickness of the septum during bacteria cell division. In Bacillus subtilis, SepF polymerization involves two distinct interfaces, the ß-ß and α-α interfaces, which define the assembly unit and contact interfaces, respectively. However, the mechanism of curvature formation in this step is not yet fully understood. In this study, we employed solid-state NMR (SSNMR) to compare the structures of cyclic wild-type SepF assemblies with linear assemblies resulting from a mutation of G137 on the ß-ß interface. Our results demonstrate that while the sequence differences arise from the internal assembly unit, the dramatic changes in the shape of the assemblies depend on the α-α interface between the units. We further provide atomic-level insights into how the angular variation of the α2 helix on the α-α interface affects the curvature of the assemblies, using a combination of SSNMR, cryo-electron microscopy, and simulation methods. Our findings shed light on the shape control of protein assemblies and emphasize the importance of interhelical contacts in retaining curvature.

OSGN-1 is a conserved flavin-containing monooxygenase required to stabilize the intercellular bridge in late cytokinesis.

Cytokinesis is the last step of cell division and is regulated by the small GTPase RhoA. RhoA activity is required for all steps of cytokinesis, including prior to abscission when daughter cells are ultimately physically separated. Like germ cells in all animals, the Caenorhabditis elegans embryonic germline founder cell initiates cytokinesis but does not complete abscission, leaving a stable intercellular bridge between the two daughter cells. Here, we identify and characterize C. elegans OSGN-1 as a cytokinetic regulator that promotes RhoA activity during late cytokinesis. Sequence analyses and biochemical reconstitutions reveal that OSGN-1 is a flavin-containing monooxygenase (MO). Genetic analyses indicate that the MO activity of OSGN-1 is required to maintain active RhoA at the end of cytokinesis in the germline founder cell and to stabilize the intercellular bridge. Deletion of OSGIN1 in human cells results in an increase in binucleation as a result of cytokinetic furrow regression, and this phenotype can be rescued by expressing a catalytically active form of C. elegans OSGN-1, indicating that OSGN-1 and OSGIN1 are functional orthologs. We propose that OSGN-1 and OSGIN1 are conserved MO enzymes required to maintain RhoA activity at the intercellular bridge during late cytokinesis and thus favor its stability, enabling proper abscission in human cells and bridge stabilization in C. elegans germ cells.

Cell-intrinsic mechanical regulation of plasma membrane accumulation at the cytokinetic furrow.

Cytokinesis is the process where the mother cell's cytoplasm separates into daughter cells. This is driven by an actomyosin contractile ring that produces cortical contractility and drives cleavage furrow ingression, resulting in the formation of a thin intercellular bridge. While cytoskeletal reorganization during cytokinesis has been extensively studied, less is known about the spatiotemporal dynamics of the plasma membrane. Here, we image and model plasma membrane lipid and protein dynamics on the cell surface during leukemia cell cytokinesis. We reveal an extensive accumulation and folding of the plasma membrane at the cleavage furrow and the intercellular bridge, accompanied by a depletion and unfolding of the plasma membrane at the cell poles. These membrane dynamics are caused by two actomyosin-driven biophysical mechanisms: the radial constriction of the cleavage furrow causes local compression of the apparent cell surface area and accumulation of the plasma membrane at the furrow, while actomyosin cortical flows drag the plasma membrane toward the cell division plane as the furrow ingresses. The magnitude of these effects depends on the plasma membrane fluidity, cortex adhesion, and cortical contractility. Overall, our work reveals cell-intrinsic mechanical regulation of plasma membrane accumulation at the cleavage furrow that is likely to generate localized differences in membrane tension across the cytokinetic cell. This may locally alter endocytosis, exocytosis, and mechanotransduction, while also serving as a self-protecting mechanism against cytokinesis failures that arise from high membrane tension at the intercellular bridge.

A cytokinetic ring-driven cell rotation achieves Hertwig's rule in early development.

Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems.

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.

Microtubule reorganization during mitotic cell division in the dinoflagellate Ostreospis cf. ovata.

Dinoflagellates are marine organisms that undergo seasonal proliferation events known as algal blooms. Vegetative cell proliferation is a main contributing factor in these events. However, mechanistical understanding of mitosis and cytokinesis in dinoflagellates remains rudimentary. Using an optimized immunofluorescence protocol, we analysed changes in microtubule organization occurring during the mitotic cycle of the toxic dinoflagellate Ostreopsis cf. ovata. We find that the flagella and the cortical microtubule array persist throughout the mitotic cycle. Two cytoplasmic microtubule bundles originate from the ventral area, where the basal bodies are located - a cortical bundle and a cytoplasmic bundle. The latter associates with the nucleus in the cell centre before mitosis and with the acentrosomal extranuclear spindle during mitosis. Analysis of tubulin post-translational modifications identifies two populations of spindle microtubules - polar acetylated microtubules, whose length is constant, and central tyrosinated microtubules, which elongate during chromosome segregation. During cell division a microtubule-rich structure forms along the dorsal-ventral axis, associated with the site of cytokinesis, consistent with a cytokinetic mechanism that is independent of the actomyosin ring typical of animal and yeast cells.

A kinesin-13 family kinesin in Trypanosoma brucei regulates cytokinesis and cytoskeleton morphogenesis by promoting microtubule bundling.

The early branching eukaryote Trypanosoma brucei divides uni-directionally along the longitudinal cell axis from the cell anterior toward the cell posterior, and the cleavage furrow ingresses along the cell division plane between the new and the old flagella of a dividing bi-flagellated cell. Regulation of cytokinesis in T. brucei involves actomyosin-independent machineries and trypanosome-specific signaling pathways, but the molecular mechanisms underlying cell division plane positioning remain poorly understood. Here we report a kinesin-13 family protein, KIN13-5, that functions downstream of FPRC in the cytokinesis regulatory pathway and determines cell division plane placement. KIN13-5 localizes to multiple cytoskeletal structures, interacts with FPRC, and depends on FPRC for localization to the site of cytokinesis initiation. Knockdown of KIN13-5 causes loss of microtubule bundling at both ends of the cell division plane, leading to mis-placement of the cleavage furrow and unequal cytokinesis, and at the posterior cell tip, causing the formation of a blunt posterior. In vitro biochemical assays demonstrate that KIN13-5 bundles microtubules, providing mechanistic insights into the role of KIN13-5 in cytokinesis and posterior morphogenesis. Altogether, KIN13-5 promotes microtubule bundle formation to ensure cleavage furrow placement and to maintain posterior cytoskeleton morphology in T. brucei.

Activating the abscission checkpoint: Top2α senses chromatin bridges in cytokinesis: Top2α binds to DNA knots on chromatin bridges to activate the abscission checkpoint in human cells.

How chromatin bridges are detected by the abscission checkpoint during mammalian cell division is unknown. Here, we discuss recent findings from our lab showing that the DNA topoisomerase IIα (Top2α) enzyme binds to catenated ("knotted") DNA next to the midbody and forms abortive Top2-DNA cleavage complexes (Top2ccs) on chromatin bridges. Top2ccs are then processed by the proteasome to promote localization of the DNA damage sensor protein Rad17 to Top2-generated double-strand DNA ends on DNA knots. In turn, Rad17 promotes local recruitment of the MRN protein complex and downstream ATM-Chk2-INCENP signaling to delay abscission and prevent chromatin bridge breakage in cytokinesis.

Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variants.

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn's disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.

A neurodevelopmental disorder associated with a loss-of-function missense mutation in RAB35.

Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder.

From biosynthesis and beyond-Loss or overexpression of the cytokinin synthesis gene, iptA, alters cytokinesis and mitochondrial and amino acid metabolism in Dictyostelium discoideum.

Cytokinins (CKs) are a class of growth-promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA-IPTs). In this study, the social amoeba, Dictyostelium discoideum, was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate-IPT gene, iptA. The life cycle of D. discoideum is unique and possesses both single- and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum. To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA-deficiency results in cytokinesis defects, and both iptA-deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development.

Racgap1 knockdown results in cells with multiple cilia due to cytokinesis failure.

Most mammalian cells have a single primary cilium that acts as a signalling hub in mediating cellular functions. However, little is known about the mechanisms that result in aberrant supernumerary primary cilia per cell. In this study, we re-analysed a previously published whole-genome siRNA-based reverse genetic screen for genes mediating ciliogenesis to identify knockdowns that permit multi-ciliation. We identified siRNA knockdowns that caused significant formation of supernumerary cilia, validated candidate hits in different cell-lines and confirmed that RACGAP1, a component of the centralspindlin complex, was the strongest candidate hit at the whole-genome level. Following loss of RACGAP1, mother centrioles were specified correctly prior to ciliogenesis and the cilia appeared normal. Live cell imaging revealed that increased cilia incidence was caused by cytokinesis failure which led to the formation of multinucleate cells with supernumerary cilia. This suggests that the signalling mechanisms for ciliogenesis are unable to identify supernumerary centrosomes and therefore allow ciliation of duplicated centrosomes as if they were in a new diploid daughter cell. These results, demonstrating that aberrant ciliogenesis is de-coupled from cell cycle regulation, have functional implications in diseases marked by centrosomal amplification.

The molecular mechanisms driving Plasmodium cell division.

Malaria, a vector borne disease, is a major global health and socioeconomic problem caused by the apicomplexan protozoan parasite Plasmodium. The parasite alternates between mosquito vector and vertebrate host, with meiosis in the mosquito and proliferative mitotic cell division in both hosts. In the canonical eukaryotic model, cell division is either by open or closed mitosis and karyokinesis is followed by cytokinesis; whereas in Plasmodium closed mitosis is not directly accompanied by concomitant cell division. Key molecular players and regulatory mechanisms of this process have been identified, but the pivotal role of certain protein complexes and the post-translational modifications that modulate their actions are still to be deciphered. Here, we discuss recent evidence for the function of known proteins in Plasmodium cell division and processes that are potential novel targets for therapeutic intervention. We also identify key questions to open new and exciting research to understand divergent Plasmodium cell division.

Four-dimensional quantitative analysis of cell plate development in Arabidopsis using lattice light sheet microscopy identifies robust transition points between growth phases.

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis.

KIFC3 is a member of Kinesin-14 family motor proteins, which play a variety of roles such as centrosome cohesion, cytokinesis, vesicles transportation and cell proliferation in mitosis. Here, we investigated the functional roles of KIFC3 in meiosis. Our findings demonstrated that KIFC3 exhibited expression and localization at centromeres during metaphase I, followed by translocation to the midbody at telophase I throughout mouse oocyte meiosis. Disruption of KIFC3 activity resulted in defective polar body extrusion. We observed aberrant meiotic spindles and misaligned chromosomes, accompanied by the loss of kinetochore-microtubule attachment, which might be due to the failed recruitment of BubR1/Bub3. Coimmunoprecipitation data revealed that KIFC3 plays a crucial role in maintaining the acetylated tubulin level mediated by Sirt2, thereby influencing microtubule stability. Additionally, our findings demonstrated an interaction between KIFC3 and PRC1 in regulating midbody formation during telophase I, which is involved in cytokinesis regulation. Collectively, these results underscore the essential contribution of KIFC3 to spindle assembly and cytokinesis during mouse oocyte meiosis.

Cytokinetic engineering enhances the secretory production of recombinant human lysozyme in Komagataella phaffii.

BACKGROUND: Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast. RESULTS: Here through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ. CONCLUSIONS: Our work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.